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Sample records for bacterial ribosomal proteins

  1. Ribosome reinitiation at leader peptides increases translation of bacterial proteins.

    Science.gov (United States)

    Korolev, Semen A; Zverkov, Oleg A; Seliverstov, Alexandr V; Lyubetsky, Vassily A

    2016-01-01

    Short leader genes usually do not encode stable proteins, although their importance in expression control of bacterial genomes is widely accepted. Such genes are often involved in the control of attenuation regulation. However, the abundance of leader genes suggests that their role in bacteria is not limited to regulation. Specifically, we hypothesize that leader genes increase the expression of protein-coding (structural) genes via ribosome reinitiation at the leader peptide in the case of a short distance between the stop codon of the leader gene and the start codon of the structural gene. For instance, in Actinobacteria, the frequency of leader genes at a distance of 10-11 bp is about 70 % higher than the mean frequency within the 1 to 65 bp range; and it gradually decreases as the range grows longer. A pronounced peak of this frequency-distance relationship is also observed in Proteobacteria, Bacteroidetes, Spirochaetales, Acidobacteria, the Deinococcus-Thermus group, and Planctomycetes. In contrast, this peak falls to the distance of 15-16 bp and is not very pronounced in Firmicutes; and no such peak is observed in cyanobacteria and tenericutes. Generally, this peak is typical for many bacteria. Some leader genes located close to a structural gene probably play a regulatory role as well. PMID:27084079

  2. Mutations in the bacterial ribosomal protein l3 and their association with antibiotic resistance

    DEFF Research Database (Denmark)

    Klitgaard, Rasmus N; Ntokou, Eleni; Nørgaard, Katrine;

    2015-01-01

    Different groups of antibiotics bind to the peptidyl transferase center (PTC) in the large subunit of the bacterial ribosome. Resistance to these groups of antibiotics has often been linked with mutations or methylations of the 23S rRNA. In recent years, there has been a rise in the number of...... studies where mutations have been found in the ribosomal protein L3 in bacterial strains resistant to PTC-targeting antibiotics but there is often no evidence that these mutations actually confer antibiotic resistance. In this study, a plasmid exchange system was used to replace plasmid-carried wild...

  3. Horizontal gene transfer of zinc and non-zinc forms of bacterial ribosomal protein S4

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    Luthey-Schulten Zaida

    2009-07-01

    Full Text Available Abstract Background The universal ribosomal protein S4 is essential for the initiation of small subunit ribosomal assembly and translational accuracy. Being part of the information processing machinery of the cell, the gene for S4 is generally thought of as being inherited vertically and has been used in concatenated gene phylogenies. Here we report the evolution of ribosomal protein S4 in relation to a broad sharing of zinc/non-zinc forms of the gene and study the scope of horizontal gene transfer (HGT of S4 during bacterial evolution. Results In this study we present the complex evolutionary history of ribosomal protein S4 using 660 bacterial genomes from 16 major bacterial phyla. According to conserved characteristics in the sequences, S4 can be classified into C+ (zinc-binding and C- (zinc-free variants, with 26 genomes (mainly from the class Clostridia containing genes for both. A maximum likelihood phylogenetic tree of the S4 sequences was incongruent with the standard bacterial phylogeny, indicating a departure from strict vertical inheritance. Further analysis using the genome content near the S4 genes, which are usually located in a conserved gene cluster, showed not only that HGT of the C- gene had occurred at various stages of bacterial evolution, but also that both the C- and C+ genes were present before the individual phyla diverged. To explain the latter, we theorize that a gene pool existed early in bacterial evolution from which bacteria could sample S4 gene variants, according to environmental conditions. The distribution of the C+/- variants for seven other zinc-binding ribosomal proteins in these 660 bacterial genomes is consistent with that seen for S4 and may shed light on the evolutionary pressures involved. Conclusion The complex history presented for "core" protein S4 suggests the existence of a gene pool before the emergence of bacterial lineages and reflects the pervasive nature of HGT in subsequent bacterial evolution

  4. A protein residing at the subunit interface of the bacterial ribosome.

    Science.gov (United States)

    Agafonov, D E; Kolb, V A; Nazimov, I V; Spirin, A S

    1999-10-26

    Surface labeling of Escherichia coli ribosomes with the use of the tritium bombardment technique has revealed a minor unidentified ribosome-bound protein (spot Y) that is hidden in the 70S ribosome and becomes highly labeled on dissociation of the 70S ribosome into subunits. In the present work, the N-terminal sequence of the protein Y was determined and its gene was identified as yfia, an ORF located upstream the phe operon of E. coli. This 12.7-kDa protein was isolated and characterized. An affinity of the purified protein Y for the 30S subunit, but not for the 50S ribosomal subunit, was shown. The protein proved to be exposed on the surface of the 30S subunit. The attachment of the 50S subunit resulted in hiding the protein Y, thus suggesting the protein location at the subunit interface in the 70S ribosome. The protein was shown to stabilize ribosomes against dissociation. The possible role of the protein Y as ribosome association factor in translation is discussed. PMID:10535924

  5. Horizontal gene transfer of zinc and non-zinc forms of bacterial ribosomal protein S4

    OpenAIRE

    Luthey-Schulten Zaida; Roberts Elijah; Chen Ke

    2009-01-01

    Abstract Background The universal ribosomal protein S4 is essential for the initiation of small subunit ribosomal assembly and translational accuracy. Being part of the information processing machinery of the cell, the gene for S4 is generally thought of as being inherited vertically and has been used in concatenated gene phylogenies. Here we report the evolution of ribosomal protein S4 in relation to a broad sharing of zinc/non-zinc forms of the gene and study the scope of horizontal gene tr...

  6. Bacterial effector binding to ribosomal protein s3 subverts NF-kappaB function.

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    Xiaofei Gao

    2009-12-01

    Full Text Available Enteric bacterial pathogens cause food borne disease, which constitutes an enormous economic and health burden. Enterohemorrhagic Escherichia coli (EHEC causes a severe bloody diarrhea following transmission to humans through various means, including contaminated beef and vegetable products, water, or through contact with animals. EHEC also causes a potentially fatal kidney disease (hemolytic uremic syndrome for which there is no effective treatment or prophylaxis. EHEC and other enteric pathogens (e.g., enteropathogenic E. coli (EPEC, Salmonella, Shigella, Yersinia utilize a type III secretion system (T3SS to inject virulence proteins (effectors into host cells. While it is known that T3SS effectors subvert host cell function to promote diarrheal disease and bacterial transmission, in many cases, the mechanisms by which these effectors bind to host proteins and disrupt the normal function of intestinal epithelial cells have not been completely characterized. In this study, we present evidence that the E. coli O157:H7 nleH1 and nleH2 genes encode T3SS effectors that bind to the human ribosomal protein S3 (RPS3, a subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB transcriptional complexes. NleH1 and NleH2 co-localized with RPS3 in the cytoplasm, but not in cell nuclei. The N-terminal region of both NleH1 and NleH2 was required for binding to the N-terminus of RPS3. NleH1 and NleH2 are autophosphorylated Ser/Thr protein kinases, but their binding to RPS3 is independent of kinase activity. NleH1, but not NleH2, reduced the nuclear abundance of RPS3 without altering the p50 or p65 NF-kappaB subunits or affecting the phosphorylation state or abundance of the inhibitory NF-kappaB chaperone IkappaBalpha NleH1 repressed the transcription of a RPS3/NF-kappaB-dependent reporter plasmid, but did not inhibit the transcription of RPS3-independent reporters. In contrast, NleH2 stimulated RPS3-dependent transcription, as well

  7. Ribosome-inactivating proteins

    OpenAIRE

    Walsh, Matthew J; Dodd, Jennifer E; Hautbergue, Guillaume M.

    2013-01-01

    Ribosome-inactivating proteins (RIPs) were first isolated over a century ago and have been shown to be catalytic toxins that irreversibly inactivate protein synthesis. Elucidation of atomic structures and molecular mechanism has revealed these proteins to be a diverse group subdivided into two classes. RIPs have been shown to exhibit RNA N-glycosidase activity and depurinate the 28S rRNA of the eukaryotic 60S ribosomal subunit. In this review, we compare archetypal RIP family members with oth...

  8. Plant Ribosomal Proteins, RPL12 and RPL19, Play a Role in Nonhost Disease Resistance against Bacterial Pathogens.

    Science.gov (United States)

    Nagaraj, Satish; Senthil-Kumar, Muthappa; Ramu, Vemanna S; Wang, Keri; Mysore, Kirankumar S

    2015-01-01

    Characterizing the molecular mechanism involved in nonhost disease resistance is important to understand the adaptations of plant-pathogen interactions. In this study, virus-induced gene silencing (VIGS)-based forward genetics screen was utilized to identify genes involved in nonhost resistance in Nicotiana benthamiana. Genes encoding ribosomal proteins, RPL12 and RPL19, were identified in the screening. These genes when silenced in N. benthamiana caused a delay in nonhost bacteria induced hypersensitive response (HR) with concurrent increase in nonhost bacterial multiplication. Arabidopsis mutants of AtRPL12 and AtRPL19 also compromised nonhost resistance. The studies on NbRPL12 and NbRPL19 double silenced plants suggested that both RPL12 and RPL19 act in the same pathway to confer nonhost resistance. Our work suggests a role for RPL12 and RPL19 in nonhost disease resistance in N. benthamiana and Arabidopsis. In addition, we show that these genes also play a minor role in basal resistance against virulent pathogens. PMID:26779226

  9. Plant Ribosomal Proteins, RPL12 and RPL19, Play a Role in Nonhost Disease Resistance against Bacterial Pathogens

    Science.gov (United States)

    Nagaraj, Satish; Senthil-Kumar, Muthappa; Ramu, Vemanna S.; Wang, Keri; Mysore, Kirankumar S.

    2016-01-01

    Characterizing the molecular mechanism involved in nonhost disease resistance is important to understand the adaptations of plant-pathogen interactions. In this study, virus-induced gene silencing (VIGS)-based forward genetics screen was utilized to identify genes involved in nonhost resistance in Nicotiana benthamiana. Genes encoding ribosomal proteins, RPL12 and RPL19, were identified in the screening. These genes when silenced in N. benthamiana caused a delay in nonhost bacteria induced hypersensitive response (HR) with concurrent increase in nonhost bacterial multiplication. Arabidopsis mutants of AtRPL12 and AtRPL19 also compromised nonhost resistance. The studies on NbRPL12 and NbRPL19 double silenced plants suggested that both RPL12 and RPL19 act in the same pathway to confer nonhost resistance. Our work suggests a role for RPL12 and RPL19 in nonhost disease resistance in N. benthamiana and Arabidopsis. In addition, we show that these genes also play a minor role in basal resistance against virulent pathogens. PMID:26779226

  10. Inter-Protein Sequence Co-Evolution Predicts Known Physical Interactions in Bacterial Ribosomes and the Trp Operon.

    Science.gov (United States)

    Feinauer, Christoph; Szurmant, Hendrik; Weigt, Martin; Pagnani, Andrea

    2016-01-01

    Interaction between proteins is a fundamental mechanism that underlies virtually all biological processes. Many important interactions are conserved across a large variety of species. The need to maintain interaction leads to a high degree of co-evolution between residues in the interface between partner proteins. The inference of protein-protein interaction networks from the rapidly growing sequence databases is one of the most formidable tasks in systems biology today. We propose here a novel approach based on the Direct-Coupling Analysis of the co-evolution between inter-protein residue pairs. We use ribosomal and trp operon proteins as test cases: For the small resp. large ribosomal subunit our approach predicts protein-interaction partners at a true-positive rate of 70% resp. 90% within the first 10 predictions, with areas of 0.69 resp. 0.81 under the ROC curves for all predictions. In the trp operon, it assigns the two largest interaction scores to the only two interactions experimentally known. On the level of residue interactions we show that for both the small and the large ribosomal subunit our approach predicts interacting residues in the system with a true positive rate of 60% and 85% in the first 20 predictions. We use artificial data to show that the performance of our approach depends crucially on the size of the joint multiple sequence alignments and analyze how many sequences would be necessary for a perfect prediction if the sequences were sampled from the same model that we use for prediction. Given the performance of our approach on the test data we speculate that it can be used to detect new interactions, especially in the light of the rapid growth of available sequence data. PMID:26882169

  11. Inter-Protein Sequence Co-Evolution Predicts Known Physical Interactions in Bacterial Ribosomes and the Trp Operon.

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    Christoph Feinauer

    Full Text Available Interaction between proteins is a fundamental mechanism that underlies virtually all biological processes. Many important interactions are conserved across a large variety of species. The need to maintain interaction leads to a high degree of co-evolution between residues in the interface between partner proteins. The inference of protein-protein interaction networks from the rapidly growing sequence databases is one of the most formidable tasks in systems biology today. We propose here a novel approach based on the Direct-Coupling Analysis of the co-evolution between inter-protein residue pairs. We use ribosomal and trp operon proteins as test cases: For the small resp. large ribosomal subunit our approach predicts protein-interaction partners at a true-positive rate of 70% resp. 90% within the first 10 predictions, with areas of 0.69 resp. 0.81 under the ROC curves for all predictions. In the trp operon, it assigns the two largest interaction scores to the only two interactions experimentally known. On the level of residue interactions we show that for both the small and the large ribosomal subunit our approach predicts interacting residues in the system with a true positive rate of 60% and 85% in the first 20 predictions. We use artificial data to show that the performance of our approach depends crucially on the size of the joint multiple sequence alignments and analyze how many sequences would be necessary for a perfect prediction if the sequences were sampled from the same model that we use for prediction. Given the performance of our approach on the test data we speculate that it can be used to detect new interactions, especially in the light of the rapid growth of available sequence data.

  12. Chloroplast ribosomes and protein synthesis.

    OpenAIRE

    Harris, E. H.; Boynton, J E; Gillham, N W

    1994-01-01

    Consistent with their postulated origin from endosymbiotic cyanobacteria, chloroplasts of plants and algae have ribosomes whose component RNAs and proteins are strikingly similar to those of eubacteria. Comparison of the secondary structures of 16S rRNAs of chloroplasts and bacteria has been particularly useful in identifying highly conserved regions likely to have essential functions. Comparative analysis of ribosomal protein sequences may likewise prove valuable in determining their roles i...

  13. Cisplatin Targeting of Bacterial Ribosomal RNA Hairpins

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    Gayani N. P. Dedduwa-Mudalige

    2015-09-01

    Full Text Available Cisplatin is a clinically important chemotherapeutic agent known to target purine bases in nucleic acids. In addition to major deoxyribonucleic acid (DNA intrastrand cross-links, cisplatin also forms stable adducts with many types of ribonucleic acid (RNA including siRNA, spliceosomal RNAs, tRNA, and rRNA. All of these RNAs play vital roles in the cell, such as catalysis of protein synthesis by rRNA, and therefore serve as potential drug targets. This work focused on platination of two highly conserved RNA hairpins from E. coli ribosomes, namely pseudouridine-modified helix 69 from 23S rRNA and the 790 loop of helix 24 from 16S rRNA. RNase T1 probing, MALDI mass spectrometry, and dimethyl sulfate mapping revealed platination at GpG sites. Chemical probing results also showed platination-induced RNA structural changes. These findings reveal solvent and structural accessibility of sites within bacterial RNA secondary structures that are functionally significant and therefore viable targets for cisplatin as well as other classes of small molecules. Identifying target preferences at the nucleotide level, as well as determining cisplatin-induced RNA conformational changes, is important for the design of more potent drug molecules. Furthermore, the knowledge gained through studies of RNA-targeting by cisplatin is applicable to a broad range of organisms from bacteria to human.

  14. Neuron-Like Networks Between Ribosomal Proteins Within the Ribosome.

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    Poirot, Olivier; Timsit, Youri

    2016-01-01

    From brain to the World Wide Web, information-processing networks share common scale invariant properties. Here, we reveal the existence of neural-like networks at a molecular scale within the ribosome. We show that with their extensions, ribosomal proteins form complex assortative interaction networks through which they communicate through tiny interfaces. The analysis of the crystal structures of 50S eubacterial particles reveals that most of these interfaces involve key phylogenetically conserved residues. The systematic observation of interactions between basic and aromatic amino acids at the interfaces and along the extension provides new structural insights that may contribute to decipher the molecular mechanisms of signal transmission within or between the ribosomal proteins. Similar to neurons interacting through "molecular synapses", ribosomal proteins form a network that suggest an analogy with a simple molecular brain in which the "sensory-proteins" innervate the functional ribosomal sites, while the "inter-proteins" interconnect them into circuits suitable to process the information flow that circulates during protein synthesis. It is likely that these circuits have evolved to coordinate both the complex macromolecular motions and the binding of the multiple factors during translation. This opens new perspectives on nanoscale information transfer and processing. PMID:27225526

  15. Structures of the Bacterial Ribosome in Classical and Hybrid States of tRNA Binding

    Energy Technology Data Exchange (ETDEWEB)

    Dunkle, Jack A.; Wang, Leyi; Feldman, Michael B.; Pulk, Arto; Chen, Vincent B.; Kapral, Gary J.; Noeske, Jonas; Richardson, Jane S.; Blanchard, Scott C.; Cate, Jamie H. Doudna (Cornell); (UCB); (Duke)

    2011-09-06

    During protein synthesis, the ribosome controls the movement of tRNA and mRNA by means of large-scale structural rearrangements. We describe structures of the intact bacterial ribosome from Escherichia coli that reveal how the ribosome binds tRNA in two functionally distinct states, determined to a resolution of {approx}3.2 angstroms by means of x-ray crystallography. One state positions tRNA in the peptidyl-tRNA binding site. The second, a fully rotated state, is stabilized by ribosome recycling factor and binds tRNA in a highly bent conformation in a hybrid peptidyl/exit site. The structures help to explain how the ratchet-like motion of the two ribosomal subunits contributes to the mechanisms of translocation, termination, and ribosome recycling.

  16. Synthetic peptides and ribosomal proteins as substrate for 60S ribosomal protein kinase from yeast cells

    DEFF Research Database (Denmark)

    Grankowski, N; Gasior, E; Issinger, O G

    1993-01-01

    Kinetic studies on the 60S protein kinase were conducted with synthetic peptides and ribosomal proteins as substrate. Peptide RRREEESDDD proved to be the best synthetic substrate for this enzyme. The peptide has a sequence of amino acids which most closely resembles the structure of potential...... phosphorylation sites in natural substrates, i.e., acidic ribosomal proteins. The superiority of certain kinetic parameters for 60S kinase obtained with the native whole 80S ribosomes over those of the isolated fraction of acidic ribosomal proteins indicates that the affinity of 60S kinase to the specific protein...

  17. Novel Accurate Bacterial Discrimination by MALDI-Time-of-Flight MS Based on Ribosomal Proteins Coding in S10-spc-alpha Operon at Strain Level S10-GERMS

    Science.gov (United States)

    Tamura, Hiroto; Hotta, Yudai; Sato, Hiroaki

    2013-08-01

    Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is one of the most widely used mass-based approaches for bacterial identification and classification because of the simple sample preparation and extremely rapid analysis within a few minutes. To establish the accurate MALDI-TOF MS bacterial discrimination method at strain level, the ribosomal subunit proteins coded in the S 10-spc-alpha operon, which encodes half of the ribosomal subunit protein and is highly conserved in eubacterial genomes, were selected as reliable biomarkers. This method, named the S10-GERMS method, revealed that the strains of genus Pseudomonas were successfully identified and discriminated at species and strain levels, respectively; therefore, the S10-GERMS method was further applied to discriminate the pathovar of P. syringae. The eight selected biomarkers (L24, L30, S10, S12, S14, S16, S17, and S19) suggested the rapid discrimination of P. syringae at the strain (pathovar) level. The S10-GERMS method appears to be a powerful tool for rapid and reliable bacterial discrimination and successful phylogenetic characterization. In this article, an overview of the utilization of results from the S10-GERMS method is presented, highlighting the characterization of the Lactobacillus casei group and discrimination of the bacteria of genera Bacillus and Sphingopyxis despite only two and one base difference in the 16S rRNA gene sequence, respectively.

  18. RPG: the Ribosomal Protein Gene database

    OpenAIRE

    Nakao, Akihiro; Yoshihama, Maki; Kenmochi, Naoya

    2004-01-01

    RPG (http://ribosome.miyazaki-med.ac.jp/) is a new database that provides detailed information about ribosomal protein (RP) genes. It contains data from humans and other organisms, including Drosophila melanogaster, Caenorhabditis elegans, Saccharo myces cerevisiae, Methanococcus jannaschii and Escherichia coli. Users can search the database by gene name and organism. Each record includes sequences (genomic, cDNA and amino acid sequences), intron/exon structures, genomic locations and informa...

  19. A comparative study of ribosomal proteins: linkage between amino acid distribution and ribosomal assembly

    International Nuclear Information System (INIS)

    Assembly of the ribosome from its protein and RNA constituents must occur quickly and efficiently in order to synthesize the proteins necessary for all cellular activity. Since the early 1960’s, certain characteristics of possible assembly pathways have been elucidated, yet the mechanisms that govern the precise recognition events remain unclear. We utilize a comparative analysis to investigate the amino acid composition of ribosomal proteins (r-proteins) with respect to their role in the assembly process. We compared small subunit (30S) r-protein sequences to those of other housekeeping proteins from 560 bacterial species and searched for correlations between r-protein amino acid content and factors such as assembly binding order, environmental growth temperature, protein size, and contact with ribosomal RNA (rRNA) in the 30S complex. We find r-proteins have a significantly high percent of positive residues, which are highly represented at rRNA contact sites. An inverse correlation between the percent of positive residues and r-protein size was identified and is mainly due to the content of Lysine residues, rather than Arginine. Nearly all r-proteins carry a net positive charge, but no statistical correlation between the net charge and the binding order was detected. Thermophilic (high-temperature) r-proteins contain increased Arginine, Isoleucine, and Tyrosine, and decreased Serine and Threonine compared to mesophilic (lower-temperature), reflecting a known distinction between thermophiles and mesophiles, possibly to account for protein thermostability. However, this difference in amino acid content does not extend to rRNA contact sites, as the proportions of thermophilic and mesophilic contact residues are not significantly different. Given the significantly higher level of positively charged residues in r-proteins and at contact sites, we conclude that ribosome assembly relies heavily on an electrostatic component of interaction. However, the binding order of

  20. A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

    OpenAIRE

    Schütz, Sabina; Fischer, Ute; Altvater, Martin; Nerurkar, Purnima; Peña, Cohue; Gerber, Michaela; Chang, Yiming; Caesar, Stefanie; Schubert, Olga T; Schlenstedt, Gabriel; Panse, Vikram G.

    2014-01-01

    eLife digest The production of a protein in a cell starts with a region of DNA being transcribed to produce a molecule of messenger RNA. A large molecular machine called ribosome then reads the information in the messenger RNA molecule to produce a protein. Ribosomes themselves are made of RNA and several different proteins called r-proteins. The construction of a ribosome starts with the assembly of a pre-ribosome inside the cell nucleus, and the ribosome is completed in the cytosol of the c...

  1. Interrelationships between yeast ribosomal protein assembly events and transient ribosome biogenesis factors interactions in early pre-ribosomes.

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    Steffen Jakob

    Full Text Available Early steps of eukaryotic ribosome biogenesis require a large set of ribosome biogenesis factors which transiently interact with nascent rRNA precursors (pre-rRNA. Most likely, concomitant with that initial contacts between ribosomal proteins (r-proteins and ribosome precursors (pre-ribosomes are established which are converted into robust interactions between pre-rRNA and r-proteins during the course of ribosome maturation. Here we analysed the interrelationship between r-protein assembly events and the transient interactions of ribosome biogenesis factors with early pre-ribosomal intermediates termed 90S pre-ribosomes or small ribosomal subunit (SSU processome in yeast cells. We observed that components of the SSU processome UTP-A and UTP-B sub-modules were recruited to early pre-ribosomes independently of all tested r-proteins. On the other hand, groups of SSU processome components were identified whose association with early pre-ribosomes was affected by specific r-protein assembly events in the head-platform interface of the SSU. One of these components, Noc4p, appeared to be itself required for robust incorporation of r-proteins into the SSU head domain. Altogether, the data reveal an emerging network of specific interrelationships between local r-protein assembly events and the functional interactions of SSU processome components with early pre-ribosomes. They point towards some of these components being transient primary pre-rRNA in vivo binders and towards a role for others in coordinating the assembly of major SSU domains.

  2. The Bacterial Translocon SecYEG Opens upon Ribosome Binding*

    OpenAIRE

    Knyazev, Denis G.; Lents, Alexander; Krause, Eberhard; Ollinger, Nicole; Siligan, Christine; Papinski, Daniel; Winter, Lukas; Horner, Andreas; Pohl, Peter

    2013-01-01

    In co-translational translocation, the ribosome funnel and the channel of the protein translocation complex SecYEG are aligned. For the nascent chain to enter the channel immediately after synthesis, a yet unidentified signal triggers displacement of the SecYEG sealing plug from the pore. Here, we show that ribosome binding to the resting SecYEG channel triggers this conformational transition. The purified and reconstituted SecYEG channel opens to form a large ion-conducting channel, which ha...

  3. Ribosome Inactivating Proteins from Plants Inhibiting Viruses

    Institute of Scientific and Technical Information of China (English)

    Inderdeep Kaur; R C Gupta; Munish Puri

    2011-01-01

    Many plants contain ribosome inactivating proteins (RIPs) with N-glycosidase activity,which depurinate large ribosomal RNA and arrest protein synthesis.RIPs so far tested inhibit replication of mRNA as well as DNA viruses and these proteins,isolated from plants,are found to be effective against a broad range of viruses such as human immunodeficiency virus (HIV),hepatitis B virus (HBV) and herpes simplex virus (HSV).Most of the research work related to RIPs has been focused on antiviral activity against HIV; however,the exact mechanism of antiviral activity is still not clear.The mechanism of antiviral activity was thought to follow inactivation of the host cell ribosome,leading to inhibition of viral protein translation and host cell death.Enzymatic activity of RIPs is not hmited to depurination of the large rRNA,in addition they can depurinate viral DNA as well as RNA.Recently,Phase Ⅰ/Ⅱ clinical trials have demonstrated the potential use of RIPs for treating patients with HIV disease.The aim of this review is to focus on various RIPs from plants associated with anti-HIV activity.

  4. The bacterial translocon SecYEG opens upon ribosome binding.

    Science.gov (United States)

    Knyazev, Denis G; Lents, Alexander; Krause, Eberhard; Ollinger, Nicole; Siligan, Christine; Papinski, Daniel; Winter, Lukas; Horner, Andreas; Pohl, Peter

    2013-06-21

    In co-translational translocation, the ribosome funnel and the channel of the protein translocation complex SecYEG are aligned. For the nascent chain to enter the channel immediately after synthesis, a yet unidentified signal triggers displacement of the SecYEG sealing plug from the pore. Here, we show that ribosome binding to the resting SecYEG channel triggers this conformational transition. The purified and reconstituted SecYEG channel opens to form a large ion-conducting channel, which has the conductivity of the plug deletion mutant. The number of ion-conducting channels inserted into the planar bilayer per fusion event roughly equals the number of SecYEG channels counted by fluorescence correlation spectroscopy in a single proteoliposome. Thus, the open probability of the channel must be close to unity. To prevent the otherwise lethal proton leak, a closed post-translational conformation of the SecYEG complex bound to a ribosome must exist. PMID:23645666

  5. The Bacterial Translocon SecYEG Opens upon Ribosome Binding*

    Science.gov (United States)

    Knyazev, Denis G.; Lents, Alexander; Krause, Eberhard; Ollinger, Nicole; Siligan, Christine; Papinski, Daniel; Winter, Lukas; Horner, Andreas; Pohl, Peter

    2013-01-01

    In co-translational translocation, the ribosome funnel and the channel of the protein translocation complex SecYEG are aligned. For the nascent chain to enter the channel immediately after synthesis, a yet unidentified signal triggers displacement of the SecYEG sealing plug from the pore. Here, we show that ribosome binding to the resting SecYEG channel triggers this conformational transition. The purified and reconstituted SecYEG channel opens to form a large ion-conducting channel, which has the conductivity of the plug deletion mutant. The number of ion-conducting channels inserted into the planar bilayer per fusion event roughly equals the number of SecYEG channels counted by fluorescence correlation spectroscopy in a single proteoliposome. Thus, the open probability of the channel must be close to unity. To prevent the otherwise lethal proton leak, a closed post-translational conformation of the SecYEG complex bound to a ribosome must exist. PMID:23645666

  6. Structural and Functional Studies of Ribosome-inactivating Proteins and Ribosomal RNA

    Institute of Scientific and Technical Information of China (English)

    LIU Wangyi; ZHANG Jinsong; LIU Renshui; HE Wenjun; LING Jun

    2007-01-01

    @@ A plant's ribosome-inactivating proteins (RIPs) are a group of toxic proteins. Theoretically, they can be employed as a tool enzyme in the exploration of the structure and function of the ribosomal RNA; in practical application, they can be used as an insecticide in agriculture, for preparation of immuno-toxic protein to kill cancer cells or against viral infection in medicine.

  7. Ribosome-Inactivating and Related Proteins

    OpenAIRE

    Joachim Schrot; Alexander Weng; Matthias F. Melzig

    2015-01-01

    Ribosome-inactivating proteins (RIPs) are toxins that act as N-glycosidases (EC 3.2.2.22). They are mainly produced by plants and classified as type 1 RIPs and type 2 RIPs. There are also RIPs and RIP related proteins that cannot be grouped into the classical type 1 and type 2 RIPs because of their different sizes, structures or functions. In addition, there is still not a uniform nomenclature or classification existing for RIPs. In this review, we give the current status of all known plant ...

  8. Protein-protein interactions within late pre-40S ribosomes.

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    Melody G Campbell

    Full Text Available Ribosome assembly in eukaryotic organisms requires more than 200 assembly factors to facilitate and coordinate rRNA transcription, processing, and folding with the binding of the ribosomal proteins. Many of these assembly factors bind and dissociate at defined times giving rise to discrete assembly intermediates, some of which have been partially characterized with regards to their protein and RNA composition. Here, we have analyzed the protein-protein interactions between the seven assembly factors bound to late cytoplasmic pre-40S ribosomes using recombinant proteins in binding assays. Our data show that these factors form two modules: one comprising Enp1 and the export adaptor Ltv1 near the beak structure, and the second comprising the kinase Rio2, the nuclease Nob1, and a regulatory RNA binding protein Dim2/Pno1 on the front of the head. The GTPase-like Tsr1 and the universally conserved methylase Dim1 are also peripherally connected to this second module. Additionally, in an effort to further define the locations for these essential proteins, we have analyzed the interactions between these assembly factors and six ribosomal proteins: Rps0, Rps3, Rps5, Rps14, Rps15 and Rps29. Together, these results and previous RNA-protein crosslinking data allow us to propose a model for the binding sites of these seven assembly factors. Furthermore, our data show that the essential kinase Rio2 is located at the center of the pre-ribosomal particle and interacts, directly or indirectly, with every other assembly factor, as well as three ribosomal proteins required for cytoplasmic 40S maturation. These data suggest that Rio2 could play a central role in regulating cytoplasmic maturation steps.

  9. The architecture of mammalian ribosomal protein promoters

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    Perry Robert P

    2005-02-01

    Full Text Available Abstract Background Mammalian ribosomes contain 79 different proteins encoded by widely scattered single copy genes. Coordinate expression of these genes at transcriptional and post-transcriptional levels is required to ensure a roughly equimolar accumulation of ribosomal proteins. To date, detailed studies of only a very few ribosomal protein (rp promoters have been made. To elucidate the general features of rp promoter architecture, I made a detailed sequence comparison of the promoter regions of the entire set of orthologous human and mouse rp genes. Results A striking evolutionarily conserved feature of most rp genes is the separation by an intron of the sequences involved in transcriptional and translational regulation from the sequences with protein encoding function. Another conserved feature is the polypyrimidine initiator, which conforms to the consensus (Y2C+1TY(T2(Y3. At least 60 % of the rp promoters contain a largely conserved TATA box or A/T-rich motif, which should theoretically have TBP-binding capability. A remarkably high proportion of the promoters contain conserved binding sites for transcription factors that were previously implicated in rp gene expression, namely upstream GABP and Sp1 sites and downstream YY1 sites. Over 80 % of human and mouse rp genes contain a transposable element residue within 900 bp of 5' flanking sequence; very little sequence identity between human and mouse orthologues was evident more than 200 bp upstream of the transcriptional start point. Conclusions This analysis has provided some valuable insights into the general architecture of mammalian rp promoters and has identified parameters that might coordinately regulate the transcriptional activity of certain subsets of rp genes.

  10. The role of human ribosomal proteins in the maturation of rRNA and ribosome production

    OpenAIRE

    Robledo, Sara; Rachel A Idol; Crimmins, Dan L.; Ladenson, Jack H.; Mason, Philip J.; Bessler, Monica

    2008-01-01

    Production of ribosomes is a fundamental process that occurs in all dividing cells. It is a complex process consisting of the coordinated synthesis and assembly of four ribosomal RNAs (rRNA) with about 80 ribosomal proteins (r-proteins) involving more than 150 nonribosomal proteins and other factors. Diamond Blackfan anemia (DBA) is an inherited red cell aplasia caused by mutations in one of several r-proteins. How defects in r-proteins, essential for proliferation in all cells, lead to a hum...

  11. A comparative study of ribosomal proteins: linkage between amino acid distribution and ribosomal assembly

    OpenAIRE

    Lott, Brittany Burton; Wang, Yongmei; Nakazato, Takuya

    2013-01-01

    Background Assembly of the ribosome from its protein and RNA constituents must occur quickly and efficiently in order to synthesize the proteins necessary for all cellular activity. Since the early 1960’s, certain characteristics of possible assembly pathways have been elucidated, yet the mechanisms that govern the precise recognition events remain unclear. We utilize a comparative analysis to investigate the amino acid composition of ribosomal proteins (r-proteins) with respect to their role...

  12. The ribosome can prevent aggregation of partially folded protein intermediates: studies using the Escherichia coli ribosome.

    Directory of Open Access Journals (Sweden)

    Bani Kumar Pathak

    Full Text Available BACKGROUND: Molecular chaperones that support de novo folding of proteins under non stress condition are classified as chaperone 'foldases' that are distinct from chaperone' holdases' that provide high affinity binding platform for unfolded proteins and prevent their aggregation specifically under stress conditions. Ribosome, the cellular protein synthesis machine can act as a foldase chaperone that can bind unfolded proteins and release them in folding competent state. The peptidyl transferase center (PTC located in the domain V of the 23S rRNA of Escherichia coli ribosome (bDV RNA is the chaperoning center of the ribosome. It has been proposed that via specific interactions between the RNA and refolding proteins, the chaperone provides information for the correct folding of unfolded polypeptide chains. RESULTS: We demonstrate using Escherichia coli ribosome and variants of its domain V RNA that the ribosome can bind to partially folded intermediates of bovine carbonic anhydrase II (BCAII and lysozyme and suppress aggregation during their refolding. Using mutants of domain V RNA we demonstrate that the time for which the chaperone retains the bound protein is an important factor in determining its ability to suppress aggregation and/or support reactivation of protein. CONCLUSION: The ribosome can behave like a 'holdase' chaperone and has the ability to bind and hold back partially folded intermediate states of proteins from participating in the aggregation process. Since the ribosome is an essential organelle that is present in large numbers in all living cells, this ability of the ribosome provides an energetically inexpensive way to suppress cellular aggregation. Further, this ability of the ribosome might also be crucial in the context that the ribosome is one of the first chaperones to be encountered by a large nascent polypeptide chains that have a tendency to form partially folded intermediates immediately following their synthesis.

  13. Ribosome-Inactivating and Related Proteins

    Directory of Open Access Journals (Sweden)

    Joachim Schrot

    2015-05-01

    Full Text Available Ribosome-inactivating proteins (RIPs are toxins that act as N-glycosidases (EC 3.2.2.22. They are mainly produced by plants and classified as type 1 RIPs and type 2 RIPs. There are also RIPs and RIP related proteins that cannot be grouped into the classical type 1 and type 2 RIPs because of their different sizes, structures or functions. In addition, there is still not a uniform nomenclature or classification existing for RIPs. In this review, we give the current status of all known plant RIPs and we make a suggestion about how to unify those RIPs and RIP related proteins that cannot be classified as type 1 or type 2 RIPs.

  14. YsxC, an essential protein in Staphylococcus aureus crucial for ribosome assembly/stability

    Directory of Open Access Journals (Sweden)

    García-Lara Jorge

    2009-12-01

    Full Text Available Abstract Background Bacterial growth and division requires a core set of essential proteins, several of which are still of unknown function. They are also attractive targets for the development of new antibiotics. YsxC is a member of a family of GTPases highly conserved across eubacteria with a possible ribosome associated function. Results Here, we demonstrate by the creation of a conditional lethal mutant that ysxC is apparently essential for growth in S. aureus. To begin to elucidate YsxC function, a translational fusion of YsxC to the CBP-ProteinA tag in the staphylococcal chromosome was made, enabling Tandem Affinity Purification (TAP of YsxC-interacting partners. These included the ribosomal proteins S2, S10 and L17, as well as the β' subunit of the RNA polymerase. YsxC was then shown to copurify with ribosomes as an accessory protein specifically localizing to the 50 S subunit. YsxC depletion led to a decrease in the presence of mature ribosomes, indicating a role in ribosome assembly and/or stability in S. aureus. Conclusions In this study we demonstrate that YsxC of S. aureus localizes to the ribosomes, is crucial for ribosomal stability and is apparently essential for the life of S. aureus.

  15. Analysis of ribosomal protein gene structures: implications for intron evolution.

    Directory of Open Access Journals (Sweden)

    2006-03-01

    Full Text Available Many spliceosomal introns exist in the eukaryotic nuclear genome. Despite much research, the evolution of spliceosomal introns remains poorly understood. In this paper, we tried to gain insights into intron evolution from a novel perspective by comparing the gene structures of cytoplasmic ribosomal proteins (CRPs and mitochondrial ribosomal proteins (MRPs, which are held to be of archaeal and bacterial origin, respectively. We analyzed 25 homologous pairs of CRP and MRP genes that together had a total of 527 intron positions. We found that all 12 of the intron positions shared by CRP and MRP genes resulted from parallel intron gains and none could be considered to be "conserved," i.e., descendants of the same ancestor. This was supported further by the high frequency of proto-splice sites at these shared positions; proto-splice sites are proposed to be sites for intron insertion. Although we could not definitively disprove that spliceosomal introns were already present in the last universal common ancestor, our results lend more support to the idea that introns were gained late. At least, our results show that MRP genes were intronless at the time of endosymbiosis. The parallel intron gains between CRP and MRP genes accounted for 2.3% of total intron positions, which should provide a reliable estimate for future inferences of intron evolution.

  16. A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly.

    Science.gov (United States)

    Schütz, Sabina; Fischer, Ute; Altvater, Martin; Nerurkar, Purnima; Peña, Cohue; Gerber, Michaela; Chang, Yiming; Caesar, Stefanie; Schubert, Olga T; Schlenstedt, Gabriel; Panse, Vikram G

    2014-01-01

    Within a single generation time a growing yeast cell imports ∼14 million ribosomal proteins (r-proteins) into the nucleus for ribosome production. After import, it is unclear how these intrinsically unstable and aggregation-prone proteins are targeted to the ribosome assembly site in the nucleolus. Here, we report the discovery of a conserved nuclear carrier Tsr2 that coordinates transfer of the r-protein eS26 to the earliest assembling pre-ribosome, the 90S. In vitro studies revealed that Tsr2 efficiently dissociates importin:eS26 complexes via an atypical RanGTP-independent mechanism that terminates the import process. Subsequently, Tsr2 binds the released eS26, shields it from proteolysis, and ensures its safe delivery to the 90S pre-ribosome. We anticipate similar carriers-termed here escortins-to securely connect the nuclear import machinery with pathways that deposit r-proteins onto developing pre-ribosomal particles. PMID:25144938

  17. Phosphorylation of acidic ribosomal proteins from rabbit reticulocytes by a ribosome-associated casein kinase

    DEFF Research Database (Denmark)

    Issinger, O G

    1977-01-01

    Two acidic proteins from 80-S ribosomes were isolated and purified to homogeneity. The purified acidic proteins could be phosphorylated by casein kinase using [gamma-32P]ATP and [gamma-32P]GTP as a phosphoryl donor. The proteins became phosphorylated in situ, too. Sodium dodecyl sulfate...

  18. Molecular mechanisms of ribosomal protein gene coregulation.

    Science.gov (United States)

    Reja, Rohit; Vinayachandran, Vinesh; Ghosh, Sujana; Pugh, B Franklin

    2015-09-15

    The 137 ribosomal protein genes (RPGs) of Saccharomyces provide a model for gene coregulation. We examined the positional and functional organization of their regulators (Rap1 [repressor activator protein 1], Fhl1, Ifh1, Sfp1, and Hmo1), the transcription machinery (TFIIB, TFIID, and RNA polymerase II), and chromatin at near-base-pair resolution using ChIP-exo, as RPGs are coordinately reprogrammed. Where Hmo1 is enriched, Fhl1, Ifh1, Sfp1, and Hmo1 cross-linked broadly to promoter DNA in an RPG-specific manner and demarcated by general minor groove widening. Importantly, Hmo1 extended 20-50 base pairs (bp) downstream from Fhl1. Upon RPG repression, Fhl1 remained in place. Hmo1 dissociated, which was coupled to an upstream shift of the +1 nucleosome, as reflected by the Hmo1 extension and core promoter region. Fhl1 and Hmo1 may create two regulatable and positionally distinct barriers, against which chromatin remodelers position the +1 nucleosome into either an activating or a repressive state. Consistent with in vitro studies, we found that specific TFIID subunits, in addition to cross-linking at the core promoter, made precise cross-links at Rap1 sites, which we interpret to reflect native Rap1-TFIID interactions. Our findings suggest how sequence-specific DNA binding regulates nucleosome positioning and transcription complex assembly >300 bp away and how coregulation coevolved with coding sequences. PMID:26385964

  19. Conservation of ribosomal protein gene ordering in 16 complete genomes

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The organization of ribosomal proteins in 16 prokaryotic genomes was studied as an example of comparative genome analyses of gene systems. Hypothetical ribosomal protein-containing operons were constructed. These operons also contained putative genes and other non-ribosomal genes. The correspondences among these genes across different organisms were clarified by sequence homology computations. In this way a cross tabulation of 70 ribosomal proteins genes was constructed. On average, these were organized into 9-14 operons in each genome. There were also 25 non-ribosomal or putative genes in these mainly ribosomal protein operons. Hence the table contains 95 genes in total. It was found that: (i) the conservation of the block of about 20 r-proteins in the L3 and L4 operons across almost the entire eubacteria and archaebacteria is remarkable; (ii) some operons only belong to eubacteria or archaebacteria; (iii) although the ribosomal protein operons are highly conserved within domain, there are fine variations in some operons across different organisms within each domain, and these variations are informative on the evolutionary relations among the organisms. This method provides a new potential for studying the origin and evolution of old species.

  20. Conservation of ribosomal protein gene ordering in 16 complete genomes

    Institute of Scientific and Technical Information of China (English)

    王宁; 陈润生; 王永雄

    2000-01-01

    The organization of ribosomal proteins in 16 prokaryotic genomes was studied as an example of comparative genome analyses of gene systems. Hypothetical ribosomal protein-containing operons were constructed. These operons also contained putative genes and other non-ribosomal genes. The correspondences among these genes across different organisms were clarified by sequence homology computations. In this way a cross tabulation of 70 ribosomal proteins genes was constructed. On average, these were organized into 9-14 operons in each genome. There were also 25 non-ribosomal or putative genes in these mainly ribosomal protein operons. Hence the table contains 95 genes in total. It was found that: (i) the conservation of the block of about 20 r-proteins in the L3 and L4 operons across almost the entire eubacteria and ar-chaebacteria is remarkable; (ii) some operons only belong to eubacteria or archaebacte-ria; (iii) although the ribosomal protein operons are highly conserved within domain, there are fine variat

  1. Disassembly of yeast 80S ribosomes into subunits is a concerted action of ribosome-assisted folding of denatured protein.

    Science.gov (United States)

    Chakraborty, Biprashekhar; Bhakta, Sayan; Sengupta, Jayati

    2016-01-22

    It has been shown by several groups that ribosome can assist folding of denatured protein in vitro and the process is conserved across the species. Domain V of large ribosomal rRNA which occupies the intersubunit side of the large subunit was identified as the key player responsible for chaperoning the folding process. Thus, it is conceivable that denatured protein needs to access the intersubunit space of the ribosome in order to get folded. In this study, we have investigated the mechanism of release of the protein from the eukaryotic ribosome following reactivation. We have observed significant splitting of yeast 80S ribosome when incubated with the denatured BCAII protein. Energy-free disassembly mechanism functions in low Mg(+2) ion concentration for prokaryotic ribosomes. Eukaryotic ribosomes do not show significant splitting even at low Mg(+2) ion concentration. In this respect, denatured protein-induced disassembly of eukaryotic ribosome without the involvement of any external energy source is intriguing. For prokaryotic ribosomes, it was reported that the denatured protein induces ribosome splitting into subunits in order to access domain V-rRNA. In contrast, our results suggest an alternative mechanism for eukaryotic ribosomal rRNA-mediated protein folding and subsequent separation of the subunits by which release of the activated-protein occurs. PMID:26723252

  2. A-Site Residues Move Independently from P-Site Residues in all-Atom Molecular Dynamics Simulations of the 70S Bacterial Ribosome

    OpenAIRE

    Relly Brandman; Yigal Brandman; Pande, Vijay S.

    2012-01-01

    The ribosome is a large macromolecular machine, and correlated motion between residues is necessary for coordinating function across multiple protein and RNA chains. We ran two all-atom, explicit solvent molecular dynamics simulations of the bacterial ribosome and calculated correlated motion between residue pairs by using mutual information. Because of the short timescales of our simulation (ns), we expect that dynamics are largely local fluctuations around the crystal structure. We hypothes...

  3. Effects of ribosomal exit tunnel on protein's cotranslational folding

    International Nuclear Information System (INIS)

    In vivo, folding of many proteins occurs during their synthesis in the ribosome and continues after they have escaped from the ribosomal exit tunnel. In this research, we investigate the confinement effects of the ribosome on the cotranslational folding of three proteins, of PDB codes 1PGA, 1CRN and 2RJX, by using a coarse-grained model and molecular dynamics simulation. The exit tunnel is modeled as a hollow cylinder attached to a flat wall, whereas a Go-like model is adopted for the proteins. Our results show that the exit tunnel has a strong effect on the folding mechanism by setting an order by which the secondary and tertiary structures are formed. For protein 1PGA, the folding follows two different folding routes. The presence of the tunnel also improves the foldability of protein. (author)

  4. A ribosome-inactivating protein in a Drosophila defensive symbiont.

    Science.gov (United States)

    Hamilton, Phineas T; Peng, Fangni; Boulanger, Martin J; Perlman, Steve J

    2016-01-12

    Vertically transmitted symbionts that protect their hosts against parasites and pathogens are well known from insects, yet the underlying mechanisms of symbiont-mediated defense are largely unclear. A striking example of an ecologically important defensive symbiosis involves the woodland fly Drosophila neotestacea, which is protected by the bacterial endosymbiont Spiroplasma when parasitized by the nematode Howardula aoronymphium. The benefit of this defense strategy has led to the rapid spread of Spiroplasma throughout the range of D. neotestacea, although the molecular basis for this protection has been unresolved. Here, we show that Spiroplasma encodes a ribosome-inactivating protein (RIP) related to Shiga-like toxins from enterohemorrhagic Escherichia coli and that Howardula ribosomal RNA (rRNA) is depurinated during Spiroplasma-mediated protection of D. neotestacea. First, we show that recombinant Spiroplasma RIP catalyzes depurination of 28S rRNAs in a cell-free assay, as well as Howardula rRNA in vitro at the canonical RIP target site within the α-sarcin/ricin loop (SRL) of 28S rRNA. We then show that Howardula parasites in Spiroplasma-infected flies show a strong signal of rRNA depurination consistent with RIP-dependent modification and large decreases in the proportion of 28S rRNA intact at the α-sarcin/ricin loop. Notably, host 28S rRNA is largely unaffected, suggesting targeted specificity. Collectively, our study identifies a novel RIP in an insect defensive symbiont and suggests an underlying RIP-dependent mechanism in Spiroplasma-mediated defense. PMID:26712000

  5. Bacterial Protein Synthesis as a Target for Antibiotic Inhibition.

    Science.gov (United States)

    Arenz, Stefan; Wilson, Daniel N

    2016-01-01

    Protein synthesis occurs on macromolecular machines, called ribosomes. Bacterial ribosomes and the translational machinery represent one of the major targets for antibiotics in the cell. Therefore, structural and biochemical investigations into ribosome-targeting antibiotics provide not only insight into the mechanism of action and resistance of antibiotics, but also insight into the fundamental process of protein synthesis. This review summarizes the recent advances in our understanding of protein synthesis, particularly with respect to X-ray and cryoelectron microscopy (cryo-EM) structures of ribosome complexes, and highlights the different steps of translation that are targeted by the diverse array of known antibiotics. Such findings will be important for the ongoing development of novel and improved antimicrobial agents to combat the rapid emergence of multidrug resistant pathogenic bacteria. PMID:27481773

  6. The ribosomal protein Rpl22 controls ribosome composition by directly repressing expression of its own paralog, Rpl22l1.

    Directory of Open Access Journals (Sweden)

    Monique N O'Leary

    Full Text Available Most yeast ribosomal protein genes are duplicated and their characterization has led to hypotheses regarding the existence of specialized ribosomes with different subunit composition or specifically-tailored functions. In yeast, ribosomal protein genes are generally duplicated and evidence has emerged that paralogs might have specific roles. Unlike yeast, most mammalian ribosomal proteins are thought to be encoded by a single gene copy, raising the possibility that heterogenous populations of ribosomes are unique to yeast. Here, we examine the roles of the mammalian Rpl22, finding that Rpl22(-/- mice have only subtle phenotypes with no significant translation defects. We find that in the Rpl22(-/- mouse there is a compensatory increase in Rpl22-like1 (Rpl22l1 expression and incorporation into ribosomes. Consistent with the hypothesis that either ribosomal protein can support translation, knockdown of Rpl22l1 impairs growth of cells lacking Rpl22. Mechanistically, Rpl22 regulates Rpl22l1 directly by binding to an internal hairpin structure and repressing its expression. We propose that ribosome specificity may exist in mammals, providing evidence that one ribosomal protein can influence composition of the ribosome by regulating its own paralog.

  7. Genomic location of the major ribosomal protein gene locus determines Vibrio cholerae global growth and infectivity.

    OpenAIRE

    Soler-Bistué, Alfonso; Mondotte, Juan A; Bland, Michael Jason; Val, Marie-Eve; Saleh, María-Carla; Mazel, Didier

    2015-01-01

    The effects on cell physiology of gene order within the bacterial chromosome are poorly understood. In silico approaches have shown that genes involved in transcription and translation processes, in particular ribosomal protein (RP) genes, localize near the replication origin (oriC) in fast-growing bacteria suggesting that such a positional bias is an evolutionarily conserved growth-optimization strategy. Such genomic localization could either provide a higher dosage of these genes during fas...

  8. Mechanism of Tetracycline Resistance by Ribosomal Protection Protein Tet(O)

    Science.gov (United States)

    Li, Wen; Atkinson, Gemma C.; Thakor, Nehal S.; Allas, Ülar; Lu, Chuao-chao; Chan, Kwok-Yan; Tenson, Tanel; Schulten, Klaus; Wilson, Kevin S.; Hauryliuk, Vasili; Frank, Joachim

    2013-01-01

    Tetracycline resistance protein Tet(O), which protects the bacterial ribosome from binding the antibiotic tetracycline, is a translational GTPase with significant similarity in both sequence and structure to the elongation factor EF-G. Here, we present an atomic model of the Tet(O)-bound 70S ribosome based on our cryo-electron microscopic reconstruction at 9.6 Å resolution. This atomic model allowed us to identify the Tet(O)-ribosome binding sites, which involve three characteristic loops in domain 4 of Tet(O). Replacements of the three-amino acid tips of these loops by a single glycine residue result in loss of Tet(O)-mediated tetracycline resistance. On the basis of these findings, the mechanism of Tet(O)-mediated tetracycline resistance can be explained in molecular detail. PMID:23403578

  9. Mechanism of fusidic acid inhibition of RRF- and EF-G-dependent splitting of the bacterial post-termination ribosome

    OpenAIRE

    Borg, Anneli; Pavlov, Michael; Ehrenberg, Måns

    2016-01-01

    The antibiotic drug fusidic acid (FA) is commonly used in the clinic against gram-positive bacterial infections. FA targets ribosome-bound elongation factor G (EF-G), a translational GTPase that accelerates both messenger RNA (mRNA) translocation and ribosome recycling. How FA inhibits translocation was recently clarified, but FA inhibition of ribosome recycling by EF-G and ribosome recycling factor (RRF) has remained obscure. Here we use fast kinetics techniques to estimate mean times of rib...

  10. Ribosomal protein uS19 mutants reveal its role in coordinating ribosome structure and function.

    Science.gov (United States)

    Bowen, Alicia M; Musalgaonkar, Sharmishtha; Moomau, Christine A; Gulay, Suna P; Mirvis, Mary; Dinman, Jonathan D

    2015-01-01

    Prior studies identified allosteric information pathways connecting functional centers in the large ribosomal subunit to the decoding center in the small subunit through the B1a and B1b/c intersubunit bridges in yeast. In prokaryotes a single SSU protein, uS13, partners with H38 (the A-site finger) and uL5 to form the B1a and B1b/c bridges respectively. In eukaryotes, the SSU component was split into 2 separate proteins during the course of evolution. One, also known as uS13, participates in B1b/c bridge with uL5 in eukaryotes. The other, called uS19 is the SSU partner in the B1a bridge with H38. Here, polyalanine mutants of uS19 involved in the uS19/uS13 and the uS19/H38 interfaces were used to elucidate the important amino acid residues involved in these intersubunit communication pathways. Two key clusters of amino acids were identified: one located at the junction between uS19 and uS13, and a second that appears to interact with the distal tip of H38. Biochemical analyses reveal that these mutations shift the ribosomal rotational equilibrium toward the unrotated state, increasing ribosomal affinity for tRNAs in the P-site and for ternary complex in the A-site, and inhibit binding of the translocase, eEF2. These defects in turn affect specific aspects of translational fidelity. These findings suggest that uS19 plays a critical role as a conduit of information exchange between the large and small ribosomal subunits directly through the B1a, and indirectly through the B1b/c bridges. PMID:26824029

  11. Comparative analysis of ribosomal proteins in complete genomes: an example of reductive evolution at the domain scale

    OpenAIRE

    Lecompte, Odile; Ripp, Raymond; Thierry, Jean-Claude; Moras, Dino; Poch, Olivier

    2002-01-01

    A comprehensive investigation of ribosomal genes in complete genomes from 66 different species allows us to address the distribution of r-proteins between and within the three primary domains. Thirty-four r-protein families are represented in all domains but 33 families are specific to Archaea and Eucarya, providing evidence for specialisation at an early stage of evolution between the bacterial lineage and the lineage leading to Archaea and Eukaryotes. With only one specific r-protein, the a...

  12. The ribosome and the mechanism of protein synthesis

    International Nuclear Information System (INIS)

    In virtually all forms of life on earth, proteins in each cell are made according to a genetic blueprint, in the form of DNA. The translation of copies of this genetic blueprint (in the form of messenger RNA) into polypeptides is performed on the ribosome, a highly complex molecular machine composed of RNAs and proteins. To this end, special adaptor molecules called transfer RNAs are lined up by the ribosome in the sequence dictated by the genetic code, such that the amino acids carried by these molecules can be linked into a polypeptide. Several cofactors are involved in these processes, some of which require energy freed up by GTP hydrolysis. Although the ribosome was discovered more than 50 years ago, its structure has only been solved recently by X-ray crystallography. Another technique, cryo-electron microscopy, is starting to contribute toward our understanding of the ribosome's function, by portraying its conformational changes and binding interactions with the cofactors and tRNA

  13. Crystal structure of ribosomal protein L1 from the bacterium Aquifex aeolicus

    Science.gov (United States)

    Nikonova, E. Yu.; Tishchenko, S. V.; Gabdulkhakov, A. G.; Shklyaeva, A. A.; Garber, M. B.; Nikonov, S. V.; Nevskaya, N. A.

    2011-07-01

    The crystal structure of ribosomal protein L1 from the bacterium Aquifex aeolicus was solved by the molecular-replacement method and refined to R cryst = 19.4% and R free = 25.1% at 2.1 Å protein consists of two domains linked together by a flexible hinge region. In the structure under consideration, the domains are in close proximity and adopt a closed conformation. Earlier, this conformation has been found in the structure of protein L1 from the bacterium Thermus thermophilus, whereas the structures of archaeal L1 proteins and the structures of all L1 proteins in the RNA-bound form have an open conformation. The fact that a closed conformation was found in the structures of two L1 proteins which crystallize in different space groups and belong to different bacteria suggests that this conformation is a characteristic feature of L1 bacterial proteins in the free form.

  14. Can we estimate bacterial growth rates from ribosomal RNA content?

    Energy Technology Data Exchange (ETDEWEB)

    Kemp, P.F.

    1995-12-31

    Several studies have demonstrated a strong relationship between the quantity of RNA in bacterial cells and their growth rate under laboratory conditions. It may be possible to use this relationship to provide information on the activity of natural bacterial communities, and in particular on growth rate. However, if this approach is to provide reliably interpretable information, the relationship between RNA content and growth rate must be well-understood. In particular, a requisite of such applications is that the relationship must be universal among bacteria, or alternately that the relationship can be determined and measured for specific bacterial taxa. The RNA-growth rate relationship has not been used to evaluate bacterial growth in field studies, although RNA content has been measured in single cells and in bulk extracts of field samples taken from coastal environments. These measurements have been treated as probable indicators of bacterial activity, but have not yet been interpreted as estimators of growth rate. The primary obstacle to such interpretations is a lack of information on biological and environmental factors that affect the RNA-growth rate relationship. In this paper, the available data on the RNA-growth rate relationship in bacteria will be reviewed, including hypotheses regarding the regulation of RNA synthesis and degradation as a function of growth rate and environmental factors; i.e. the basic mechanisms for maintaining RNA content in proportion to growth rate. An assessment of the published laboratory and field data, the current status of this research area, and some of the remaining questions will be presented.

  15. Structure determination of archaea-specific ribosomal protein L46a reveals a novel protein fold

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Yingang, E-mail: fengyg@qibebt.ac.cn [Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101 (China); Song, Xiaxia [Department of Biological Science and Engineering, School of Chemical and Biological Engineering, University of Science and Technology Beijing, Beijing 100083 (China); Lin, Jinzhong [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Xuan, Jinsong [Department of Biological Science and Engineering, School of Chemical and Biological Engineering, University of Science and Technology Beijing, Beijing 100083 (China); Cui, Qiu [Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101 (China); Wang, Jinfeng [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

    2014-07-18

    Highlights: • The archaea-specific ribosomal protein L46a has no homology to known proteins. • Three dimensional structure and backbone dynamics of L46a were determined by NMR. • The structure of L46a represents a novel protein fold. • A potential rRNA-binding surface on L46a was identified. • The potential position of L46a on the ribosome was proposed. - Abstract: Three archaea-specific ribosomal proteins recently identified show no sequence homology with other known proteins. Here we determined the structure of L46a, the most conserved one among the three proteins, from Sulfolobus solfataricus P2 using NMR spectroscopy. The structure presents a twisted β-sheet formed by the N-terminal part and two helices at the C-terminus. The L46a structure has a positively charged surface which is conserved in the L46a protein family and is the potential rRNA-binding site. Searching homologous structures in Protein Data Bank revealed that the structure of L46a represents a novel protein fold. The backbone dynamics identified by NMR relaxation experiments reveal significant flexibility at the rRNA binding surface. The potential position of L46a on the ribosome was proposed by fitting the structure into a previous electron microscopy map of the ribosomal 50S subunit, which indicated that L46a contacts to domain I of 23S rRNA near a multifunctional ribosomal protein L7ae.

  16. Structure determination of archaea-specific ribosomal protein L46a reveals a novel protein fold

    International Nuclear Information System (INIS)

    Highlights: • The archaea-specific ribosomal protein L46a has no homology to known proteins. • Three dimensional structure and backbone dynamics of L46a were determined by NMR. • The structure of L46a represents a novel protein fold. • A potential rRNA-binding surface on L46a was identified. • The potential position of L46a on the ribosome was proposed. - Abstract: Three archaea-specific ribosomal proteins recently identified show no sequence homology with other known proteins. Here we determined the structure of L46a, the most conserved one among the three proteins, from Sulfolobus solfataricus P2 using NMR spectroscopy. The structure presents a twisted β-sheet formed by the N-terminal part and two helices at the C-terminus. The L46a structure has a positively charged surface which is conserved in the L46a protein family and is the potential rRNA-binding site. Searching homologous structures in Protein Data Bank revealed that the structure of L46a represents a novel protein fold. The backbone dynamics identified by NMR relaxation experiments reveal significant flexibility at the rRNA binding surface. The potential position of L46a on the ribosome was proposed by fitting the structure into a previous electron microscopy map of the ribosomal 50S subunit, which indicated that L46a contacts to domain I of 23S rRNA near a multifunctional ribosomal protein L7ae

  17. Evolution of Drosophila ribosomal protein gene core promoters

    OpenAIRE

    Ma, Xiaotu; Zhang, Kangyu; Li, Xiaoman

    2008-01-01

    The coordinated expression of ribosomal protein genes (RPGs) has been well documented in many species. Previous analyses of RPG promoters focus only on Fungi and mammals. Recognizing this gap and using a comparative genomics approach, we utilize a motif-finding algorithm that incorporates cross-species conservation to identify several significant motifs in Drosophila RPG promoters. As a result, significant differences of the enriched motifs in RPG promoter are found among Drosophila, Fungi, a...

  18. Preferential translation of chloroplast ribosomal proteins in Chlamydomonas reinhardtti

    International Nuclear Information System (INIS)

    The nuclear cr-1 mutant of C. reinhardtii is deficient in the 30S subunit of the chloroplast (cp) ribosome and in cp protein synthesis. The cp spectinomycin resistant mutant, spr-u-1-27-3, has a normal level of 70S ribosomes but only a low rate of cp protein synthesis with spectinomycin present. In both mutants there is little accumulation of the large subunit of ribulose 1,5-bisphosphate carboxylase (Rubisco LSU), but near wild-type levels of cp synthesized r-proteins. In cells pulse-labelled with 35SO4 and immunoprecipitated with specific antisera, the ratio of the rate of synthesis of cp r-proteins to that of Rubisco LSU is 7 times greater in both mutants than in wild-type. No difference in the rate of turnover between r-proteins and Rubisco LSU in mutant and wild-type cells was observed during a one hour chase. The mRNA levels for r-protein L1 and Rubisco LSU actually increase slightly in the mutants. These data suggest that C. reinhardtii has a translation mechanism for preferential synthesis of cp r-proteins that operates under conditions of reduced total cp protein synthesis

  19. Combined eukaryotic and bacterial community fingerprinting of natural freshwater biofilms using automated ribosomal intergenic spacer analysis

    OpenAIRE

    2010-01-01

    Biofilms are complex communities playing an important role in aquatic ecosystems. Automated ribosomal intergenic spacer analysis (ARISA) has been used successfully to explore biofilm bacterial diversity. However, a gap remains to be filled as regards its application to biofilm eukaryotic populations. The aim of this study is to use ARISA to detect eukaryotic population shifts in biofilm. We designed a new set of primers to focus specifically on the ITS1-5.8S-ITS2 region of diatoms and tested ...

  20. Impact of P-Site tRNA and antibiotics on ribosome mediated protein folding: studies using the Escherichia coli ribosome.

    Directory of Open Access Journals (Sweden)

    Surojit Mondal

    Full Text Available BACKGROUND: The ribosome, which acts as a platform for mRNA encoded polypeptide synthesis, is also capable of assisting in folding of polypeptide chains. The peptidyl transferase center (PTC that catalyzes peptide bond formation resides in the domain V of the 23S rRNA of the bacterial ribosome. Proper positioning of the 3' -CCA ends of the A- and P-site tRNAs via specific interactions with the nucleotides of the PTC are crucial for peptidyl transferase activity. This RNA domain is also the center for ribosomal chaperoning activity. The unfolded polypeptide chains interact with the specific nucleotides of the PTC and are released in a folding competent form. In vitro transcribed RNA corresponding to this domain (bDV RNA also displays chaperoning activity. RESULTS: The present study explores the effects of tRNAs, antibiotics that are A- and P-site PTC substrate analogs (puromycin and blasticidin and macrolide antibiotics (erythromycin and josamycin on the chaperoning ability of the E. coli ribosome and bDV RNA. Our studies using mRNA programmed ribosomes show that a tRNA positioned at the P-site effectively inhibits the ribosome's chaperoning function. We also show that the antibiotic blasticidin (that mimics the interaction between 3'-CCA end of P/P-site tRNA with the PTC is more effective in inhibiting ribosome and bDV RNA chaperoning ability than either puromycin or the macrolide antibiotics. Mutational studies of the bDV RNA could identify the nucleotides U2585 and G2252 (both of which interact with P-site tRNA to be important for its chaperoning ability. CONCLUSION: Both protein synthesis and their proper folding are crucial for maintenance of a functional cellular proteome. The PTC of the ribosome is attributed with both these abilities. The silencing of the chaperoning ability of the ribosome in the presence of P-site bound tRNA might be a way to segregate these two important functions.

  1. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten;

    2010-01-01

    Bacteria and Eukarya share essentially the same family of protein-serine/threonine kinases, also known as the Hanks-type kinases. However, when it comes to protein-tyrosine phosphorylation, bacteria seem to have gone their own way. Bacterial protein-tyrosine kinases (BY-kinases) are bacterial...... enzymes that are unique in exploiting the ATP/GTP-binding Walker motif to catalyze phosphorylation of protein tyrosine residues. Characterized for the first time only a decade ago, BY-kinases have now come to the fore. Important regulatory roles have been linked with these enzymes, via their involvement...... in exopolysaccharide production, virulence, DNA metabolism, stress response and other key functions of the bacterial cell. BY-kinases act through autophosphorylation (mainly in exopolysaccharide production) and phosphorylation of other proteins, which have in most cases been shown to be activated by...

  2. Ribosomes containing mutants of L4 ribosomal protein from Thermus thermophilus display multiple defects in ribosomal functions and sensitivity against erythromycin

    Science.gov (United States)

    TSAGKALIA, AIKATERINI; LEONTIADOU, FOTINI; XAPLANTERI, MARIA A.; PAPADOPOULOS, GEORGIOS; KALPAXIS, DIMITRIOS L.; CHOLI-PAPADOPOULOU, THEODORA

    2005-01-01

    Protein L4 from Thermus thermophilus (TthL4) was heterologously overproduced in Escherichia coli cells. To study the implication of the extended loop of TthL4 in the exit-tunnel and peptidyltransferase functions, the highly conserved E56 was replaced by D or Q, while the semiconserved G55 was changed to E or S. Moreover, the sequence -G55E56- was inverted to -E55G56-. When we incorporated these mutants into E. coli ribosomes and investigated their impact on poly(Phe) synthesis, high variations in the synthetic activity and response to erythromycin of the resulting ribosomes were observed. In the absence of erythromycin, ribosomes harboring mutations G55E and E56D in TthL4 protein were characterized by low activity in synthesizing poly(Phe) and decreased capability in binding tRNA at the A site. On the other hand, ribosomes possessing mutations G55E, G55S, G55E-E56G, or E56Q in TthL4 protein were unexpectedly more sensitive to erythromycin. Evidence in support of these findings was drawn by in vivo experiments, assessing the erythromycin sensitivity of E. coli cells expressing wild-type or mutant TthL4 proteins. Our results emphasize the role of the extended loop of L4 ribosomal protein in the exit-tunnel and peptidyltransferase center functions. PMID:16244130

  3. Mechanism of fusidic acid inhibition of RRF- and EF-G-dependent splitting of the bacterial post-termination ribosome.

    Science.gov (United States)

    Borg, Anneli; Pavlov, Michael; Ehrenberg, Måns

    2016-04-20

    The antibiotic drug fusidic acid (FA) is commonly used in the clinic against gram-positive bacterial infections. FA targets ribosome-bound elongation factor G (EF-G), a translational GTPase that accelerates both messenger RNA (mRNA) translocation and ribosome recycling. How FA inhibits translocation was recently clarified, but FA inhibition of ribosome recycling by EF-G and ribosome recycling factor (RRF) has remained obscure. Here we use fast kinetics techniques to estimate mean times of ribosome splitting and the stoichiometry of GTP hydrolysis by EF-G at varying concentrations of FA, EF-G and RRF. These mean times together with previous data on uninhibited ribosome recycling were used to clarify the mechanism of FA inhibition of ribosome splitting. The biochemical data on FA inhibition of translocation and recycling were used to model the growth inhibitory effect of FA on bacterial populations. We conclude that FA inhibition of translocation provides the dominant cause of bacterial growth reduction, but that FA inhibition of ribosome recycling may contribute significantly to FA-induced expression of short regulatory open reading frames, like those involved in FA resistance. PMID:27001509

  4. Use of Quantitative 16S Ribosomal DNA Detection for Diagnosis of Central Vascular Catheter-Associated Bacterial Infection

    OpenAIRE

    Warwick, S.; Wilks, M; Hennessy, E.; Powell-Tuck, J; Small, M.; Sharp, J.; Millar, M R

    2004-01-01

    Many central vascular catheters (CVCs) are removed unnecessarily because current diagnostic methods for CVC-associated infection are unreliable. A quantitative PCR assay using primers and probe targeted to bacterial 16S ribosomal DNA was used to measure the levels of bacterial DNA in blood samples drawn through the CVC in a population of patients receiving intravenous nutrition. Bacterial DNA concentrations were raised in 16 of 16 blood samples taken during episodes of probable bacterial CVC-...

  5. Genomic location of the major ribosomal protein gene locus determines Vibrio cholerae global growth and infectivity.

    Science.gov (United States)

    Soler-Bistué, Alfonso; Mondotte, Juan A; Bland, Michael Jason; Val, Marie-Eve; Saleh, María-Carla; Mazel, Didier

    2015-04-01

    The effects on cell physiology of gene order within the bacterial chromosome are poorly understood. In silico approaches have shown that genes involved in transcription and translation processes, in particular ribosomal protein (RP) genes, localize near the replication origin (oriC) in fast-growing bacteria suggesting that such a positional bias is an evolutionarily conserved growth-optimization strategy. Such genomic localization could either provide a higher dosage of these genes during fast growth or facilitate the assembly of ribosomes and transcription foci by keeping physically close the many components of these macromolecular machines. To explore this, we used novel recombineering tools to create a set of Vibrio cholerae strains in which S10-spec-α (S10), a locus bearing half of the ribosomal protein genes, was systematically relocated to alternative genomic positions. We show that the relative distance of S10 to the origin of replication tightly correlated with a reduction of S10 dosage, mRNA abundance and growth rate within these otherwise isogenic strains. Furthermore, this was accompanied by a significant reduction in the host-invasion capacity in Drosophila melanogaster. Both phenotypes were rescued in strains bearing two S10 copies highly distal to oriC, demonstrating that replication-dependent gene dosage reduction is the main mechanism behind these alterations. Hence, S10 positioning connects genome structure to cell physiology in Vibrio cholerae. Our results show experimentally for the first time that genomic positioning of genes involved in the flux of genetic information conditions global growth control and hence bacterial physiology and potentially its evolution. PMID:25875621

  6. Genomic location of the major ribosomal protein gene locus determines Vibrio cholerae global growth and infectivity.

    Directory of Open Access Journals (Sweden)

    Alfonso Soler-Bistué

    2015-04-01

    Full Text Available The effects on cell physiology of gene order within the bacterial chromosome are poorly understood. In silico approaches have shown that genes involved in transcription and translation processes, in particular ribosomal protein (RP genes, localize near the replication origin (oriC in fast-growing bacteria suggesting that such a positional bias is an evolutionarily conserved growth-optimization strategy. Such genomic localization could either provide a higher dosage of these genes during fast growth or facilitate the assembly of ribosomes and transcription foci by keeping physically close the many components of these macromolecular machines. To explore this, we used novel recombineering tools to create a set of Vibrio cholerae strains in which S10-spec-α (S10, a locus bearing half of the ribosomal protein genes, was systematically relocated to alternative genomic positions. We show that the relative distance of S10 to the origin of replication tightly correlated with a reduction of S10 dosage, mRNA abundance and growth rate within these otherwise isogenic strains. Furthermore, this was accompanied by a significant reduction in the host-invasion capacity in Drosophila melanogaster. Both phenotypes were rescued in strains bearing two S10 copies highly distal to oriC, demonstrating that replication-dependent gene dosage reduction is the main mechanism behind these alterations. Hence, S10 positioning connects genome structure to cell physiology in Vibrio cholerae. Our results show experimentally for the first time that genomic positioning of genes involved in the flux of genetic information conditions global growth control and hence bacterial physiology and potentially its evolution.

  7. Differential effects of ribosomal proteins and Mg2+ ions on a conformational switch during 30S ribosome 5'-domain assembly.

    Science.gov (United States)

    Abeysirigunawardena, Sanjaya C; Woodson, Sarah A

    2015-11-01

    Ribosomal protein S4 nucleates assembly of the 30S ribosome 5' and central domains, which is crucial for the survival of cells. Protein S4 changes the structure of its 16S rRNA binding site, passing through a non-native intermediate complex before forming native S4-rRNA contacts. Ensemble FRET was used to measure the thermodynamic stability of non-native and native S4 complexes in the presence of Mg(2+) ions and other 5'-domain proteins. Equilibrium titrations of Cy3-labeled 5'-domain RNA with Cy5-labeled protein S4 showed that Mg(2+) ions preferentially stabilize the native S4-rRNA complex. In contrast, ribosomal proteins S20 and S16 act by destabilizing the non-native S4-rRNA complex. The full cooperative switch to the native complex requires S4, S16, and S20 and is achieved to a lesser degree by S4 and S16. The resulting thermodynamic model for assembly of the 30S body illustrates how ribosomal proteins selectively bias the equilibrium between alternative rRNA conformations, increasing the cooperativity of rRNA folding beyond what can be achieved by Mg(2+) ions alone. PMID:26354770

  8. Ribosome-Inactivating Proteins: From Plant Defense to Tumor Attack

    Directory of Open Access Journals (Sweden)

    Maria Serena Fabbrini

    2010-11-01

    Full Text Available Ribosome-inactivating proteins (RIPs are EC3.2.32.22 N-glycosidases that recognize a universally conserved stem-loop structure in 23S/25S/28S rRNA, depurinating a single adenine (A4324 in rat and irreversibly blocking protein translation, leading finally to cell death of intoxicated mammalian cells. Ricin, the plant RIP prototype that comprises a catalytic A subunit linked to a galactose-binding lectin B subunit to allow cell surface binding and toxin entry in most mammalian cells, shows a potency in the picomolar range. The most promising way to exploit plant RIPs as weapons against cancer cells is either by designing molecules in which the toxic domains are linked to selective tumor targeting domains or directly delivered as suicide genes for cancer gene therapy. Here, we will provide a comprehensive picture of plant RIPs and discuss successful designs and features of chimeric molecules having therapeutic potential.

  9. Rice Ribosomal Protein Large Subunit Genes and Their Spatio-temporal and Stress Regulation.

    Science.gov (United States)

    Moin, Mazahar; Bakshi, Achala; Saha, Anusree; Dutta, Mouboni; Madhav, Sheshu M; Kirti, P B

    2016-01-01

    Ribosomal proteins (RPs) are well-known for their role in mediating protein synthesis and maintaining the stability of the ribosomal complex, which includes small and large subunits. In the present investigation, in a genome-wide survey, we predicted that the large subunit of rice ribosomes is encoded by at least 123 genes including individual gene copies, distributed throughout the 12 chromosomes. We selected 34 candidate genes, each having 2-3 identical copies, for a detailed characterization of their gene structures, protein properties, cis-regulatory elements and comprehensive expression analysis. RPL proteins appear to be involved in interactions with other RP and non-RP proteins and their encoded RNAs have a higher content of alpha-helices in their predicted secondary structures. The majority of RPs have binding sites for metal and non-metal ligands. Native expression profiling of 34 ribosomal protein large (RPL) subunit genes in tissues covering the major stages of rice growth shows that they are predominantly expressed in vegetative tissues and seedlings followed by meiotically active tissues like flowers. The putative promoter regions of these genes also carry cis-elements that respond specifically to stress and signaling molecules. All the 34 genes responded differentially to the abiotic stress treatments. Phytohormone and cold treatments induced significant up-regulation of several RPL genes, while heat and H2O2 treatments down-regulated a majority of them. Furthermore, infection with a bacterial pathogen, Xanthomonas oryzae, which causes leaf blight also induced the expression of 80% of the RPL genes in leaves. Although the expression of RPL genes was detected in all the tissues studied, they are highly responsive to stress and signaling molecules indicating that their encoded proteins appear to have roles in stress amelioration besides house-keeping. This shows that the RPL gene family is a valuable resource for manipulation of stress tolerance in rice

  10. Ribosome Profiling Provides Evidence that Large Noncoding RNAs Do Not Encode Proteins

    OpenAIRE

    Guttman, Mitchell; Russell, Pamela; Ingolia, Nicholas T.; Weissman, Jonathan S.; Lander, Eric S.; Lander, Eric S.

    2013-01-01

    Large noncoding RNAs are emerging as an important component in cellular regulation. Considerable evidence indicates that these transcripts act directly as functional RNAs rather than through an encoded protein product. However, a recent study of ribosome occupancy reported that many large intergenic ncRNAs (lincRNAs) are bound by ribosomes, raising the possibility that they are translated into proteins. Here, we show that classical noncoding RNAs and 5′ UTRs show the same ribosome occupancy a...

  11. Effects of electroacupuncture on the expression of p70 ribosomal protein S6 kinase and ribosomal protein S6 in the hippocampus of rats with vascular dementia

    Institute of Scientific and Technical Information of China (English)

    Yanzhen Zhu; Xuan Wang; Xiaobao Ye; Changhua Gao; Wei Wang

    2012-01-01

    This study investigated the mechanism underlying electroacupuncture therapy for vascular dementia through electroacupuncture at the acupoints of Baihui (DU20), Dazhui (DU14), and bilateral Shenshu (BL23) in a rat model of vascular dementia produced by bilateral middle cerebral artery occlusion. Morris water maze test showed that electroacupuncture improved the learning ability of vascular dementia rats. Western blot assay revealed that the expression of p70 ribosomal protein S6 kinase and ribosomal protein S6 in vascular dementia rats was significantly increased after electroacupuncture, compared with the model group that was not treated with acupuncture. The average escape latency was also shortened after electroacupuncture, and escape strategies in the spatial probe test improved from edge and random searches, to linear and trending swim pathways. The experimental findings indicate that electroacupuncture improves learning and memory ability by up-regulating expression of p70 ribosomal protein S6 kinase and ribosomal protein S6 in the hippocampus of vascular dementia rats.

  12. Ribosomal protein methylation in Escherichia coli: the gene prmA, encoding the ribosomal protein L11 methyltransferase, is dispensable.

    Science.gov (United States)

    Vanet, A; Plumbridge, J A; Guérin, M F; Alix, J H

    1994-12-01

    The prmA gene, located at 72 min on the Escherichia coli chromosome, is the genetic determinant of ribosomal protein L11-methyltransferase activity. Mutations at this locus, prmA1 and prmA3, result in a severely undermethylated form of L11. No effect, other than the lack of methyl groups on L11, has been ascribed to these mutations. DNA sequence analysis of the mutant alleles prmA1 and prmA3 detected point mutations near the C-terminus of the protein and plasmids overproducing the wild-type and the two mutant proteins have been constructed. The wild-type PrmA protein could be crosslinked to its radiolabelled substrate, S-adenosyl-L-methionine (SAM), by u.v. irradiation indicating that it is the gene for the methyltransferase rather than a regulatory protein. One of the mutant proteins, PrmA3, was also weakly crosslinked to SAM. Both mutant enzymes when expressed from the overproducing plasmids were capable of catalysing the incorporation of 3H-labelled methyl groups from SAM to L11 in vitro. This confirmed the observation that the mutant proteins possess significant residual activity which could account for their lack of growth phenotype. However, a strain carrying an in vitro-constructed null mutation of the prmA gene, transferred to the E. coli chromosome by homologous recombination, was perfectly viable. PMID:7715456

  13. Autogenous Regulation of Splicing of the Transcript of a Yeast Ribosomal Protein Gene

    Science.gov (United States)

    Dabeva, Mariana D.; Post-Beittenmiller, Martha A.; Warner, Jonathan R.

    1986-08-01

    The gene for a yeast ribosomal protein, RPL32, contains a single intron. The product of this gene appears to participate in feedback control of the splicing of the intron from the transcript. This autogenous regulation of splicing provides a striking analogy to the autogenous regulation of translation of ribosomal proteins in Escherichia coli.

  14. Autogenous regulation of splicing of the transcript of a yeast ribosomal protein gene.

    OpenAIRE

    Dabeva, M. D.; Post-Beittenmiller, M A; Warner, J R

    1986-01-01

    The gene for a yeast ribosomal protein, RPL32, contains a single intron. The product of this gene appears to participate in feedback control of the splicing of the intron from the transcript. This autogenous regulation of splicing provides a striking analogy to the autogenous regulation of translation of ribosomal proteins in Escherichia coli.

  15. Folding and escape of nascent proteins at ribosomal exit tunnel

    Science.gov (United States)

    Bui, Phuong Thuy; Hoang, Trinh Xuan

    2016-03-01

    We investigate the interplay between post-translational folding and escape of two small single-domain proteins at the ribosomal exit tunnel by using Langevin dynamics with coarse-grained models. It is shown that at temperatures lower or near the temperature of the fastest folding, folding proceeds concomitantly with the escape process, resulting in vectorial folding and enhancement of foldability of nascent proteins. The concomitance between the two processes, however, deteriorates as temperature increases. Our folding simulations as well as free energy calculation by using umbrella sampling show that, at low temperatures, folding at the tunnel follows one or two specific pathways without kinetic traps. It is shown that the escape time can be mapped to a one-dimensional diffusion model with two different regimes for temperatures above and below the folding transition temperature. Attractive interactions between amino acids and attractive sites on the tunnel wall lead to a free energy barrier along the escape route of the protein. It is suggested that this barrier slows down the escape process and consequently promotes correct folding of the released nascent protein.

  16. tmRNA-mediated trans-translation as the major ribosome rescue system in a bacterial cell

    Directory of Open Access Journals (Sweden)

    Hyouta eHimeno

    2014-04-01

    Full Text Available tmRNA (transfer messenger RNA; also known as 10Sa RNA or SsrA RNA is a small RNA molecule that is conserved among bacteria. It has structural and functional similarities to tRNA: it has an upper half of the tRNA-like structure, its 5’ end is processed by RNase P, it has typical tRNA-specific base modifications, it is aminoacylated with alanine, it binds to EF-Tu after aminoacylation and it enters the ribosome with EF-Tu and GTP. However, tmRNA lacks an anticodon, and instead it has a coding sequence for a short peptide called tag-peptide. An elaborate interplay of actions of tmRNA as both tRNA and mRNA with the help of a tmRNA-binding protein, SmpB, facilitates trans-translation, which produces a single polypeptide from two mRNA molecules. Initially alanyl-tmRNA in complex with EF-Tu and SmpB enters the vacant A-site of the stalled ribosome like aminoacyl-tRNA but without a codon-anticodon interaction, and subsequently truncated mRNA is replaced with the tag-encoding region of tmRNA. During these processes, not only tmRNA but also SmpB structurally and functionally mimics both tRNA and mRNA. Thus trans-translation rescues the stalled ribosome, thereby allowing recycling of the ribosome. Since the tag-peptide serves as a target of AAA+ proteases, the trans-translation products are preferentially degraded so that they do not accumulate in the cell. Although alternative rescue systems have recently been revealed, trans-translation is the only system that universally exists in bacteria. Furthermore, it is unique in that it employs a small RNA and that it prevents accumulation of nonfunctional proteins from truncated mRNA in the cell. It might play the major role in rescuing the stalled translation in the bacterial cell.

  17. Mammalian sperm translate nuclear-encoded proteins by mitochondrial-type ribosomes

    OpenAIRE

    Gur, Yael; Breitbart, Haim

    2006-01-01

    It is widely accepted that spermatozoa are translationally silent. The present study demonstrates, for the first time, incorporation of labeled amino acids into polypeptides during sperm capacitation, which was completely inhibited by mitochondrial translation inhibitors but not by the cytoplasmic translation inhibitor. Unlike 80S cytoplasmic ribosomes, 55S mitochondrial ribosomes were present in polysomal fractions, indicating that these ribosomes are actively involved in protein translation...

  18. Properties of the ribosome-inactivating proteins gelonin, Momordica charantia inhibitor, and dianthins.

    Science.gov (United States)

    Falasca, A; Gasperi-Campani, A; Abbondanza, A; Barbieri, L; Stirpe, F

    1982-12-01

    The amino acid and sugar compositions of four ribosome-inactivating proteins (gelonin, Momordica charantia inhibitor, dianthin 30 and dianthin 32) were determined. The proteins are all basic glycoproteins (pI greater than 8) containing mannose (more abundant in gelonin), glucose, xylose, fucose (absent from gelonin) and glucosamine. The ribosome-inactivating properties of the proteins examined are not modified by pretreatment with N-ethylmaleimide. Precipitating and inactivating antibodies can be raised against ribosome-inactivating proteins; a weak cross-reaction was observed only between dianthin 30 and dianthin 32. PMID:6819861

  19. Changes in ribosomal proteins in wheat embryos in the course of grain development and maturation

    Directory of Open Access Journals (Sweden)

    Stanisław Weidner

    2014-02-01

    Full Text Available It was found, by comparing the densitometric profiles of ribosomal proteins of wheat embryos in milk and full grain ripeness, that in the process of development and ripening of caryopses the percentual proportion of low molecular weight proteins increases at the cost of those of high molecular weight. This concerns both acidic and basic proteins. In electrophoretic separation of ribosomal proteins from embryos of fully ripe seeds by the method of two-dimensional electrophoresis the appearance of three new low molecular weight proteins - an acidic one and two basic ones - was observed. These proteins were not found in the embryos of caryopses of milk ripeness. These results indicate that with development and ripening of wheat caryopses new low molecular weight ribosomal proteins are built into the ribosomes in the embryo. These changes are both quantitative and qualitative.

  20. Traffic of interacting ribosomes: effects of single-machine mechano-chemistry on protein synthesis

    CERN Document Server

    Basu, A; Basu, Aakash; Chowdhury, Debashish

    2006-01-01

    Many ribosomes simultaneously move on the same messenger RNA (mRNA), each synthesizing a protein. Earlier models of ribosome traffic represent each ribosome by a ``self-propelled particle'' and capture the dynamics by an extension of the totally asymmetric simple exclusion process. In contrast, here we develope a ``unified'' theoretical model that not only incorporates the mutual exclusions of the interacting ribosomes, but also describes explicitly the mechano-chemistry of each of these individual cyclic machines during protein synthesis. Using a combination of analytical and numerical techniques of non-equilibrium statistical mechanics, we analyze this model and illustrate its power by making experimentally testable predictions on the rate of protein synthesis and the density profile of the ribosomes on some mRNAs in E-Coli.

  1. Production of ribosome-inactivating protein from hairy root cultures of Luffa cylindrica (L.) Roem.

    Science.gov (United States)

    di Toppi, L S; Gorini, P; Properzi, G; Barbieri, L; Spanò, L

    1996-09-01

    Transformed root lines of Luffa cylindrica (L.) Roem. (Cucurbitaceae) were established by inoculation of in vitro grown plantlets with wild type Agrobacterium rhizogenes strain 1855. Cloned lines of hairy roots were tested for the presence of ribosome-inactivating proteins; crude extracts inhibited protein synthesis in a reaction mixture based on rabbit reticulocyte lysate. Inhibitory activity increased during culture period, reaching a maximum value in the stationary phase. No activity could be detected in the culture medium, nor in extracts from callus and/or suspension cultures. A ribosome-inactivating protein having specific activity of 62,100 U mg protein(-1) and a molecular mass of 26-28,000 Da was purified to homogeneity. The protein showed N-glycosidase activity on rat liver ribosomes. The results demonstrate that hairy root cultures can be successfully utilized for the in vitro production of ribosome-inactivating proteins. PMID:24178273

  2. Generation of monoclonal antibodies for the assessment of protein purification by recombinant ribosomal coupling

    DEFF Research Database (Denmark)

    Kristensen, Janni; Sperling-Petersen, Hans Uffe; Mortensen, Kim Kusk;

    2005-01-01

    We recently described a conceptually novel method for the purification of recombinant proteins with a propensity to form inclusion bodies in the cytoplasm of Escherichia coli. Recombinant proteins were covalently coupled to the E. coli ribosome by fusing them to ribosomal protein 23 (rpL23....... We therefore purified rpL23-GFP-His, rpL23-His and GFP from E. coli recombinants using affinity, ion exchange and hydrophobic interaction chromatography. These proteins could be purified with yields of 150, 150 and 1500 microg per gram cellular wet weight, respectively. However, rpL23-GFP-His could...... purified by Protein G Sepharose affinity chromatography. The purified antibodies were used to evaluate the separation of ribosomes from GFP, streptavidin, murine interleukin-6, a phagedisplay antibody and yeast elongation factor 1A by centrifugation, when ribosomes with covalently coupled target protein...

  3. Studies on the Coordination of Ribosomal Protein Assembly Events Involved in Processing and Stabilization of Yeast Early Large Ribosomal Subunit Precursors.

    Directory of Open Access Journals (Sweden)

    Uli Ohmayer

    Full Text Available Cellular production of ribosomes involves the formation of highly defined interactions between ribosomal proteins (r-proteins and ribosomal RNAs (rRNAs. Moreover in eukaryotic cells, efficient ribosome maturation requires the transient association of a large number of ribosome biogenesis factors (RBFs with newly forming ribosomal subunits. Here, we investigated how r-protein assembly events in the large ribosomal subunit (LSU rRNA domain II are coordinated with each other and with the association of RBFs in early LSU precursors of the yeast Saccharomyces cerevisiae. Specific effects on the pre-ribosomal association of RBFs could be observed in yeast mutants blocked in LSU rRNA domain II assembly. Moreover, formation of a cluster of r-proteins was identified as a downstream event in LSU rRNA domain II assembly. We analyzed in more detail the functional relevance of eukaryote specific bridges established by this r-protein cluster between LSU rRNA domain II and VI and discuss how they can support the stabilization and efficient processing of yeast early LSU precursor RNAs.

  4. Analysis of the protein-protein interactions between the human acidic ribosomal P-proteins: evaluation by the two hybrid system

    DEFF Research Database (Denmark)

    Tchórzewski, M; Boldyreff, B; Issinger, O;

    2000-01-01

    function of these proteins, we are the first to have precisely analyzed mutual interactions among human P-proteins, employing the two hybrid system. The human acidic ribosomal P-proteins, (P1 or P2,) were fused to the GAL4 binding domain (BD) as well as the activation domain (AD), and analyzed in yeast...... forms the 60 S ribosomal stalk: P0-(P1/P2)(2). Additionally, mutual interactions among human and yeast P-proteins were analyzed. Heterodimer formation could be observed between human P2 and yeast P1 proteins.......The surface acidic ribosomal proteins (P-proteins), together with ribosomal core protein P0 form a multimeric lateral protuberance on the 60 S ribosomal subunit. This structure, also called stalk, is important for efficient translational activity of the ribosome. In order to shed more light on the...

  5. Bacterial cell division proteins as antibiotic targets

    NARCIS (Netherlands)

    T. den Blaauwen; J.M. Andreu; O. Monasterio

    2014-01-01

    Proteins involved in bacterial cell division often do not have a counterpart in eukaryotic cells and they are essential for the survival of the bacteria. The genetic accessibility of many bacterial species in combination with the Green Fluorescence Protein revolution to study localization of protein

  6. The subcellular distribution of the human ribosomal "stalk" components: P1, P2 and P0 proteins

    DEFF Research Database (Denmark)

    Tchórzewski, Marek; Krokowski, Dawid; Rzeski, Wojciech;

    2003-01-01

    The ribosomal "stalk" structure is a distinct lateral protuberance located on the large ribosomal subunit in prokaryotic, as well as in eukaryotic cells. In eukaryotes, this ribosomal structure is composed of the acidic ribosomal P proteins, forming two hetero-dimers (P1/P2) attached to the ribos......The ribosomal "stalk" structure is a distinct lateral protuberance located on the large ribosomal subunit in prokaryotic, as well as in eukaryotic cells. In eukaryotes, this ribosomal structure is composed of the acidic ribosomal P proteins, forming two hetero-dimers (P1/P2) attached...... to the ribosome through the P0 protein. The "stalk" is essential for the ribosome activity, taking part in the interaction with elongation factors.In this report, we have shown that the subcellular distribution of the human P proteins does not fall into standard behavior of regular ribosomal proteins. We have...... used two approaches to assess the distribution of the P proteins, in vivo experiments with GFP fusion proteins and in vitro one with anti-P protein antibodies. In contrast to standard r-proteins, the P1 and P2 proteins are not actively transported into the nucleus compartment, remaining predominantly...

  7. Structural and Functional Characterization of Ribosomal Protein Gene Introns in Sponges

    OpenAIRE

    Perina, Dragutin; Korolija, Marina; Mikoč, Andreja; Roller Milošević, Maša; Pleše, Bruna; Imešek, Mirna; Morrow, Christine; Batel, Renato; Ćetković, Helena

    2012-01-01

    Ribosomal protein genes (RPGs) are a powerful tool for studying intron evolution. They exist in all three domains of life and are much conserved. Accumulating genomic data suggest that RPG introns in many organisms abound with non-protein-coding-RNAs (ncRNAs). These ancient ncRNAs are small nucleolar RNAs (snoRNAs) essential for ribosome assembly. They are also mobile genetic elements and therefore probably important in diversification and enrichment of transcriptomes through various mechanis...

  8. Evidence that ribosomal protein S10 participates in control of transcription termination.

    OpenAIRE

    Friedman, D I; Schauer, A T; Baumann, M R; Baron, L S; Adhya, S L

    1981-01-01

    We report the isolation of an Escherichia coli K-12 strain with a mutation, nusE71, that results in a change in ribosomal protein S10. Phage lambda fails to grow in hosts carrying the nusE71 mutation because the lambda N gene product is not active. The N product regulates phage gene expression by altering transcription complexes so that they can overcome termination barriers. This suggests that a ribosomal protein is involved in antitermination of transcription.

  9. Involvement of human ribosomal proteins in nucleolar structure and p53-dependent nucleolar stress

    OpenAIRE

    Nicolas, Emilien; Parisot, Pascaline; Pinto-Monteiro, Celina; de Walque, Roxane; De Vleeschouwer, Christophe; Lafontaine, Denis L. J.

    2016-01-01

    The nucleolus is a potent disease biomarker and a target in cancer therapy. Ribosome biogenesis is initiated in the nucleolus where most ribosomal (r-) proteins assemble onto precursor rRNAs. Here we systematically investigate how depletion of each of the 80 human r-proteins affects nucleolar structure, pre-rRNA processing, mature rRNA accumulation and p53 steady-state level. We developed an image-processing programme for qualitative and quantitative discrimination of normal from altered nucl...

  10. Auxin-induced changes in the incorporation of /sup 3/H-amino acids into soybean ribosomal proteins

    Energy Technology Data Exchange (ETDEWEB)

    Travis, R.L.; Key, J.L.

    1976-06-01

    Auxin-induced activation of 80S ribosomes and polyribosome formation in mature soybean (Glycine max var. Hawkeye) hypocotyl (R. L. Travis, J. M. Anderson, and J. L. Key. 1973. Plant Physiol. 52: 608-612) in the presence of a mixture of radioactive amino acids correlates with an increased specific radioactivity of at least three ribosomal proteins; the labeling of one of these increased severalfold above the control level. Results of experiments with 5-fluorouracil and cycloheximide indicated that the proteins in question were synthesized in response to auxin and became associated with pre-existing ribosomes. Ribosome dissociation experiments indicated that these proteins were associated with the 60S ribosome subunit.

  11. Metabolism of ribosomal proteins microinjected into the oocytes of Xenopus laevis

    International Nuclear Information System (INIS)

    When the total proteins from Xenopus laevis 60 S ribosomal subunits (TP60) were 3H-labeled in vitro and injected back into X. laevis oocytes, most 3H-TP60 are integrated into the cytoplasmic 60 S subunits via the nucleus during 16 h of incubation. In the oocytes whose rRNA synthesis is inhibited, 3H-TP60 are rapidly degraded with a half-life of 2-3 h. This degradation ceased as soon as rRNA synthesis was resumed, suggesting that ribosomal proteins unassociated with nascent rRNA are unstable in the oocytes. The degradation of 3H-TP60 in the absence of RNA synthesis was inhibited by iodoacetamide, a cysteine protease inhibitor, resulting in the accumulation of 3H-TP60 in the nucleus reaching about a threefold concentration in the cytoplasm. Considering the results with enucleated oocytes, we suggest that the X. laevis nucleus has a limited capacity to accumulate ribosomal proteins in an active manner but that those ribosomal proteins accumulated in excess over rRNA synthesis are degraded by a cysteine protease in the nucleus. By contrast, ribosomal proteins from Escherichia coli only equilibrate between the nucleus and the cytoplasm and are degraded by serine protease(s) in the cytoplasm without being integrated in the form of ribosomes in the nucleus

  12. Regulatory elements of Caenorhabditis elegans ribosomal protein genes

    Directory of Open Access Journals (Sweden)

    Sleumer Monica C

    2012-08-01

    Full Text Available Abstract Background Ribosomal protein genes (RPGs are essential, tightly regulated, and highly expressed during embryonic development and cell growth. Even though their protein sequences are strongly conserved, their mechanism of regulation is not conserved across yeast, Drosophila, and vertebrates. A recent investigation of genomic sequences conserved across both nematode species and associated with different gene groups indicated the existence of several elements in the upstream regions of C. elegans RPGs, providing a new insight regarding the regulation of these genes in C. elegans. Results In this study, we performed an in-depth examination of C. elegans RPG regulation and found nine highly conserved motifs in the upstream regions of C. elegans RPGs using the motif discovery algorithm DME. Four motifs were partially similar to transcription factor binding sites from C. elegans, Drosophila, yeast, and human. One pair of these motifs was found to co-occur in the upstream regions of 250 transcripts including 22 RPGs. The distance between the two motifs displayed a complex frequency pattern that was related to their relative orientation. We tested the impact of three of these motifs on the expression of rpl-2 using a series of reporter gene constructs and showed that all three motifs are necessary to maintain the high natural expression level of this gene. One of the motifs was similar to the binding site of an orthologue of POP-1, and we showed that RNAi knockdown of pop-1 impacts the expression of rpl-2. We further determined the transcription start site of rpl-2 by 5’ RACE and found that the motifs lie 40–90 bases upstream of the start site. We also found evidence that a noncoding RNA, contained within the outron of rpl-2, is co-transcribed with rpl-2 and cleaved during trans-splicing. Conclusions Our results indicate that C. elegans RPGs are regulated by a complex novel series of regulatory elements that is evolutionarily distinct from

  13. Recent advances in bacterial heme protein biochemistry

    OpenAIRE

    Mayfield, Jeffery A.; Dehner, Carolyn A.; Dubois, Jennifer L.

    2011-01-01

    Recent progress in genetics, fed by the burst in genome sequence data, has led to the identification of a host of novel bacterial heme proteins that are now being characterized in structural and mechanistic terms. The following short review highlights very recent work with bacterial heme proteins involved in the uptake, biosynthesis, degradation, and use of heme in respiration and sensing.

  14. Abiotic Stress Resistance, a Novel Moonlighting Function of Ribosomal Protein RPL44 in the Halophilic Fungus Aspergillus glaucus

    OpenAIRE

    LIU, XIAO-DAN; Xie, Lixia; Wei, Yi; Zhou, Xiaoyang; Jia, Baolei; Liu, Jinliang; Zhang, Shihong

    2014-01-01

    Ribosomal proteins are highly conserved components of basal cellular organelles, primarily involved in the translation of mRNA leading to protein synthesis. However, certain ribosomal proteins moonlight in the development and differentiation of organisms. In this study, the ribosomal protein L44 (RPL44), associated with salt resistance, was screened from the halophilic fungus Aspergillus glaucus (AgRPL44), and its activity was investigated in Saccharomyces cerevisiae and Nicotiana tabacum. Se...

  15. Affinity chromatography and capillary electrophoresis for analysis of the yeast ribosomal proteins

    Directory of Open Access Journals (Sweden)

    Miriam S. Goyder

    2012-04-01

    Full Text Available We present a top down separation platform for yeast ribosomalproteins using affinity chromatography and capillary electrophoresiswhich is designed to allow deposition of proteins ontoa substrate. FLAG tagged ribosomes were affinity purified, andrRNA acid precipitation was performed on the ribosomes followedby capillary electrophoresis to separate the ribosomalproteins. Over 26 peaks were detected with excellent reproducibility(<0.5% RSD migration time. This is the first reportedseparation of eukaryotic ribosomal proteins using capillaryelectrophoresis. The two stages in this workflow, affinity chromatographyand capillary electrophoresis, share the advantagesthat they are fast, flexible and have small sample requirementsin comparison to more commonly used techniques. This methodis a remarkably quick route from cell to separation that hasthe potential to be coupled to high throughput readout platformsfor studies of the ribosomal proteome. [BMB reports2012; 45(4: 233-238

  16. Ribosome-dependent ATPase interacts with conserved membrane protein in Escherichia coli to modulate protein synthesis and oxidative phosphorylation.

    Directory of Open Access Journals (Sweden)

    Mohan Babu

    Full Text Available Elongation factor RbbA is required for ATP-dependent deacyl-tRNA release presumably after each peptide bond formation; however, there is no information about the cellular role. Proteomic analysis in Escherichia coli revealed that RbbA reciprocally co-purified with a conserved inner membrane protein of unknown function, YhjD. Both proteins are also physically associated with the 30S ribosome and with members of the lipopolysaccharide transport machinery. Genome-wide genetic screens of rbbA and yhjD deletion mutants revealed aggravating genetic interactions with mutants deficient in the electron transport chain. Cells lacking both rbbA and yhjD exhibited reduced cell division, respiration and global protein synthesis as well as increased sensitivity to antibiotics targeting the ETC and the accuracy of protein synthesis. Our results suggest that RbbA appears to function together with YhjD as part of a regulatory network that impacts bacterial oxidative phosphorylation and translation efficiency.

  17. Label- and amplification-free electrochemical detection of bacterial ribosomal RNA.

    Science.gov (United States)

    Henihan, Grace; Schulze, Holger; Corrigan, Damion K; Giraud, Gerard; Terry, Jonathan G; Hardie, Alison; Campbell, Colin J; Walton, Anthony J; Crain, Jason; Pethig, Ronald; Templeton, Kate E; Mount, Andrew R; Bachmann, Till T

    2016-07-15

    Current approaches to molecular diagnostics rely heavily on PCR amplification and optical detection methods which have restrictions when applied to point of care (POC) applications. Herein we describe the development of a label-free and amplification-free method of pathogen detection applied to Escherichia coli which overcomes the bottleneck of complex sample preparation and has the potential to be implemented as a rapid, cost effective test suitable for point of care use. Ribosomal RNA is naturally amplified in bacterial cells, which makes it a promising target for sensitive detection without the necessity for prior in vitro amplification. Using fluorescent microarray methods with rRNA targets from a range of pathogens, an optimal probe was selected from a pool of probe candidates identified in silico. The specificity of probes was investigated on DNA microarray using fluorescently labeled 16S rRNA target. The probe yielding highest specificity performance was evaluated in terms of sensitivity and a LOD of 20 pM was achieved on fluorescent glass microarray. This probe was transferred to an EIS end point format and specificity which correlated to microarray data was demonstrated. Excellent sensitivity was facilitated by the use of uncharged PNA probes and large 16S rRNA target and investigations resulted in an LOD of 50 pM. An alternative kinetic EIS assay format was demonstrated with which rRNA could be detected in a species specific manner within 10-40min at room temperature without wash steps. PMID:27016627

  18. Dual Targeting of the Protein Methyltransferase PrmA Contributes to Both Chloroplastic and Mitochondrial Ribosomal Protein L11 Methylation in Arabidopsis.

    Science.gov (United States)

    Mazzoleni, Meryl; Figuet, Sylvie; Martin-Laffon, Jacqueline; Mininno, Morgane; Gilgen, Annabelle; Leroux, Mélanie; Brugière, Sabine; Tardif, Marianne; Alban, Claude; Ravanel, Stéphane

    2015-09-01

    Methylation of ribosomal proteins has long been described in prokaryotes and eukaryotes, but our knowledge about the enzymes responsible for these modifications in plants is scarce. The bacterial protein methyltransferase PrmA catalyzes the trimethylation of ribosomal protein L11 (RPL11) at three distinct sites. The role of these modifications is still unknown. Here, we show that PrmA from Arabidopsis thaliana (AtPrmA) is dually targeted to chloroplasts and mitochondria. Mass spectrometry and enzymatic assays indicated that the enzyme methylates RPL11 in plasto- and mitoribosomes in vivo. We determined that the Arabidopsis and Escherichia coli PrmA enzymes share similar product specificity, making trimethylated residues, but, despite an evolutionary relationship, display a difference in substrate site specificity. In contrast to the bacterial enzyme that trimethylates the ε-amino group of two lysine residues and the N-terminal α-amino group, AtPrmA methylates only one lysine in the MAFCK(D/E)(F/Y)NA motif of plastidial and mitochondrial RPL11. The plant enzyme possibly methylates the N-terminus of plastidial RPL11, whereas mitochondrial RPL11 is N-α-acetylated by an unknown acetyltransferase. Lastly, we found that an Arabidopsis prma-null mutant is viable in standard environmental conditions and no molecular defect could be associated with a lack of RPL11 methylation in leaf chloroplasts or mitochondria. However, the conservation of PrmA during the evolution of photosynthetic eukaryotes together with the location of methylated residues at the binding site of translation factors to ribosomes suggests that RPL11 methylation in plant organelles could be involved, in combination with other post-translational modifications, in optimizing ribosome function. PMID:26116422

  19. Developmentally regulated phosphorylation-dephosphorylation of ribosomal proteins from maize embryonic axes

    International Nuclear Information System (INIS)

    Previous work in our lab suggests translational control during maize germination. To test this possibility the present research focuses on the phosphorylated status of the ribosomal proteins of maize axes during germination. Ribosomes from embryonic axes incubated for different periods were in vitro or in vivo labeled by 1h-pulse for 32P orthophosphate. Electrophoretic analysis of the ribosomal proteins and autoradiographs revealed: (a) in vitro, several 32P bands and very similar patterns for all stages tested (0 to 24h); in vivo, less number of labeled bands and a changing pattern from 3 to 24h of incubation. A protein of 30,900 MW did not appear phosphorylated until 8h of incubation, while a 17,000 MW protein was strongly labeled at 3h and fastly dephosphorylated toward 24h. Phosphorylated proteins belong to both the small and the large subunits. The implication of this process will be discussed

  20. Nucleotide sequence and characterization of the transcript of a Dictyostelium ribosomal protein gene.

    OpenAIRE

    Steel, L F; Smyth, A; A. Jacobson

    1987-01-01

    Dictyostelium ribosomal protein mRNAs are subject to developmental regulation of both their translation and their stability. In order to consider whether such post-transcriptional regulation can be attributed to structural features of the mRNAs, we have cloned and sequenced a 1.9 kb EcoRI genomic DNA fragment which contains the gene for the Dictyostelium ribosomal protein 1024 (rp1024). The rp1024 gene contains a single intron of 350 bp which begins just after the fourth codon of protein codi...

  1. Synthesis and Evaluation of a Library of Fluorescent Dipeptidomimetic Analogues as Substrates for Modified Bacterial Ribosomes.

    Science.gov (United States)

    Chowdhury, Sandipan Roy; Chauhan, Pradeep S; Dedkova, Larisa M; Bai, Xiaoguang; Chen, Shengxi; Talukder, Poulami; Hecht, Sidney M

    2016-05-01

    Described herein are the synthesis and photophysical characterization of a library of aryl-substituted oxazole- and thiazole-based dipeptidomimetic analogues, and their incorporation into position 66 of green fluorescent protein (GFP) in lieu of the natural fluorophore. These fluorescent analogues resemble the fluorophore formed naturally by GFP. As anticipated, the photophysical properties of the analogues varied as a function of the substituents at the para position of the phenyl ring. The fluorescence emission wavelength maxima of compounds in the library varied from ∼365 nm (near-UV region) to ∼490 nm (visible region). The compounds also exhibited a large range of quantum yields (0.01-0.92). The analogues were used to activate a suppressor tRNACUA and were incorporated into position 66 of GFP using an in vitro protein biosynthesizing system that employed engineered ribosomes selected for their ability to incorporate dipeptides. Four analogues with interesting photophysical properties and reasonable suppression yields were chosen, and the fluorescent proteins (FPs) containing these fluorophores were prepared on a larger scale for more detailed study. When the FPs were compared with the respective aminoacyl-tRNAs and the actual dipeptide analogues, the FPs exhibited significantly enhanced fluorescence intensities at the same concentrations. Part of this was shown to be due to the presence of the fluorophores as an intrinsic element of the protein backbone. There were also characteristic shifts in the emission maxima, indicating the environmental sensitivity of these probes. Acridon-2-ylalanine and oxazole 1a were incorporated into positions 39 and 66 of GFP, respectively, and were shown to form an efficient Förster resonance energy transfer (FRET) pair, demonstrating that the analogues can be used as FRET probes. PMID:27050631

  2. Binding site of ribosomal proteins on prokaryotic 5S ribonucleic acids: a study with ribonucleases

    DEFF Research Database (Denmark)

    Douthwaite, S; Christensen, A; Garrett, R A

    1982-01-01

    stearothermophilus 5S RNA. Several protein-induced changes in the RNA structures were identified; some are possibly allosteric in nature. The two prokaryotic 5S RNAs were also incubated with total 50S subunit proteins from E. coli and B. stearothermophilus ribosomes. Homologous and heterologous reconstitution...

  3. Phosphorylation in vivo of non-ribosomal proteins from native 40 S ribosomal particles of Krebs II mouse ascites-tumour cells

    DEFF Research Database (Denmark)

    Schuck, J; Reichert, G; Issinger, O G

    1981-01-01

    Four non-ribosomal proteins from native 40 S ribosomal subunits with mol.wts. of 110 000, 84 000, 68 000 and 26 000 were phosphorylated in vivo when ascites cells were incubated in the presence of [32P]Pi. The 110 000-, 84 000- and 26 000-dalton proteins are identical with phosphorylated products...... from native 40 S subunits after phosphorylation in vitro by a cyclic nucleotide-independent protein kinase. Phosphoserine was the major phosphorylated amino acid of the proteins phosphorylated in vivo and in vitro....

  4. Ribosomal P3 protein AtP3B of Arabidopsis acts as both protein and RNA chaperone to increase tolerance of heat and cold stresses.

    Science.gov (United States)

    Kang, Chang Ho; Lee, Young Mee; Park, Joung Hun; Nawkar, Ganesh M; Oh, Hun Taek; Kim, Min Gab; Lee, Soo In; Kim, Woe Yeon; Yun, Dae-Jin; Lee, Sang Yeol

    2016-07-01

    The P3 proteins are plant-specific ribosomal P-proteins; however, their molecular functions have not been characterized. In a screen for components of heat-stable high-molecular weight (HMW) complexes, we isolated the P3 protein AtP3B from heat-treated Arabidopsis suspension cultures. By size-exclusion chromatography (SEC), SDS-PAGE and native PAGE followed by immunoblotting with anti-AtP3B antibody, we showed that AtP3B was stably retained in HMW complexes following heat shock. The level of AtP3B mRNA increased in response to both high- and low-temperature stresses. Bacterially expressed recombinant AtP3B protein exhibited both protein and RNA chaperone activities. Knockdown of AtP3B by RNAi made plants sensitive to both high- and low-temperature stresses, whereas overexpression of AtP3B increased tolerance of both conditions. Together, our results suggest that AtP3B protects cells against both high- and low-temperature stresses. These findings provide novel insight into the molecular functions and in vivo roles of acidic ribosomal P-proteins, thereby expanding our knowledge of the protein production machinery. PMID:27004478

  5. Differential Effects of Replacing Escherichia coli Ribosomal Protein L27 with Its Homologue from Aquifex aeolicus

    OpenAIRE

    Maguire, Bruce A.; Manuilov, Anton V; Zimmermann, Robert A.

    2001-01-01

    The rpmA gene, which encodes 50S ribosomal subunit protein L27, was cloned from the extreme thermophile Aquifex aeolicus, and the protein was overexpressed and purified. Comparison of the A. aeolicus protein with its homologue from Escherichia coli by circular dichroism analysis and proton nuclear magnetic resonance spectroscopy showed that it readily adopts some structure in solution that is very stable, whereas the E. coli protein is unstructured under the same conditions. A mutant of E. co...

  6. Targeting and plasticity of mitochondrial proteins revealed by proximity-specific ribosome profiling

    OpenAIRE

    Williams, Christopher C.; Jan, Calvin H.; Weissman, Jonathan S.

    2014-01-01

    Nearly all mitochondrial proteins are nuclear-encoded and are targeted to their mitochondrial destination from the cytosol. Here, we used proximity-specific ribosome profiling to comprehensively measure translation at the mitochondrial surface in yeast. Most inner membrane proteins were co-translationally targeted to mitochondria, reminiscent of proteins entering the endoplasmic reticulum (ER). Comparison between mitochondrial and ER localization demonstrated that the vast majority of protein...

  7. Ribosome-inhibiting proteins from in vitro cultures of Phytolacca dodecandra

    DEFF Research Database (Denmark)

    Thomsen, S.; Hansen, Harald S.; Nyman, U.

    1991-01-01

    initiated from leaf and cotyledon exhibited protein synthesis-inhibiting activity. Ribosome-inhibiting proteins were purified at least 14 times from suspension cells of P. dodecandra. The purified protein fraction contained two proteins as seen by sodium dodecyl sulphate polyacrylamide gel electrophoresis....... The relative molecular masses were 30,000 and 31,000 and they showed a pI > 9.3. These new RIP's were shown to be different from dodecandrin with respect to molecular mass....

  8. Expression of Muscle-Specific Ribosomal Protein L3-Like Impairs Myotube Growth.

    Science.gov (United States)

    Chaillou, Thomas; Zhang, Xiping; McCarthy, John J

    2016-09-01

    The ribosome has historically been considered to have no cell-specific function but rather serve in a "housekeeping" capacity. This view is being challenged by evidence showing that heterogeneity in the protein composition of the ribosome can lead to the functional specialization of the ribosome. Expression profiling of different tissues revealed that ribosomal protein large 3-like (Rpl3l) is exclusively expressed in striated muscle. In response to a hypertrophic stimulus, Rpl3l expression in skeletal muscle was significantly decreased by 82% whereas expression of the ubiquitous paralog Rpl3 was significantly increased by ∼fivefold. Based on these findings, we developed the hypothesis that Rpl3l functions as a negative regulator of muscle growth. To test this hypothesis, we used the Tet-On system to express Rpl3l in myoblasts during myotube formation. In support of our hypothesis, RPL3L expression significantly impaired myotube growth as assessed by myotube diameter (-23%) and protein content (-14%). Further analysis showed that the basis of this impairment was caused by a significant decrease in myoblast fusion as the fusion index was significantly lower (-17%) with RPL3L expression. These findings are the first evidence to support the novel concept of ribosome specialization in skeletal muscle and its role in the regulation of skeletal muscle growth. J. Cell. Physiol. 231: 1894-1902, 2016. © 2015 Wiley Periodicals, Inc. PMID:26684695

  9. Localisation of the ribosomal proteins L2, L14 and L24 of the E. coli ribosome by means of nuclear spin contrast variation

    International Nuclear Information System (INIS)

    Ribosomes are ribonucleo-protein complexes, which are the site of the protein synthesis, one of the most important steps during the translation of the genetic code from the DNA to the proteins. The proteins L2, L14 and L24 in the 50S subunit and the protein L2 in the 70S ribosome of the E. coli bacterium were localised by means of nuclear spin contrast variation. The contrast created by isotopic substitution is increased by almost a factor of three provided that polarised neutrons are scattered by polarised hydrogen spins of the sample. To get additional information about the in situ structure of the proteins the shapes were described by a two or three sphere model. Within the project of mapping the 50S subunit of the E. coli ribosome the protein L24 is localised for the first time. L24 is one of the initiator protein for the assembly of the 50S subunit therefore the knowledge of the position is of great interest for the understanding of this process. With the localisation of the protein L14, which is one of the most conserved protein in the ribosome, new hints are given in respect of the protein function which was until now unknown. The localisation of the protein L2, which is one of the candidates of the area of the central step in protein synthesis was done both in the 50S subunit and in the whole 70S ribosome. This allows to investigate conformational changes during the association of the subunits. (orig.)

  10. Protein-RNA cross-linking in the ribosomes of yeast under oxidative stress.

    Science.gov (United States)

    Mirzaei, Hamid; Regnier, Fred

    2006-12-01

    Living systems have efficient degradative pathways for dealing with the fact that reactive oxygen species (ROS) derived from cellular metabolism and the environment oxidatively damage proteins and DNA. But aggregation and cross-linking can occur as well, leading to a series of problems including disruption of cellular regulation, mutations, and even cell death. The mechanism(s) by which protein aggregation occurs and the macromolecular species involved are poorly understood. In the study reported here, evidence is provided for a new type of aggregate between proteins and RNA in ribosomes. While studying the effect of oxidative stress induced in the yeast proteome it was noted that ribosomal proteins were widely oxidized. Eighty six percent of the proteins in yeast ribosomes were found to be carbonylated after stressing yeast cell cultures with hydrogen peroxide. Moreover, many of these proteins appeared to be cross-linked based on their coelution patterns during RPC separation. Since they were not in direct contact, it was not clear how this could occur unless it was through the RNA separating them in the ribosome. This was confirmed in a multiple-step process, the first being derivatization of all carbonylated proteins in cell lysates with biotin hydrazide through Schiff base formation. Following reduction of Schiff bases with sodium cyanoborohydride, biotinylated proteins were selected from cell lysates with avidin affinity chromatography. Oxidized proteins thus captured were then selected again using boronate affinity chromatography to capture vicinal diol-containing proteins. This would include proteins cross-linked to an RNA fragment containing a ribose residue with 2',3'-hydroxyl groups. Some glycoproteins would also be selected by this process. LC/MS/MS analyses of tryptic peptides derived from proteins captured by this process along with MASCOT searches resulted in the identification of 37 ribosomal proteins that appear to be cross-linked to RNA

  11. Over-represented localized sequence motifs in ribosomal protein gene promoters of basal metazoans.

    Science.gov (United States)

    Perina, Drago; Korolija, Marina; Roller, Maša; Harcet, Matija; Jeličić, Branka; Mikoč, Andreja; Cetković, Helena

    2011-07-01

    Equimolecular presence of ribosomal proteins (RPs) in the cell is needed for ribosome assembly and is achieved by synchronized expression of ribosomal protein genes (RPGs) with promoters of similar strengths. Over-represented motifs of RPG promoter regions are identified as targets for specific transcription factors. Unlike RPs, those motifs are not conserved between mammals, drosophila, and yeast. We analyzed RPGs proximal promoter regions of three basal metazoans with sequenced genomes: sponge, cnidarian, and placozoan and found common features, such as 5'-terminal oligopyrimidine tracts and TATA-boxes. Furthermore, we identified over-represented motifs, some of which displayed the highest similarity to motifs abundant in human RPG promoters and not present in Drosophila or yeast. Our results indicate that humans over-represented motifs, as well as corresponding domains of transcription factors, were established very early in metazoan evolution. The fast evolving nature of RPGs regulatory network leads to formation of other, lineage specific, over-represented motifs. PMID:21457775

  12. Direct ribosome isolation from soil to extract bacterial rRNA for community analysis.

    OpenAIRE

    Felske, A; B. Engelen; Nübel, U; Backhaus, H

    1996-01-01

    A simple method that combines an adapted ribosome isolation method and a common RNA extraction step has been developed for selective recovery of intact rRNA from natural microbial communities in soil. After mechanical cell lysis, ribosomes are separated by centrifugation steps, avoiding massive humic acid contamination and RNA degradation. The protocol accommodates the complex composition of soils by blocking adsorbing surfaces and humic acids with polyvinylpyrrolidone and bovine serum albumi...

  13. Ribosome-associated pentatricopeptide repeat proteins function as translational activators in mitochondria of trypanosomes.

    Science.gov (United States)

    Aphasizheva, Inna; Maslov, Dmitri A; Qian, Yu; Huang, Lan; Wang, Qi; Costello, Catherine E; Aphasizhev, Ruslan

    2016-03-01

    Mitochondrial ribosomes of Trypanosoma brucei are composed of 9S and 12S rRNAs, eubacterial-type ribosomal proteins, polypeptides lacking discernible motifs and approximately 20 pentatricopeptide repeat (PPR) RNA binding proteins. Several PPRs also populate the polyadenylation complex; among these, KPAF1 and KPAF2 function as general mRNA 3' adenylation/uridylation factors. The A/U-tail enables mRNA binding to the small ribosomal subunit and is essential for translation. The presence of A/U-tail also correlates with requirement for translation of certain mRNAs in mammalian and insect parasite stages. Here, we inquired whether additional PPRs activate translation of individual mRNAs. Proteomic analysis identified KRIPP1 and KRIPP8 as components of the small ribosomal subunit in mammalian and insect forms, but also revealed their association with the polyadenylation complex in the latter. RNAi knockdowns demonstrated essential functions of KRIPP1 and KRIPP8 in the actively respiring insect stage, but not in the mammalian stage. In the KRIPP1 knockdown, A/U-tailed mRNA encoding cytochrome c oxidase subunit 1 declined concomitantly with the de novo synthesis of this subunit whereas polyadenylation and translation of cyb mRNA were unaffected. In contrast, the KRIPP8 knockdown inhibited A/U-tailing and translation of both CO1 and cyb mRNAs. Our findings indicate that ribosome-associated PPRs may selectively activate mRNAs for translation. PMID:26713541

  14. Cloning and expression of antiviral/ribosome-inactivating protein from Bougainvillea xbuttiana

    Indian Academy of Sciences (India)

    Nandlal Choudhary; Harish C Kapoor; Madan L Lodha

    2008-03-01

    A full-length cDNA encoding ribosome-inactivating/antiviral protein (RIP/AVP) from the leaves of Bougainvillea xbuttiana was isolated. The cDNA consisted of 1364 nucleotides with an open reading frame (ORF) of 960 nucleotides encoding a 35.49 kDa protein of 319 amino acids. The deduced amino acid sequence has a putative active domain conserved in RIPs/AVPs and shows a varying phylogenetic relationship to the RIPs from other plant species. The deduced protein has been designated BBAP1 (Bougainvillea xbuttiana antiviral protein1). The ORF was cloned into an expression vector and expressed in E. coli as a fusion protein of ∼78 kDa. The cleaved and purified recombinant BBAP1 exhibited ribosome-inhibiting rRNA -glycosidase activity, and imparted a high level of resistance against the tobacco mosaic virus (TMV).

  15. The antibiotic thiostrepton inhibits a functional transition within protein L11 at the ribosomal GTPase centre

    DEFF Research Database (Denmark)

    Porse, B T; Leviev, I; Mankin, A S;

    1998-01-01

    reduced for the mutant L11-rRNA complexes. These results indicate that although, as shown earlier, thiostrepton binds primarily to 23 S rRNA, the drug probably inhibits peptide elongation by impeding a conformational change within protein L11 that is important for the function of the ribosomal GTPase...

  16. Synaptic Activation of Ribosomal Protein S6 Phosphorylation Occurs Locally in Activated Dendritic Domains

    Science.gov (United States)

    Pirbhoy, Patricia Salgado; Farris, Shannon; Steward, Oswald

    2016-01-01

    Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylation of ribosomal protein S6 (rpS6) in postsynaptic neurons, but the functional significance of rpS6 phosphorylation is poorly understood. Here, we show that synaptic stimulation that induces perforant path LTP triggers phosphorylation of rpS6 (p-rpS6)…

  17. Development of Soft Tissue Sarcomas in Ribosomal Proteins L5 and S24 Heterozygous Mice

    Czech Academy of Sciences Publication Activity Database

    Kazerounian, S.; Ciarlini, P.D.S.C.; Yuan, D.; Ghazvinian, R.; Alberich-Jorda, Meritxell; Joshi, M.; Zhang, H.; Beggs, A.H.; Gazda, H.T.

    2016-01-01

    Roč. 7, č. 1 (2016), s. 32-36. ISSN 1837-9664 R&D Projects: GA MŠk LK21307 Institutional support: RVO:68378050 Keywords : Ribosomal proteins RPL5 and RPS24 * Diamond-Blackfan anemia * Soft tissue sarcoma Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.271, year: 2014

  18. Protein Folding Activity of the Ribosome (PFAR –– A Target for Antiprion Compounds

    Directory of Open Access Journals (Sweden)

    Debapriya Banerjee

    2014-10-01

    Full Text Available Prion diseases are fatal neurodegenerative diseases affecting mammals. Prions are misfolded amyloid aggregates of the prion protein (PrP, which form when the alpha helical, soluble form of PrP converts to an aggregation-prone, beta sheet form. Thus, prions originate as protein folding problems. The discovery of yeast prion(s and the development of a red-/white-colony based assay facilitated safe and high-throughput screening of antiprion compounds. With this assay three antiprion compounds; 6-aminophenanthridine (6AP, guanabenz acetate (GA, and imiquimod (IQ have been identified. Biochemical and genetic studies reveal that these compounds target ribosomal RNA (rRNA and inhibit specifically the protein folding activity of the ribosome (PFAR. The domain V of the 23S/25S/28S rRNA of the large ribosomal subunit constitutes the active site for PFAR. 6AP and GA inhibit PFAR by competition with the protein substrates for the common binding sites on the domain V rRNA. PFAR inhibition by these antiprion compounds opens up new possibilities for understanding prion formation, propagation and the role of the ribosome therein. In this review, we summarize and analyze the correlation between PFAR and prion processes using the antiprion compounds as tools.

  19. Pentatricopeptide repeat proteins in Trypanosoma brucei function in mitochondrial ribosomes

    OpenAIRE

    Pusnik, Mascha; Small, Ian; Read, Laurie K.; Fabbro, Thomas; Schneider, André

    2008-01-01

    The pentatricopeptide repeat (PPR), a degenerate 35-amino-acid motif, defines a novel eukaryotic protein family. Plants have 400 to 500 distinct PPR proteins, whereas other eukaryotes generally have fewer than 5. The few PPR proteins that have been studied have roles in organellar gene expression, probably via direct interaction with RNA. Here we show that the parasitic protozoan Trypanosoma brucei encodes 28 distinct PPR proteins, an extraordinarily high number for a nonplant organism. A com...

  20. Research Techniques Made Simple: Bacterial 16S Ribosomal RNA Gene Sequencing in Cutaneous Research.

    Science.gov (United States)

    Jo, Jay-Hyun; Kennedy, Elizabeth A; Kong, Heidi H

    2016-03-01

    Skin serves as a protective barrier and also harbors numerous microorganisms collectively comprising the skin microbiome. As a result of recent advances in sequencing (next-generation sequencing), our understanding of microbial communities on skin has advanced substantially. In particular, the 16S ribosomal RNA gene sequencing technique has played an important role in efforts to identify the global communities of bacteria in healthy individuals and patients with various disorders in multiple topographical regions over the skin surface. Here, we describe basic principles, study design, and a workflow of 16S ribosomal RNA gene sequencing methodology, primarily for investigators who are not familiar with this approach. This article will also discuss some applications and challenges of 16S ribosomal RNA sequencing as well as directions for future development. PMID:26902128

  1. Ribosomal Protein P2 from apicomplexan parasite Toxoplasma gondii is intrinsically a molten globule.

    Science.gov (United States)

    Mishra, Pushpa; Choudhary, Sinjan; Hosur, Ramakrishna V

    2015-01-01

    Toxoplasma gondii is an apicomplexan parasite, which causes toxoplasmosis. Toxoplasma P2 (TgP2) is a ribosomal protein and exists as supramolecular assembly with other proteins in the ribosome. It is also shown that TgP2 is involved in some extra ribosomal functions. However, till date the protein has evaded structural characterization by any of the known techniques. In this background, we report here a systematic study using a variety of biophysical techniques and NMR, under different conditions of pH and temperature, and deduce that TgP2 consists of only helices and unstructured regions, is a monomer at low pH but forms multimers at higher pH, and has intrinsically a molten globule structure. The C-terminal half is flexible and the helices are concentrated in the N-terminal half of the chain. The dynamism inherent to the molten globule structure may have functional implications for its extra-ribosomal functions. which is contrast to that of human P2. PMID:25866913

  2. From DNA to proteins via the ribosome: Structural insights into the workings of the translation machinery

    OpenAIRE

    Agirrezabala Xabier; Frank Joachim

    2010-01-01

    Abstract Understanding protein synthesis in bacteria and humans is important for understanding the origin of many human diseases and devising treatments for them. Over the past decade, the field of structural biology has made significant advances in the visualisation of the molecular machinery involved in protein synthesis. It is now possible to discern, at least in outline, the way that interlocking ribosomal components and factors adapt their conformations throughout this process. The deter...

  3. Mimicking Ribosomal Unfolding of RNA Pseudoknot in a Protein Channel.

    Science.gov (United States)

    Zhang, Xinyue; Xu, Xiaojun; Yang, Zhiyu; Burcke, Andrew J; Gates, Kent S; Chen, Shi-Jie; Gu, Li-Qun

    2015-12-23

    Pseudoknots are a fundamental RNA tertiary structure with important roles in regulation of mRNA translation. Molecular force spectroscopic approaches such as optical tweezers can track the pseudoknot's unfolding intermediate states by pulling the RNA chain from both ends, but the kinetic unfolding pathway induced by this method may be different from that in vivo, which occurs during translation and proceeds from the 5' to 3' end. Here we developed a ribosome-mimicking, nanopore pulling assay for dissecting the vectorial unfolding mechanism of pseudoknots. The pseudoknot unfolding pathway in the nanopore, either from the 5' to 3' end or in the reverse direction, can be controlled by a DNA leader that is attached to the pseudoknot at the 5' or 3' ends. The different nanopore conductance between DNA and RNA translocation serves as a marker for the position and structure of the unfolding RNA in the pore. With this design, we provided evidence that the pseudoknot unfolding is a two-step, multistate, metal ion-regulated process depending on the pulling direction. Most notably, unfolding in both directions is rate-limited by the unzipping of the first helix domain (first step), which is Helix-1 in the 5' → 3' direction and Helix-2 in the 3' → 5' direction, suggesting that the initial unfolding step in either pulling direction needs to overcome an energy barrier contributed by the noncanonical triplex base-pairs and coaxial stacking interactions for the tertiary structure stabilization. These findings provide new insights into RNA vectorial unfolding mechanisms, which play an important role in biological functions including frameshifting. PMID:26595106

  4. The ribosome as a missing link in prebiotic evolution II: Ribosomes encode ribosomal proteins that bind to common regions of their own mRNAs and rRNAs.

    Science.gov (United States)

    Root-Bernstein, Robert; Root-Bernstein, Meredith

    2016-05-21

    We have proposed that the ribosome may represent a missing link between prebiotic chemistries and the first cells. One of the predictions that follows from this hypothesis, which we test here, is that ribosomal RNA (rRNA) must have encoded the proteins necessary for ribosomal function. In other words, the rRNA also functioned pre-biotically as mRNA. Since these ribosome-binding proteins (rb-proteins) must bind to the rRNA, but the rRNA also functioned as mRNA, it follows that rb-proteins should bind to their own mRNA as well. This hypothesis can be contrasted to a "null" hypothesis in which rb-proteins evolved independently of the rRNA sequences and therefore there should be no necessary similarity between the rRNA to which rb-proteins bind and the mRNA that encodes the rb-protein. Five types of evidence reported here support the plausibility of the hypothesis that the mRNA encoding rb-proteins evolved from rRNA: (1) the ubiquity of rb-protein binding to their own mRNAs and autogenous control of their own translation; (2) the higher-than-expected incidence of Arginine-rich modules associated with RNA binding that occurs in rRNA-encoded proteins; (3) the fact that rRNA-binding regions of rb-proteins are homologous to their mRNA binding regions; (4) the higher than expected incidence of rb-protein sequences encoded in rRNA that are of a high degree of homology to their mRNA as compared with a random selection of other proteins; and (5) rRNA in modern prokaryotes and eukaryotes encodes functional proteins. None of these results can be explained by the null hypothesis that assumes independent evolution of rRNA and the mRNAs encoding ribosomal proteins. Also noteworthy is that very few proteins bind their own mRNAs that are not associated with ribosome function. Further tests of the hypothesis are suggested: (1) experimental testing of whether rRNA-encoded proteins bind to rRNA at their coding sites; (2) whether tRNA synthetases, which are also known to bind to their

  5. C-reactive protein and bacterial meningitis

    DEFF Research Database (Denmark)

    Gerdes, Lars Ulrik; Jørgensen, P E; Nexø, E;

    1998-01-01

    The aim of the study was to review published articles on the diagnostic accuracy of C-reactive protein (CRP) tests with cerebrospinal fluid and serum in diagnosing bacterial meningitis. The literature from 1980 and onwards was searched using the electronic databases of MEDLINE, and we used summary...

  6. Mitochondrial Ribosomal Protein L41 Suppresses Cell Growth in Association with p53 and p27Kip1

    OpenAIRE

    Yoo, Young A; Kim, Mi Jin; Park, Jong Kuk; Chung, Young Min; Lee, Jong Hyeok; Chi, Sung-Gil; Kim, Jun Suk; Yoo, Young Do

    2005-01-01

    The p53 protein arrests the cell cycle at the G1 phase when stabilized by the interaction between ribosomal proteins and HDM2 under growth-inhibitory conditions. Meanwhile, p53, when translocated to the mitochondria in response to cell death signals, induces apoptosis via transcription-independent mechanisms. In this report, we demonstrate that the mitochondrial ribosomal protein L41 (MRPL41) enhances p53 stability and contributes to p53-induced apoptosis in response to growth-inhibitory cond...

  7. Expression of protein-coding genes embedded in ribosomal DNA

    DEFF Research Database (Denmark)

    Johansen, Steinar D; Haugen, Peik; Nielsen, Henrik

    2007-01-01

    encode reverse transcriptase-like genes, and group I introns and archaeal introns that encode homing endonuclease genes (HEGs). Although rDNA-embedded protein genes are widespread in nuclei, organelles and bacteria, there is surprisingly little information available on how these genes are expressed....... Exceptions include a handful of HEGs from group I introns. Recent studies have revealed unusual and essential roles of group I and group I-like ribozymes in the endogenous expression of HEGs. Here we discuss general aspects of rDNA-embedded protein genes and focus on HEG expression from group I introns in...

  8. Engineered fluorescent proteins illuminate the bacterial periplasm

    Directory of Open Access Journals (Sweden)

    Thorben Dammeyer

    2012-10-01

    Full Text Available The bacterial periplasm is of special interest whenever cell factories are designed and engineered. Recombinantely produced proteins are targeted to the periplasmic space of Gram negative bacteria to take advantage of the authentic N-termini, disulfide bridge formation and easy accessibility for purification with less contaminating cellular proteins. The oxidizing environment of the periplasm promotes disulfide bridge formation - a prerequisite for proper folding of many proteins into their active conformation. In contrast, the most popular reporter protein in all of cell biology, Green Fluorescent Protein (GFP, remains inactive if translocated to the periplasmic space prior to folding. Here, the self-catalyzed chromophore maturation is blocked by formation of covalent oligomers via interchain disulfide bonds in the oxidizing environment. However, different protein engineering approaches addressing folding and stability of GFP resulted in improved proteins with enhanced folding properties. Recent studies describe GFP variants that are not only active if translocated in their folded form via the twin-arginine translocation (Tat pathway, but actively fold in the periplasm following general secretory pathway (Sec and signal recognition particle (SRP mediated secretion. This mini-review highlights the progress that enables new insights into bacterial export and periplasmic protein organization, as well as new biotechnological applications combining the advantages of the periplasmic production and the Aequorea-based fluorescent reporter proteins.

  9. Direct TFIIA-TFIID protein contacts drive budding yeast ribosomal protein gene transcription.

    Science.gov (United States)

    Layer, Justin H; Weil, P Anthony

    2013-08-01

    We have previously shown that yeast TFIID provides coactivator function on the promoters of ribosomal protein-encoding genes (RPGs) by making direct contact with the transactivator repressor activator protein 1 (Rap1). Further, our structural studies of assemblies generated with purified Rap1, TFIID, and TFIIA on RPG enhancer-promoter DNA indicate that Rap1-TFIID interaction induces dramatic conformational rearrangements of enhancer-promoter DNA and TFIID-bound TFIIA. These data indicate a previously unknown yet critical role for yeast TFIIA in the integration of activator-TFIID contacts with promoter conformation and downstream preinitiation complex formation and/or function. Here we describe the use of systematic mutagenesis to define how specific TFIIA contacts contribute to these processes. We have verified that TFIIA is required for RPG transcription in vivo and in vitro, consistent with the existence of a critical Rap1-TFIIA-TFIID interaction network. We also identified essential points of contact for TFIIA and Rap1 within the Rap1 binding domain of the Taf4 subunit of TFIID. These data suggest a mechanism for how interactions between TFIID, TFIIA, and Rap1 contribute to the high rate of transcription initiation seen on RPGs in vivo. PMID:23814059

  10. Orsay virus utilizes ribosomal frameshifting to express a novel protein that is incorporated into virions

    International Nuclear Information System (INIS)

    Orsay virus is the first identified virus that is capable of naturally infecting Caenorhabditis elegans. Although it is most closely related to nodaviruses, Orsay virus differs from nodaviruses in its genome organization. In particular, the Orsay virus RNA2 segment encodes a putative novel protein of unknown function, termed delta, which is absent from all known nodaviruses. Here we present evidence that Orsay virus utilizes a ribosomal frameshifting strategy to express a novel fusion protein from the viral capsid (alpha) and delta ORFs. Moreover, the fusion protein was detected in purified virus fractions, demonstrating that it is most likely incorporated into Orsay virions. Furthermore, N-terminal sequencing of both the fusion protein and the capsid protein demonstrated that these proteins must be translated from a non-canonical initiation site. While the function of the alpha–delta fusion remains cryptic, these studies provide novel insights into the fundamental properties of this new clade of viruses. - Highlights: • Orsay virus encodes a novel fusion protein by a ribosomal frameshifting mechanism. • Orsay capsid and fusion protein is translated from a non-canonical initiation site. • The fusion protein is likely incorporated into Orsay virions

  11. Survey of bacterial diversity in chronic wounds using Pyrosequencing, DGGE, and full ribosome shotgun sequencing

    OpenAIRE

    Wolcott Benjamin M; Rhoads Daniel D; Secor Patrick R; Sun Yan; Dowd Scot E; James Garth A; Wolcott Randall D

    2008-01-01

    Abstract Background Chronic wound pathogenic biofilms are host-pathogen environments that colonize and exist as a cohabitation of many bacterial species. These bacterial populations cooperate to promote their own survival and the chronic nature of the infection. Few studies have performed extensive surveys of the bacterial populations that occur within different types of chronic wound biofilms. The use of 3 separate16S-based molecular amplifications followed by pyrosequencing, shotgun Sanger ...

  12. Ice nucleation protein as a bacterial surface display protein

    OpenAIRE

    Sarhan Mohammed A.A.

    2011-01-01

    Surface display technology can be defined as that phenotype (protein or peptide) which is linked to a genotype (DNA or RNA) through an appropriate anchoring motif. A bacterial surface display system is based on expressing recombinant proteins fused to sorting signals (anchoring motifs) that direct their incorporation on the cell surface.

  13. A residue substitution in the plastid ribosomal protein L12/AL1 produces defective plastid ribosome and causes early seedling lethality in rice.

    Science.gov (United States)

    Zhao, Dong-Sheng; Zhang, Chang-Quan; Li, Qian-Feng; Yang, Qing-Qing; Gu, Ming-Hong; Liu, Qiao-Quan

    2016-05-01

    The plastid ribosome is essential for chloroplast biogenesis as well as seedling formation. As the plastid ribosome closely resembles the prokaryotic 70S ribosome, many plastid ribosomal proteins (PRPs) have been identified in higher plants. However, their assembly in the chloroplast ribosome in rice remains unclear. In the present study, we identified a novel rice mutant, albino lethal 1 (al1), from a chromosome segment substitution line population. The al1 mutant displayed an albino phenotype at the seedling stage and did not survive past the three-leaf stage. No other apparent differences in plant morphology were observed in the al1 mutant. The albino phenotype of the al1 mutant was associated with decreased chlorophyll content and abnormal chloroplast morphology. Using fine mapping, AL1 was shown to encode the PRPL12, a protein localized in the chloroplasts of rice, and a spontaneous single-nucleotide mutation (C/T), resulting in a residue substitution from leucine in AL1 to phenylalanine in al1, was found to be responsible for the early seedling lethality. This point mutation is located at the L10 interface feature of the L12/AL1 protein. Yeast two-hybrid analysis showed that there was no physical interaction between al1 and PRPL10. In addition, the mutation had little effect on the transcript abundance of al1, but had a remarkable effect on the protein abundance of al1 and transcript abundance of chloroplast biogenesis-related and photosynthesis-related genes. These results provide a first glimpse into the molecular details of L12's function in rice. PMID:26873698

  14. Involvement of ribosomal protein RPL27a in meristem activity and organ development

    OpenAIRE

    Szakonyi, Dóra; Byrne, Mary E

    2011-01-01

    The plant shoot apical meristem is established early during embryogenesis and subsequently gives rise to a shoot through reiterative generation of lateral organs and axillary meristems. In our recent manuscript we reported identification and characterization of a semi-dominant mutation in ribosomal protein RPL27a, which disrupts plant growth and shoot development.1 rpl27ac-1d effects on the shoot are evident from an early stage of embryo development. During embryogenesis rpl27-1d mutants are ...

  15. Study of the radiation effect on the ribosome proteins using proteolytic enzymes

    International Nuclear Information System (INIS)

    Radiosensitivity of ribosomal proteins isolated from intact and preirradiated, at early stages of ontogenesis, wheat seeds (0.5, 5 and 40 kr) has been studied. It has been shown that γ-irradiation of airdry seeds impaires in the seedlings the firmness of the polypertide chain bonds in the molecule of the ribonucleoprotein strand, which enhances their hydrolytic dissolution under the action of proteolytic enzymes

  16. Physical and biochemical nature of the bacterial cytoplasm: movement and localization of mRNA and the 30S subunits of ribosomes.

    Science.gov (United States)

    Trevors, J T

    2012-05-01

    There is a paucity of knowledge on how mRNA transcripts in the spatially crowded, but molecularly organized bacterial cytoplasm contact the 30S ribosomal subunits. Does simple diffusion in the cytoplasm account for transcript-ribosome interactions given that a large number of ribosomes (e.g., about 72,000 in Escherichia coli during exponential growth) can be present in the cytoplasm? Or are undiscovered mechanisms present where specific transcripts are directed to specific ribosomes at specific cytoplasmic locations, while others are mobilized in a random manner? Moreover, is it possible that cytoplasmic mobilization occurs in bacteria, driven possibly by thermal infrared (IR) radiation and the generation of exclusion zone (EZ) water? These aspects will be discussed in this article and hypotheses presented. PMID:22710107

  17. Influence of commonly used primer systems on automated ribosomal intergenic spacer analysis of bacterial communities in environmental samples.

    Science.gov (United States)

    Purahong, Witoon; Stempfhuber, Barbara; Lentendu, Guillaume; Francioli, Davide; Reitz, Thomas; Buscot, François; Schloter, Michael; Krüger, Dirk

    2015-01-01

    Due to the high diversity of bacteria in many ecosystems, their slow generation times, specific but mostly unknown nutrient requirements and syntrophic interactions, isolation based approaches in microbial ecology mostly fail to describe microbial community structure. Thus, cultivation independent techniques, which rely on directly extracted nucleic acids from the environment, are a well-used alternative. For example, bacterial automated ribosomal intergenic spacer analysis (B-ARISA) is one of the widely used methods for fingerprinting bacterial communities after PCR-based amplification of selected regions of the operon coding for rRNA genes using community DNA. However, B-ARISA alone does not provide any taxonomic information and the results may be severely biased in relation to the primer set selection. Furthermore, amplified DNA stemming from mitochondrial or chloroplast templates might strongly bias the obtained fingerprints. In this study, we determined the applicability of three different B-ARISA primer sets to the study of bacterial communities. The results from in silico analysis harnessing publicly available sequence databases showed that all three primer sets tested are specific to bacteria but only two primers sets assure high bacterial taxa coverage (1406f/23Sr and ITSF/ITSReub). Considering the study of bacteria in a plant interface, the primer set ITSF/ITSReub was found to amplify (in silico) sequences of some important crop species such as Sorghum bicolor and Zea mays. Bacterial genera and plant species potentially amplified by different primer sets are given. These data were confirmed when DNA extracted from soil and plant samples were analyzed. The presented information could be useful when interpreting existing B-ARISA results and planning B-ARISA experiments, especially when plant DNA can be expected. PMID:25749323

  18. Influence of commonly used primer systems on automated ribosomal intergenic spacer analysis of bacterial communities in environmental samples.

    Directory of Open Access Journals (Sweden)

    Witoon Purahong

    Full Text Available Due to the high diversity of bacteria in many ecosystems, their slow generation times, specific but mostly unknown nutrient requirements and syntrophic interactions, isolation based approaches in microbial ecology mostly fail to describe microbial community structure. Thus, cultivation independent techniques, which rely on directly extracted nucleic acids from the environment, are a well-used alternative. For example, bacterial automated ribosomal intergenic spacer analysis (B-ARISA is one of the widely used methods for fingerprinting bacterial communities after PCR-based amplification of selected regions of the operon coding for rRNA genes using community DNA. However, B-ARISA alone does not provide any taxonomic information and the results may be severely biased in relation to the primer set selection. Furthermore, amplified DNA stemming from mitochondrial or chloroplast templates might strongly bias the obtained fingerprints. In this study, we determined the applicability of three different B-ARISA primer sets to the study of bacterial communities. The results from in silico analysis harnessing publicly available sequence databases showed that all three primer sets tested are specific to bacteria but only two primers sets assure high bacterial taxa coverage (1406f/23Sr and ITSF/ITSReub. Considering the study of bacteria in a plant interface, the primer set ITSF/ITSReub was found to amplify (in silico sequences of some important crop species such as Sorghum bicolor and Zea mays. Bacterial genera and plant species potentially amplified by different primer sets are given. These data were confirmed when DNA extracted from soil and plant samples were analyzed. The presented information could be useful when interpreting existing B-ARISA results and planning B-ARISA experiments, especially when plant DNA can be expected.

  19. Purification and properties of new ribosome-inactivating proteins with RNA N-glycosidase activity.

    Science.gov (United States)

    Bolognesi, A; Barbieri, L; Abbondanza, A; Falasca, A I; Carnicelli, D; Battelli, M G; Stirpe, F

    1990-11-30

    Ribosome-inactivating proteins (RIPs) similar to those already known (Stirpe & Barbieri (1986) FEBS Lett. 195, 1-8) were purified from the seeds of Asparagus officinalis (two proteins, asparin 1 and 2), of Citrullus colocynthis (two proteins, colocin 1 and 2), of Lychnis chalcedonica (lychnin) and of Manihot palmata (mapalmin), from the roots of Phytolacca americana (pokeweed antiviral protein from roots, PAP-R) and from the leaves of Bryonia dioica (bryodin-L). The two latter proteins can be considered as isoforms, respectively, of previously purified PAP, from the leaves of P. americana, and of bryodin-R, from the roots of B. dioica. All proteins have an Mr at approx, 30,000, and an alkaline isoelectric point. Bryodin-L, colocins, lychnin and mapalmin are glycoproteins. All RIPs inhibit protein synthesis by a rabbit reticulocyte lysate and phenylalanine polymerization by isolated ribosomes and alter rRNA in a similar manner as the A-chain of ricin and related toxins (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). PMID:2248976

  20. Transient ribosomal attenuation coordinates protein synthesis and co-translational folding.

    Science.gov (United States)

    Zhang, Gong; Hubalewska, Magdalena; Ignatova, Zoya

    2009-03-01

    Clustered codons that pair to low-abundance tRNA isoacceptors can form slow-translating regions in the mRNA and cause transient ribosomal arrest. We report that folding efficiency of the Escherichia coli multidomain protein SufI can be severely perturbed by alterations in ribosome-mediated translational attenuation. Such alterations were achieved by global acceleration of the translation rate with tRNA excess in vitro or by synonymous substitutions to codons with highly abundant tRNAs both in vitro and in vivo. Conversely, the global slow-down of the translation rate modulated by low temperature suppresses the deleterious effect of the altered translational attenuation pattern. We propose that local discontinuous translation temporally separates the translation of segments of the peptide chain and actively coordinates their co-translational folding. PMID:19198590

  1. Eliminated chromatin of Ascaris contains a gene that encodes a putative ribosomal protein.

    OpenAIRE

    Etter, A; Aboutanos, M; Tobler, H; Müller, F.

    1991-01-01

    Chromatin diminution in the nematodes Parascaris equorum and Ascaris lumbricoides leads to the formation of somatic cells that contain less DNA than the germ-line cells. We present molecular evidence for the coding potential of germ-line-specific DNA. We report on a cDNA clone that codes for a putative ribosomal protein (ALEP-1, for A. lumbricoides eliminated protein 1). That the corresponding gene is located in the eliminated portion of the genome indicates a difference in germ-line and soma...

  2. The trypanosome Pumilio-domain protein PUF7 associates with a nuclear cyclophilin and is involved in ribosomal RNA maturation.

    Science.gov (United States)

    Droll, Dorothea; Archer, Stuart; Fenn, Katelyn; Delhi, Praveen; Matthews, Keith; Clayton, Christine

    2010-03-19

    Proteins with Pumilio RNA binding domains (Puf proteins) are ubiquitous in eukaryotes. Some Puf proteins bind to the 3'-untranslated regions of mRNAs, acting to repress translation and promote degradation; others are involved in ribosomal RNA maturation. The genome of Trypanosoma brucei encodes eleven Puf proteins whose function cannot be predicted by sequence analysis. We show here that epitope-tagged TbPUF7 is located in the nucleolus, and associated with a nuclear cyclophilin-like protein, TbNCP1. RNAi targeting PUF7 reduced trypanosome growth and inhibited two steps in ribosomal RNA processing. PMID:20153321

  3. Characterization and analysis of ribosomal proteins in two marine calanoid copepods

    Science.gov (United States)

    Yang, Feifei; Xu, Donghui; Zhuang, Yunyun; Huang, Yousong; Yi, Xiaoyan; Chen, Hongju; Liu, Guangxing; Zhang, Huan

    2016-02-01

    Copepods are among the most abundant and successful metazoans in the marine ecosystem. However, genomic resources related to fundamental cellular processes are still limited in this particular group of crustaceans. Ribosomal proteins are the building blocks of ribosomes, the primary site for protein synthesis. In this study, we characterized and analyzed the cDNAs of cytoplasmic ribosomal proteins (cRPs) of two calanoid copepods, Pseudodiaptomus poplesia and Acartia pacifica. We obtained 79 cRP cDNAs from P. poplesia and 67 from A. pacifica by cDNA library construction/sequencing and rapid amplification of cDNA ends. Analysis of the nucleic acid composition showed that the copepod cRP-encoding genes had higher GC content in the protein-coding regions (CDSs) than in the untranslated regions (UTRs), and single nucleotide repeats (>3 repeats) were common, with "A" repeats being the most frequent, especially in the CDSs. The 3'-UTRs of the cRP genes were significantly longer than the 5'-UTRs. Codon usage analysis showed that the third positions of the codons were dominated by C or G. The deduced amino acid sequences of the cRPs contained high proportions of positively charged residues and had high pI values. This is the first report of a complete set of cRP-encoding genes from copepods. Our results shed light on the characteristics of cRPs in copepods, and provide fundamental data for further studies of protein synthesis in copepods. The copepod cRP information revealed in this study indicates that additional comparisons and analysis should be performed on different taxonomic categories such as orders and families.

  4. The ribosome in action: Tuning of translational efficiency and protein folding.

    Science.gov (United States)

    Rodnina, Marina V

    2016-08-01

    The cellular proteome is shaped by the combined activities of the gene expression and quality control machineries. While transcription plays an undoubtedly important role, in recent years also translation emerged as a key step that defines the composition and quality of the proteome and the functional activity of proteins in the cell. Among the different post-transcriptional control mechanisms, translation initiation and elongation provide multiple checkpoints that can affect translational efficiency. A multitude of specific signals in mRNAs can determine the frequency of translation initiation, choice of the open reading frame, global and local elongation velocities, and the folding of the emerging protein. In addition to specific signatures in the mRNAs, also variations in the global pools of translation components, including ribosomes, tRNAs, mRNAs, and translation factors can alter translational efficiencies. The cellular outcomes of phenomena such as mRNA codon bias are sometimes difficult to understand due to the staggering complexity of covariates that affect codon usage, translation, and protein folding. Here we summarize the experimental evidence on how the ribosome-together with the other components of the translational machinery-can alter translational efficiencies of mRNA at the initiation and elongation stages and how translation velocity affects protein folding. We seek to explain these findings in the context of mechanistic work on the ribosome. The results argue in favour of a new understanding of translation control as a hub that links mRNA homeostasis to production and quality control of proteins in the cell. PMID:27198711

  5. Estimates of Soil Bacterial Ribosome Content and Diversity Are Significantly Affected by the Nucleic Acid Extraction Method Employed.

    Science.gov (United States)

    Wüst, Pia K; Nacke, Heiko; Kaiser, Kristin; Marhan, Sven; Sikorski, Johannes; Kandeler, Ellen; Daniel, Rolf; Overmann, Jörg

    2016-05-01

    Modern sequencing technologies allow high-resolution analyses of total and potentially active soil microbial communities based on their DNA and RNA, respectively. In the present study, quantitative PCR and 454 pyrosequencing were used to evaluate the effects of different extraction methods on the abundance and diversity of 16S rRNA genes and transcripts recovered from three different types of soils (leptosol, stagnosol, and gleysol). The quality and yield of nucleic acids varied considerably with respect to both the applied extraction method and the analyzed type of soil. The bacterial ribosome content (calculated as the ratio of 16S rRNA transcripts to 16S rRNA genes) can serve as an indicator of the potential activity of bacterial cells and differed by 2 orders of magnitude between nucleic acid extracts obtained by the various extraction methods. Depending on the extraction method, the relative abundances of dominant soil taxa, in particularActinobacteriaandProteobacteria, varied by a factor of up to 10. Through this systematic approach, the present study allows guidelines to be deduced for the selection of the appropriate extraction protocol according to the specific soil properties, the nucleic acid of interest, and the target organisms. PMID:26896137

  6. Fluorescent sensors based on bacterial fusion proteins

    International Nuclear Information System (INIS)

    Fluorescence proteins are widely used as markers for biomedical and technological purposes. Therefore, the aim of this project was to create a fluorescent sensor, based in the green and cyan fluorescent protein, using bacterial S-layers proteins as scaffold for the fluorescent tag. We report the cloning, expression and purification of three S-layer fluorescent proteins: SgsE-EGFP, SgsE-ECFP and SgsE-13aa-ECFP, this last containing a 13-amino acid rigid linker. The pH dependence of the fluorescence intensity of the S-layer fusion proteins, monitored by fluorescence spectroscopy, showed that the ECFP tag was more stable than EGFP. Furthermore, the fluorescent fusion proteins were reassembled on silica particles modified with cationic and anionic polyelectrolytes. Zeta potential measurements confirmed the particle coatings and indicated their colloidal stability. Flow cytometry and fluorescence microscopy showed that the fluorescence of the fusion proteins was pH dependent and sensitive to the underlying polyelectrolyte coating. This might suggest that the fluorescent tag is not completely exposed to the bulk media as an independent moiety. Finally, it was found out that viscosity enhanced the fluorescence intensity of the three fluorescent S-layer proteins. (paper)

  7. Fluorescent sensors based on bacterial fusion proteins

    Science.gov (United States)

    Prats Mateu, Batirtze; Kainz, Birgit; Pum, Dietmar; Sleytr, Uwe B.; Toca-Herrera, José L.

    2014-06-01

    Fluorescence proteins are widely used as markers for biomedical and technological purposes. Therefore, the aim of this project was to create a fluorescent sensor, based in the green and cyan fluorescent protein, using bacterial S-layers proteins as scaffold for the fluorescent tag. We report the cloning, expression and purification of three S-layer fluorescent proteins: SgsE-EGFP, SgsE-ECFP and SgsE-13aa-ECFP, this last containing a 13-amino acid rigid linker. The pH dependence of the fluorescence intensity of the S-layer fusion proteins, monitored by fluorescence spectroscopy, showed that the ECFP tag was more stable than EGFP. Furthermore, the fluorescent fusion proteins were reassembled on silica particles modified with cationic and anionic polyelectrolytes. Zeta potential measurements confirmed the particle coatings and indicated their colloidal stability. Flow cytometry and fluorescence microscopy showed that the fluorescence of the fusion proteins was pH dependent and sensitive to the underlying polyelectrolyte coating. This might suggest that the fluorescent tag is not completely exposed to the bulk media as an independent moiety. Finally, it was found out that viscosity enhanced the fluorescence intensity of the three fluorescent S-layer proteins.

  8. Cloning, periplasmic expression, purification and structural characterization of human ribosomal protein L10

    International Nuclear Information System (INIS)

    The ribosomal protein L10 (RP L10) is a strong candidate to be included in the class of tumor suppressor proteins. This protein, also denominated as QM, is known to participate in the binding of ribosomal subunits 60S and 40S and the translation of mRNAs. It has a molecular weight that varies between 24 and 26 kDa and an isoelectric point of (pI) 10.5. The sequence of the protein QM is highly conserved in mammals, plants, invertebrates, insects and yeast which indicates its critical functions in a cell. As a tumor suppressor, RP L10 has been studied in strains of Wilm's tumor (WT-1) and tumor cells in the stomach, where was observed a decrease in the amount of its mRNA. More recently, the RP L10 was found in low amounts in the early stages of prostate adenoma and showed some mutation in ovarian cancer, what indicates its role as a suppressor protein in the development of these diseases. It has also been described that this protein interacts with c-Jun and c-Yes inhibiting growth factors and consequently, cell division. This work has an important role on the establishment of soluble expression of QM to give base information for further studies on expression that aim to evaluate the specific regions where it acts binding the 60S and 40S ribosomal subunits and translation, as well as its binding to proto-oncogenes. The cDNA for QM protein was amplified by PCR and cloned into periplasmic expression vector p3SN8. The QM protein was expressed in E. coli BL21 (DE3) in the region of cytoplasm and periplasm, the best condition was obtained from the expression of the recombinant plasmid QM p1813QM at 25 degree C or 30 degree C, the soluble protein was obtained with small amounts of contaminants. The assays of secondary structure showed that the QM protein is predominantly alpha-helix, but when it loses the folding, this condition changes and the protein is replaced by β- sheet feature. (author)

  9. The structure of Aquifex aeolicus ribosomal protein S8 reveals a unique subdomain that contributes to an extremely tight association with 16S rRNA.

    Science.gov (United States)

    Menichelli, Elena; Edgcomb, Stephen P; Recht, Michael I; Williamson, James R

    2012-01-20

    The assembly of ribonucleoprotein complexes occurs under a broad range of conditions, but the principles that promote assembly and allow function at high temperature are poorly understood. The ribosomal protein S8 from Aquifex aeolicus (AS8) is unique in that there is a 41-residue insertion in the consensus S8 sequence. In addition, AS8 exhibits an unusually high affinity for the 16S ribosomal RNA, characterized by a picomolar dissociation constant that is approximately 26,000-fold tighter than the equivalent interaction from Escherichia coli. Deletion analysis demonstrated that binding to the minimal site on helix 21 occurred at the same nanomolar affinity found for other bacterial species. The additional affinity required the presence of a three-helix junction between helices 20, 21, and 22. The crystal structure of AS8 was solved, revealing the helix-loop-helix geometry of the unique AS8 insertion region, while the core of the molecule is conserved with known S8 structures. The AS8 structure was modeled onto the structure of the 30S ribosomal subunit from E. coli, suggesting the possibility that the unique subdomain provides additional backbone and side-chain contacts between the protein and an unpaired base within the three-way junction of helices 20, 21, and 22. Point mutations in the protein insertion subdomain resulted in a significantly reduced RNA binding affinity with respect to wild-type AS8. These results indicate that the AS8-specific subdomain provides additional interactions with the three-way junction that contribute to the extremely tight binding to ribosomal RNA. PMID:22079365

  10. Survey of bacterial diversity in chronic wounds using Pyrosequencing, DGGE, and full ribosome shotgun sequencing.

    Science.gov (United States)

    Background: Chronic wound pathogenic biofilms are host-pathogen environments that colonize and exist as a cohabitation of many bacterial species that cooperate to promote their own survival and the chronic nature of the infection. Few studies have performed extensive surveys of the different populat...

  11. Involvement of human ribosomal proteins in nucleolar structure and p53-dependent nucleolar stress.

    Science.gov (United States)

    Nicolas, Emilien; Parisot, Pascaline; Pinto-Monteiro, Celina; de Walque, Roxane; De Vleeschouwer, Christophe; Lafontaine, Denis L J

    2016-01-01

    The nucleolus is a potent disease biomarker and a target in cancer therapy. Ribosome biogenesis is initiated in the nucleolus where most ribosomal (r-) proteins assemble onto precursor rRNAs. Here we systematically investigate how depletion of each of the 80 human r-proteins affects nucleolar structure, pre-rRNA processing, mature rRNA accumulation and p53 steady-state level. We developed an image-processing programme for qualitative and quantitative discrimination of normal from altered nucleolar morphology. Remarkably, we find that uL5 (formerly RPL11) and uL18 (RPL5) are the strongest contributors to nucleolar integrity. Together with the 5S rRNA, they form the late-assembling central protuberance on mature 60S subunits, and act as an Hdm2 trap and p53 stabilizer. Other major contributors to p53 homeostasis are also strictly late-assembling large subunit r-proteins essential to nucleolar structure. The identification of the r-proteins that specifically contribute to maintaining nucleolar structure and p53 steady-state level provides insights into fundamental aspects of cell and cancer biology. PMID:27265389

  12. Partial genomic organization of ribosomal protein S7 gene from malaria vector Anopheles stephensi

    Institute of Scientific and Technical Information of China (English)

    RAJNIKANT DIXIT; SARITA DIXIT; UPAL ROY; YOGESH S.SHOUCHE; SURENDRA GAKHAR

    2007-01-01

    In this study, we describe the partial genomic organization of ribosomal protein S7 gene isolated from the mosquito Anopheles stephensi. Initially a 558 bp partial cDNA sequence was amplified as precursor mRNA sequence containing 223 bp long intron. 5' and 3' end sequences were recovered using end specific rapid amplification of cDNA ends (RACE) polymerase chain reaction. The full-length cDNA sequence was 914 nucleotide long with an open reading frame capable of encoding 192 amino acid long protein with calculated molecular mass of 22174 Da and a pI point of 9.94. Protein homology search revealed >75% identity to other insect's S7 ribosomal proteins. Analysis of sequence alignment revealed several highly conserved domains, one of which is related to nuclear localization signal (NLS) region of human rpS7. Interestingly, intron nucleotide sequence comparison with A. gambiae showed a lesser degree of conservation as compared to coding and untranslated regions. Like this, early studies on the genomic organization and cDNA/Expressed sequence tag analysis (EST) could help in genome annotation ofA. stephensi, and would be likely to be sequenced in the future.

  13. Involvement of human ribosomal proteins in nucleolar structure and p53-dependent nucleolar stress

    Science.gov (United States)

    Nicolas, Emilien; Parisot, Pascaline; Pinto-Monteiro, Celina; de Walque, Roxane; De Vleeschouwer, Christophe; Lafontaine, Denis L. J.

    2016-01-01

    The nucleolus is a potent disease biomarker and a target in cancer therapy. Ribosome biogenesis is initiated in the nucleolus where most ribosomal (r-) proteins assemble onto precursor rRNAs. Here we systematically investigate how depletion of each of the 80 human r-proteins affects nucleolar structure, pre-rRNA processing, mature rRNA accumulation and p53 steady-state level. We developed an image-processing programme for qualitative and quantitative discrimination of normal from altered nucleolar morphology. Remarkably, we find that uL5 (formerly RPL11) and uL18 (RPL5) are the strongest contributors to nucleolar integrity. Together with the 5S rRNA, they form the late-assembling central protuberance on mature 60S subunits, and act as an Hdm2 trap and p53 stabilizer. Other major contributors to p53 homeostasis are also strictly late-assembling large subunit r-proteins essential to nucleolar structure. The identification of the r-proteins that specifically contribute to maintaining nucleolar structure and p53 steady-state level provides insights into fundamental aspects of cell and cancer biology. PMID:27265389

  14. Bryodin, a ribosome-inactivating protein from the roots of Bryonia dioica L. (white bryony).

    Science.gov (United States)

    Stirpe, F; Barbieri, L; Battelli, M G; Falasca, A I; Abbondanza, A; Lorenzoni, E; Stevens, W A

    1986-12-15

    Bryodin is a strongly basic (pI greater than or equal to 9.5) glycoprotein (neutral sugar content 6.3%) with Mr 30,000, purified from the roots of Bryonia dioica (white bryony). This protein inhibits protein synthesis by a rabbit reticulocyte lysate with and ID50 (concentration causing 50% inhibition) of 0.12 nM (3.6 ng/ml) and has much less effect on protein synthesis by whole cells, with ID50 values ranging from 46 nM to 2.27 microM (1.4-67 micrograms/ml). Bryodin acts by inactivating ribosomes, with a less-than-equimolar ratio, which suggests a catalytic action. Bryodin decreases the number of local lesions induced by tobacco mosaic virus in the leaves of Nicotiana glutinosa. From all its properties, bryodin can be considered to be a ribosome-inactivating protein, similar to those already known [reviews: Barbieri & Stirpe (1982) Cancer Surveys 1, 489-520; Stirpe & Barbieri (1986) FEBS Lett. 195, 1-8]. PMID:3827858

  15. Survey of bacterial diversity in chronic wounds using Pyrosequencing, DGGE, and full ribosome shotgun sequencing

    Directory of Open Access Journals (Sweden)

    Wolcott Benjamin M

    2008-03-01

    Full Text Available Abstract Background Chronic wound pathogenic biofilms are host-pathogen environments that colonize and exist as a cohabitation of many bacterial species. These bacterial populations cooperate to promote their own survival and the chronic nature of the infection. Few studies have performed extensive surveys of the bacterial populations that occur within different types of chronic wound biofilms. The use of 3 separate16S-based molecular amplifications followed by pyrosequencing, shotgun Sanger sequencing, and denaturing gradient gel electrophoresis were utilized to survey the major populations of bacteria that occur in the pathogenic biofilms of three types of chronic wound types: diabetic foot ulcers (D, venous leg ulcers (V, and pressure ulcers (P. Results There are specific major populations of bacteria that were evident in the biofilms of all chronic wound types, including Staphylococcus, Pseudomonas, Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia, and Serratia spp. Each of the wound types reveals marked differences in bacterial populations, such as pressure ulcers in which 62% of the populations were identified as obligate anaerobes. There were also populations of bacteria that were identified but not recognized as wound pathogens, such as Abiotrophia para-adiacens and Rhodopseudomonas spp. Results of molecular analyses were also compared to those obtained using traditional culture-based diagnostics. Only in one wound type did culture methods correctly identify the primary bacterial population indicating the need for improved diagnostic methods. Conclusion If clinicians can gain a better understanding of the wound's microbiota, it will give them a greater understanding of the wound's ecology and will allow them to better manage healing of the wound improving the prognosis of patients. This research highlights the necessity to begin evaluating, studying, and treating chronic wound pathogenic biofilms as multi-species entities in

  16. Characterisation of ribosomal proteins from HeLa and Krebs II mouse ascites tumor cells by different two-dimensional polyacrylamide gel electrophoresis techniques

    DEFF Research Database (Denmark)

    Issinger, O G; Beier, H

    1978-01-01

    Electrophoresis of ribosomal proteins according to Kaltschmidt and Wittmann, 1970a, b (pH 8.6/pH 4.5 urea system) yielded 29 proteins for the small subunits and 35 and 37 proteins for the large subunits of Krebs II ascites and HeLa ribosomes, respectively. Analysis of the proteins according to a ...

  17. The trypanosome Pumilio-domain protein PUF7 associates with a nuclear cyclophilin and is involved in ribosomal RNA maturation

    OpenAIRE

    Droll, Dorothea; Archer, Stuart; Fenn, Katelyn; Delhi, Praveen; Matthews, Keith; Clayton, Christine

    2010-01-01

    Proteins with Pumilio RNA binding domains (Puf proteins) are ubiquitous in eukaryotes. Some Puf proteins bind to the 3′-untranslated regions of mRNAs, acting to repress translation and promote degradation; others are involved in ribosomal RNA maturation. The genome of Trypanosoma brucei encodes eleven Puf proteins whose function cannot be predicted by sequence analysis. We show here that epitope-tagged TbPUF7 is located in the nucleolus, and associated with a nuclear cyclophilin-like protein,...

  18. From DNA to proteins via the ribosome: structural insights into the workings of the translation machinery.

    Science.gov (United States)

    Agirrezabala, Xabier; Frank, Joachim

    2010-04-01

    Understanding protein synthesis in bacteria and humans is important for understanding the origin of many human diseases and devising treatments for them. Over the past decade, the field of structural biology has made significant advances in the visualisation of the molecular machinery involved in protein synthesis. It is now possible to discern, at least in outline, the way that interlocking ribosomal components and factors adapt their conformations throughout this process. The determination of structures in various functional contexts, along with the application of kinetic and fluorescent resonance energy transfer approaches to the problem, has given researchers the frame of reference for what remains as the greatest challenge: the complete dynamic portrait of protein synthesis in the cell. PMID:20511136

  19. From DNA to proteins via the ribosome: Structural insights into the workings of the translation machinery

    Directory of Open Access Journals (Sweden)

    Agirrezabala Xabier

    2010-04-01

    Full Text Available Abstract Understanding protein synthesis in bacteria and humans is important for understanding the origin of many human diseases and devising treatments for them. Over the past decade, the field of structural biology has made significant advances in the visualisation of the molecular machinery involved in protein synthesis. It is now possible to discern, at least in outline, the way that interlocking ribosomal components and factors adapt their conformations throughout this process. The determination of structures in various functional contexts, along with the application of kinetic and fluorescent resonance energy transfer approaches to the problem, has given researchers the frame of reference for what remains as the greatest challenge: the complete dynamic portrait of protein synthesis in the cell.

  20. Slow formation of stable complexes during coincubation of a minimal rRNA and ribosomal protein S4

    OpenAIRE

    Mayerle, Megan; Bellur, Deepti L.; Woodson, Sarah A.

    2011-01-01

    Ribosomal protein S4 binds and stabilizes a five-helix junction in the 5’ domain of the 16S rRNA, and is one of two proteins responsible for nucleating 30S ribosome assembly. Upon binding, both protein S4 and the five-helix junction reorganize their structures. We show that labile S4 complexes rearrange to stable complexes within a few minutes at 42°C, with longer coincubation leading to an increased population of stable complexes. In contrast, prefolding the rRNA has a smaller effect on stab...

  1. Designed Regular Tetragon-Shaped RNA-Protein Complexes with Ribosomal Protein L1 for Bionanotechnology and Synthetic Biology.

    Science.gov (United States)

    Ohno, Hirohisa; Inoue, Tan

    2015-05-26

    RNA nanotechnology has been established by employing the molecular architecture of RNA structural motifs. Here, we report two designed RNA-protein complexes (RNPs) composed of ribosomal protein L1 (RPL1) and its RNA-binding motif that are square-shaped nano-objects. The formation and the shape of the objects were confirmed by gel electrophoresis analysis and atomic force microscopy, respectively. Any protein can be attached to the RNA via a fusion protein with RPL1, indicating that it can be used as a scaffold for loading a variety of functional proteins or for building higher-order structures. In summary, the RNP object will serve as a useful tool in the fields of bionanotechnology and synthetic biology. Moreover, the RNP interaction enhances the RNA stability against nucleases, rendering these complexes stable in cells. PMID:25933202

  2. Cloning a cDNA Encoding Ribosomal Protein S25 from Amaranthus cruentus L.%籽粒苋(Amaranthus cruentus L.)核糖体蛋白S25基因(cDNA)的克隆及其表达分析

    Institute of Scientific and Technical Information of China (English)

    徐芳秀; 江树业; 等

    2001-01-01

    @@ Ribosomes, the agents of protein synthesis, consist of roughly equal amounts of RNA (rRNA) and protein (r-protein). Knowledge of the ribosome and its function mainly comes from the extensive work on 70S bacterial ribosomes. There are 21 proteins in the small (30S) subunit and 30 in the large (50S) subunit in E. coil ri bosomes. The 80S eukaryotic ribosomes are more com plex than the bacterial ones and contain at least 30 pro teins in the small (40S) subunit and 40 in the large (60 S) subunit. These r-proteins are named S1 to S30 and L1 to L40 according to whether they arise from the small or large subunit, and to their mobility in gels. In plants, several ribosomal protein genes and/or cDNAs have been isolated, such as the small subunit proteins S 11, S13, S14, S16, and S19 and the large subunit proteins L2, L7, L17, and L27. Here we report the r-protein S25 cDNA, Arps25, from Amaranthus cruentus L.

  3. Structural and functional characterization of ribosomal protein gene introns in sponges.

    Science.gov (United States)

    Perina, Drago; Korolija, Marina; Mikoč, Andreja; Roller, Maša; Pleše, Bruna; Imešek, Mirna; Morrow, Christine; Batel, Renato; Ćetković, Helena

    2012-01-01

    Ribosomal protein genes (RPGs) are a powerful tool for studying intron evolution. They exist in all three domains of life and are much conserved. Accumulating genomic data suggest that RPG introns in many organisms abound with non-protein-coding-RNAs (ncRNAs). These ancient ncRNAs are small nucleolar RNAs (snoRNAs) essential for ribosome assembly. They are also mobile genetic elements and therefore probably important in diversification and enrichment of transcriptomes through various mechanisms such as intron/exon gain/loss. snoRNAs in basal metazoans are poorly characterized. We examined 449 RPG introns, in total, from four demosponges: Amphimedon queenslandica, Suberites domuncula, Suberites ficus and Suberites pagurorum and showed that RPG introns from A. queenslandica share position conservancy and some structural similarity with "higher" metazoans. Moreover, our study indicates that mobile element insertions play an important role in the evolution of their size. In four sponges 51 snoRNAs were identified. The analysis showed discrepancies between the snoRNA pools of orthologous RPG introns between S. domuncula and A. queenslandica. Furthermore, these two sponges show as much conservancy of RPG intron positions between each other as between themselves and human. Sponges from the Suberites genus show consistency in RPG intron position conservation. However, significant differences in some of the orthologous RPG introns of closely related sponges were observed. This indicates that RPG introns are dynamic even on these shorter evolutionary time scales. PMID:22880015

  4. Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription.

    Science.gov (United States)

    Knight, Britta; Kubik, Slawomir; Ghosh, Bhaswar; Bruzzone, Maria Jessica; Geertz, Marcel; Martin, Victoria; Dénervaud, Nicolas; Jacquet, Philippe; Ozkan, Burak; Rougemont, Jacques; Maerkl, Sebastian J; Naef, Félix; Shore, David

    2014-08-01

    In yeast, ribosome production is controlled transcriptionally by tight coregulation of the 138 ribosomal protein genes (RPGs). RPG promoters display limited sequence homology, and the molecular basis for their coregulation remains largely unknown. Here we identify two prevalent RPG promoter types, both characterized by upstream binding of the general transcription factor (TF) Rap1 followed by the RPG-specific Fhl1/Ifh1 pair, with one type also binding the HMG-B protein Hmo1. We show that the regulatory properties of the two promoter types are remarkably similar, suggesting that they are determined to a large extent by Rap1 and the Fhl1/Ifh1 pair. Rapid depletion experiments allowed us to define a hierarchy of TF binding in which Rap1 acts as a pioneer factor required for binding of all other TFs. We also uncovered unexpected features underlying recruitment of Fhl1, whose forkhead DNA-binding domain is not required for binding at most promoters, and Hmo1, whose binding is supported by repeated motifs. Finally, we describe unusually micrococcal nuclease (MNase)-sensitive nucleosomes at all RPG promoters, located between the canonical +1 and -1 nucleosomes, which coincide with sites of Fhl1/Ifh1 and Hmo1 binding. We speculate that these "fragile" nucleosomes play an important role in regulating RPG transcriptional output. PMID:25085421

  5. Structural and functional characterization of ribosomal protein gene introns in sponges.

    Directory of Open Access Journals (Sweden)

    Drago Perina

    Full Text Available Ribosomal protein genes (RPGs are a powerful tool for studying intron evolution. They exist in all three domains of life and are much conserved. Accumulating genomic data suggest that RPG introns in many organisms abound with non-protein-coding-RNAs (ncRNAs. These ancient ncRNAs are small nucleolar RNAs (snoRNAs essential for ribosome assembly. They are also mobile genetic elements and therefore probably important in diversification and enrichment of transcriptomes through various mechanisms such as intron/exon gain/loss. snoRNAs in basal metazoans are poorly characterized. We examined 449 RPG introns, in total, from four demosponges: Amphimedon queenslandica, Suberites domuncula, Suberites ficus and Suberites pagurorum and showed that RPG introns from A. queenslandica share position conservancy and some structural similarity with "higher" metazoans. Moreover, our study indicates that mobile element insertions play an important role in the evolution of their size. In four sponges 51 snoRNAs were identified. The analysis showed discrepancies between the snoRNA pools of orthologous RPG introns between S. domuncula and A. queenslandica. Furthermore, these two sponges show as much conservancy of RPG intron positions between each other as between themselves and human. Sponges from the Suberites genus show consistency in RPG intron position conservation. However, significant differences in some of the orthologous RPG introns of closely related sponges were observed. This indicates that RPG introns are dynamic even on these shorter evolutionary time scales.

  6. Mutation in ribosomal protein S5 leads to spectinomycin resistance in Neisseria gonorrhoeae.

    Directory of Open Access Journals (Sweden)

    Elena eIlina

    2013-07-01

    Full Text Available Spectinomycin remains a useful reserve option for therapy of gonorrhea. The emergence of multidrug-resistant Neisseria gonorrhoeae strains with decreased susceptibility to cefixime and to ceftriaxone makes it the only medicine still effective for treatment of gonorrhea infection in analogous cases. However, adoption of spectinomycin as a routinely used drug of choice was soon followed by reports of spectinomycin resistance. The main molecular mechanism of spectinomycin resistance in N. gonorrhoeae was C1192T substitution in 16S rRNA genes. Here we reported a Thr-24→Pro mutation in ribosomal protein S5 found in spectinomycin resistant clinical N. gonorrhoeae strain, which carried no changes in 16S rRNA. In a series of experiments, the transfer of rpsE gene allele encoding the mutant ribosomal protein S5 to the recipient N. gonorrhoeae strains was analyzed. The relatively high rate of transformation (ca. 10-5 CFUs indicates the possibility of spread of spectinonycin resistance within gonococcal population due to the horizontal gene transfer.

  7. Estimation of Bacterial Cell Numbers in Humic Acid-Rich Salt Marsh Sediments with Probes Directed to 16S Ribosomal DNA

    OpenAIRE

    Edgcomb, Virginia P; Mcdonald, John H.; Devereux, Richard; Smith, David W.

    1999-01-01

    The feasibility of using probes directed towards ribosomal DNAs (rDNAs) as a quantitative approach to estimating cell numbers was examined and applied to study the structure of a bacterial community in humic acid-rich salt marsh sediments. Hybridizations were performed with membrane-bound nucleic acids by using seven group-specific DNA oligonucleotide probes complementary to 16S rRNA coding regions. These included a general eubacterial probe and probes encompassing most members of the gram-ne...

  8. Identification of sirtuin and its target as the ribosomal protein S4 in Lactobacillus paracasei.

    Science.gov (United States)

    Atarashi, Hotaka; Kawasaki, Shinji; Niimura, Yoichi; Tanaka, Naoto; Okada, Sanae; Shiwa, Yuh; Endo, Akihito; Nakagawa, Junichi

    2016-01-01

    Sirtuin is a protein with an enzymatic activity of NAD(+)-dependent protein deacetylation. It was first identified in yeast and its homologous genes have been widely found in various organisms. In bacteria, sirtuin gene was first described as cobB, encoding a cobalamin processing enzyme; and later its potential involvement in regulating acetylation levels of metabolic enzymes, transcription factors, chemotactic proteins and others have been reported. In order to study its physiological relevance in probiotic lactic acid bacteria, we analyzed the whole genome of three L. paracasei strains. All strains tested had sirtuin homolog genes designated hereby as sirA, and one of them had an additional gene designated as sirB. Following confirmation of their coding sequences by individual gene cloning, corresponding recombinant proteins have been generated and purified. The enzymatic characterization revealed that the intrinsic NAD(+)-dependent deacetylation activity of LpSirA (protein encoded by sirA) is comparable to human SIRT1. Furthermore, by blocking sirtuin activity using nicotinamide in vivo, together with an in vitro deacetylation reaction using recombinant LpSirA, we identified one of the target proteins in the lactic acid bacteria as the 30S ribosomal protein S4 (rpsD product). PMID:27118078

  9. Mitochondrial ribosomal protein S18-2 evokes chromosomal instability and transforms primary rat skin fibroblasts

    KAUST Repository

    Kashuba, Elena

    2015-05-12

    We have shown earlier that overexpression of the human mitochondrial ribosomal protein MRPS18-2 (S18-2) led to immortalization of primary rat embryonic fibroblasts. The derived cells expressed the embryonic stem cell markers, and cellular pathways that control cell proliferation, oxidative phosphorylation, cellular respiration, and other redox reactions were activated in the immortalized cells. Here we report that, upon overexpression of S18-2 protein, primary rat skin fibroblasts underwent cell transformation. Cells passed more than 300 population doublings, and two out of three tested clones gave rise to tumors in experimental animals. Transformed cells showed anchorage-independent growth and loss of contact inhibition; they expressed epithelial markers, such as E-cadherin and β-catenin. Transformed cells showed increased telomerase activity, disturbance of the cell cycle, and chromosomal instability. Taken together, our data suggest that S18-2 is a newly identified oncoprotein that may be involved in cancerogenesis.

  10. Cleavage of nicotinamide adenine dinucleotide by the ribosome-inactivating protein from Momordica charantia.

    Science.gov (United States)

    Vinkovic, M; Dunn, G; Wood, G E; Husain, J; Wood, S P; Gill, R

    2015-09-01

    The interaction of momordin, a type 1 ribosome-inactivating protein from Momordica charantia, with NADP(+) and NADPH has been investigated by X-ray diffraction analysis of complexes generated by co-crystallization and crystal soaking. It is known that the proteins of this family readily cleave the adenine-ribose bond of adenosine and related nucleotides in the crystal, leaving the product, adenine, bound to the enzyme active site. Surprisingly, the nicotinamide-ribose bond of oxidized NADP(+) is cleaved, leaving nicotinamide bound in the active site in the same position but in a slightly different orientation to that of the five-membered ring of adenine. No binding or cleavage of NADPH was observed at pH 7.4 in these experiments. These observations are in accord with current views of the enzyme mechanism and may contribute to ongoing searches for effective inhibitors. PMID:26323301

  11. Ribosomal Stalk Protein Silencing Partially Corrects the ΔF508-CFTR Functional Expression Defect.

    Science.gov (United States)

    Veit, Guido; Oliver, Kathryn; Apaja, Pirjo M; Perdomo, Doranda; Bidaud-Meynard, Aurélien; Lin, Sheng-Ting; Guo, Jingyu; Icyuz, Mert; Sorscher, Eric J; Hartman Iv, John L; Lukacs, Gergely L

    2016-05-01

    The most common cystic fibrosis (CF) causing mutation, deletion of phenylalanine 508 (ΔF508 or Phe508del), results in functional expression defect of the CF transmembrane conductance regulator (CFTR) at the apical plasma membrane (PM) of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulum (ER). Deletion of phenylalanine 670 (ΔF670) in the yeast oligomycin resistance 1 gene (YOR1, an ABC transporter) of Saccharomyces cerevisiae phenocopies the ΔF508-CFTR folding and trafficking defects. Genome-wide phenotypic (phenomic) analysis of the Yor1-ΔF670 biogenesis identified several modifier genes of mRNA processing and translation, which conferred oligomycin resistance to yeast. Silencing of orthologues of these candidate genes enhanced the ΔF508-CFTR functional expression at the apical PM in human CF bronchial epithelia. Although knockdown of RPL12, a component of the ribosomal stalk, attenuated the translational elongation rate, it increased the folding efficiency as well as the conformational stability of the ΔF508-CFTR, manifesting in 3-fold augmented PM density and function of the mutant. Combination of RPL12 knockdown with the corrector drug, VX-809 (lumacaftor) restored the mutant function to ~50% of the wild-type channel in primary CFTRΔF508/ΔF508 human bronchial epithelia. These results and the observation that silencing of other ribosomal stalk proteins partially rescue the loss-of-function phenotype of ΔF508-CFTR suggest that the ribosomal stalk modulates the folding efficiency of the mutant and is a potential therapeutic target for correction of the ΔF508-CFTR folding defect. PMID:27168400

  12. Ribosomal Stalk Protein Silencing Partially Corrects the ΔF508-CFTR Functional Expression Defect

    Science.gov (United States)

    Veit, Guido; Oliver, Kathryn; Apaja, Pirjo M.; Perdomo, Doranda; Bidaud-Meynard, Aurélien; Guo, Jingyu; Icyuz, Mert; Sorscher, Eric J.; Hartman IV, John L.; Lukacs, Gergely L.

    2016-01-01

    The most common cystic fibrosis (CF) causing mutation, deletion of phenylalanine 508 (ΔF508 or Phe508del), results in functional expression defect of the CF transmembrane conductance regulator (CFTR) at the apical plasma membrane (PM) of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulum (ER). Deletion of phenylalanine 670 (ΔF670) in the yeast oligomycin resistance 1 gene (YOR1, an ABC transporter) of Saccharomyces cerevisiae phenocopies the ΔF508-CFTR folding and trafficking defects. Genome-wide phenotypic (phenomic) analysis of the Yor1-ΔF670 biogenesis identified several modifier genes of mRNA processing and translation, which conferred oligomycin resistance to yeast. Silencing of orthologues of these candidate genes enhanced the ΔF508-CFTR functional expression at the apical PM in human CF bronchial epithelia. Although knockdown of RPL12, a component of the ribosomal stalk, attenuated the translational elongation rate, it increased the folding efficiency as well as the conformational stability of the ΔF508-CFTR, manifesting in 3-fold augmented PM density and function of the mutant. Combination of RPL12 knockdown with the corrector drug, VX-809 (lumacaftor) restored the mutant function to ~50% of the wild-type channel in primary CFTRΔF508/ΔF508 human bronchial epithelia. These results and the observation that silencing of other ribosomal stalk proteins partially rescue the loss-of-function phenotype of ΔF508-CFTR suggest that the ribosomal stalk modulates the folding efficiency of the mutant and is a potential therapeutic target for correction of the ΔF508-CFTR folding defect. PMID:27168400

  13. Ribosomal Stalk Protein Silencing Partially Corrects the ΔF508-CFTR Functional Expression Defect.

    Directory of Open Access Journals (Sweden)

    Guido Veit

    2016-05-01

    Full Text Available The most common cystic fibrosis (CF causing mutation, deletion of phenylalanine 508 (ΔF508 or Phe508del, results in functional expression defect of the CF transmembrane conductance regulator (CFTR at the apical plasma membrane (PM of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulum (ER. Deletion of phenylalanine 670 (ΔF670 in the yeast oligomycin resistance 1 gene (YOR1, an ABC transporter of Saccharomyces cerevisiae phenocopies the ΔF508-CFTR folding and trafficking defects. Genome-wide phenotypic (phenomic analysis of the Yor1-ΔF670 biogenesis identified several modifier genes of mRNA processing and translation, which conferred oligomycin resistance to yeast. Silencing of orthologues of these candidate genes enhanced the ΔF508-CFTR functional expression at the apical PM in human CF bronchial epithelia. Although knockdown of RPL12, a component of the ribosomal stalk, attenuated the translational elongation rate, it increased the folding efficiency as well as the conformational stability of the ΔF508-CFTR, manifesting in 3-fold augmented PM density and function of the mutant. Combination of RPL12 knockdown with the corrector drug, VX-809 (lumacaftor restored the mutant function to ~50% of the wild-type channel in primary CFTRΔF508/ΔF508 human bronchial epithelia. These results and the observation that silencing of other ribosomal stalk proteins partially rescue the loss-of-function phenotype of ΔF508-CFTR suggest that the ribosomal stalk modulates the folding efficiency of the mutant and is a potential therapeutic target for correction of the ΔF508-CFTR folding defect.

  14. Differential phosphorylation of ribosomal acidic proteins from yeast cell by two endogenous protein kinases: casein kinase-2 and 60S kinase

    International Nuclear Information System (INIS)

    The native 80S ribosomes isolated from ''Saccharomyces cerevisiae'' (strain W303) cells was phosphorylated by two endogenous protein kinases: multifunctional casein kinase-2 (CK-2) and specific 60S kinase. Three acidic proteins within the 60S ribosomal subunit: YP1β, YP1β' and YP2α are phosphorylated by both kinases. The other two proteins: YP1α and YP2β are predominantly phosphorylated by CK-2 but not by 60S kinase. This was confirmed in the experiment with the recombinant protein, YP2β, as a substrate, which is practically not phosphorylated by specific 60S kinase. These results together with the previous data based on the target amino-acid sequences suggest that, in addition to the multifunctional casein kinase-2 and specific 60S kinase, there exist probably other protein kinase(s) which phosphorylate the ribosomal acidic proteins in the cell. (author). 23 refs, 3 figs, 1 tab

  15. Reduced expression of ribosomal proteins relieves microRNA-mediated repression.

    Science.gov (United States)

    Janas, Maja M; Wang, Eric; Love, Tara; Harris, Abigail S; Stevenson, Kristen; Semmelmann, Karlheinz; Shaffer, Jonathan M; Chen, Po-Hao; Doench, John G; Yerramilli, Subrahmanyam V B K; Neuberg, Donna S; Iliopoulos, Dimitrios; Housman, David E; Burge, Christopher B; Novina, Carl D

    2012-04-27

    MicroRNAs (miRNAs) regulate physiological and pathological processes by inducing posttranscriptional repression of target messenger RNAs (mRNAs) via incompletely understood mechanisms. To discover factors required for human miRNA activity, we performed an RNAi screen using a reporter cell line of miRNA-mediated repression of translation initiation. We report that reduced expression of ribosomal protein genes (RPGs) dissociated miRNA complexes from target mRNAs, leading to increased polysome association, translation, and stability of miRNA-targeted mRNAs relative to untargeted mRNAs. RNA sequencing of polysomes indicated substantial overlap in sets of genes exhibiting increased or decreased polysomal association after Argonaute or RPG knockdowns, suggesting similarity in affected pathways. miRNA profiling of monosomes and polysomes demonstrated that miRNAs cosediment with ribosomes. RPG knockdowns decreased miRNAs in monosomes and increased their target mRNAs in polysomes. Our data show that most miRNAs repress translation and that the levels of RPGs modulate miRNA-mediated repression of translation initiation. PMID:22541556

  16. Human ribosomal protein L9 is a Bax suppressor that promotes cell survival in yeast.

    Science.gov (United States)

    Eid, Rawan; Sheibani, Sara; Gharib, Nada; Lapointe, Jason F; Horowitz, Avital; Vali, Hojatollah; Mandato, Craig A; Greenwood, Michael T

    2014-05-01

    The identification of a human ribosomal protein L9 (hRPL9) cDNA as a sequence capable of suppressing the lethal effects of heterologously expressed murine Bax in yeast led us to investigate its antiapoptotic potential. Using growth and viability assays, we show that yeast cells heterologously expressing hRPL9 are resistant to the growth inhibitory and lethal effects of exogenously supplied copper, indicating that it has pro-survival properties. To explore potential mechanisms, we used yeast mutants defective in all three types of programmed cell death (apoptosis, necrosis, and autophagy). The ability to retain pro-survival function in all the mutants suggests that hRPL9 may regulate a common pro-death process. In contrast, the yeast RPL9 orthologues, RPL9A and RPL9B, have opposite effects when overexpressed in yeast. In effect, instead of showing resistance to stress, RPL9A and RPL9B overexpressing cells show reduced cell growth. Further analysis indicates that the effects of overexpressed RPL9A and RPL9B are not in themselves lethal, instead, they serve to increase cell doubling time. Thus, yeast RPL9s are more representative of RPs whose extra-ribosomal function is similar to that of tumor suppressors. Taken together, our results demonstrate that RPL9 represents a species- and sequence-specific regulator of cell growth and survival. PMID:24305165

  17. Translation Inhibition of Capped and Uncapped Viral RNAs Mediated by Ribosome-Inactivating Proteins.

    Science.gov (United States)

    Vivanco, Jorge M; Tumer, Nilgun E

    2003-05-01

    ABSTRACT Ribosome-inactivating proteins (RIPs) are N-glycosidases that remove specific purine residues from the sarcin/ricin (S/R) loop of the large rRNA and arrest protein synthesis at the translocation step. In addition to their enzymatic activity, RIPs have been reputed to be potent antiviral agents against many plant, animal, and human viruses. We recently showed that pokeweed antiviral protein (PAP), an RIP from pokeweed, inhibits translation in cell extracts by binding to the cap structure of eukaryotic mRNA and viral RNAs and depurinating these RNAs at multiple sites downstream of the cap structure. In this study, we examined the activity of three different RIPs against capped and uncapped viral RNAs. PAP, Mirabilis expansa RIP (ME1), and the Saponaria officinalis RIP (saporin) depurinated the capped Tobacco mosaic virus and Brome mosaic virus RNAs, but did not depurinate the uncapped luciferase RNA, indicating that other type I RIPs besides PAP can distinguish between capped and uncapped RNAs. We did not detect depurination of Alfalfa mosaic virus (AMV) RNAs at multiple sites by PAP or ME1. Because AMV RNAs are capped, these results indicate that recognition of the cap structure alone is not sufficient for depurination of the RNA at multiple sites throughout its sequence. Furthermore, PAP did not cause detectable depurination of uncapped RNAs from Tomato bushy stunt virus (TBSV), Satellite panicum mosaic virus (SPMV), and uncapped RNA containing poliovirus internal ribosome entry site (IRES). However, in vitro translation experiments showed that PAP inhibited translation of AMV, TBSV, SPMV RNAs, and poliovirus IRES dependent translation. These results demonstrate that PAP does not depurinate every capped RNA and that PAP can inhibit translation of uncapped viral RNAs in vitro without causing detectable depurination at multiple sites. Thus, the cap structure is not the only determinant for inhibition of translation by PAP. PMID:18942981

  18. Engineering the rRNA decoding site of eukaryotic cytosolic ribosomes in bacteria

    OpenAIRE

    Hobbie, S N; Kalapala, S K; Akshay, S.; Bruell, C M; S. Schmidt; Dabow, S; Vasella, A; Sander, P; Böttger, E C

    2007-01-01

    Structural and genetic studies on prokaryotic ribosomes have provided important insights into fundamental aspects of protein synthesis and translational control and its interaction with ribosomal drugs. Comparable mechanistic studies in eukaryotes are mainly hampered by the absence of both high-resolution crystal structures and efficient genetic models. To study the interaction of aminoglycoside antibiotics with selected eukaryotic ribosomes, we replaced the bacterial drug binding site in 16S...

  19. Elderberries: A Source of Ribosome-Inactivating Proteins with Lectin Activity

    Directory of Open Access Journals (Sweden)

    Jesús Tejero

    2015-01-01

    Full Text Available Sambucus (Adoxaceae species have been used for both food and medicine purposes. Among these, Sambucus nigra L. (black elder, Sambucus ebulus L. (dwarf elder, and Sambucus sieboldiana L. are the most relevant species studied. Their use has been somewhat restricted due to the presence of bioactive proteins or/and low molecular weight compounds whose ingestion could trigger deleterious effects. Over the last few years, the chemical and pharmacological characteristics of Sambucus species have been investigated. Among the proteins present in Sambucus species both type 1, and type 2 ribosome-inactivating proteins (RIPs, and hololectins have been reported. The biological role played by these proteins remains unknown, although they are conjectured to be involved in defending plants against insect predators and viruses. These proteins might have an important impact on the nutritional characteristics and food safety of elderberries. Type 2 RIPs are able to interact with gut cells of insects and mammals triggering a number of specific and mostly unknown cell signals in the gut mucosa that could significantly affect animal physiology. In this paper, we describe all known RIPs that have been isolated to date from Sambucus species, and comment on their antiviral and entomotoxic effects, as well as their potential uses.

  20. Expression of a ribosome inactivating protein (curcin 2) in Jatropha curcas is induced by stress

    Indian Academy of Sciences (India)

    Wei Qin; Huang Ming-Xing; Xu Ying; Zhang Xin-Shen; Chen Fang

    2005-06-01

    The open reading frame (ORF) encoding curcin 2 was cloned from total genomic and cDNA of Jatropha curcas leaves, which were treated by drought, temperature stress and fungal infection, by polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR amplification. The ORF has 927 bp that encodes a precursor protein of 309 amino acid residues. There are high similarities with curcin and the conserved domain of ribosome inactivating proteins (RIPs). Antiserum to curcin recognized one band of 32 kDa on Western blot of the leaves treated by temperature stresses at 4°C and 50°C and by fungal infections of Pestalotia funerea, Curvularia lunata (Walk) Boed, Gibberelle zeae (Schw.) Petch. Two bands of 32 kDa and 65 kDa were recognized on Western blot of the leaves treated by 10%–40% polyethylene glycol (PEG). In addition, the 32 kDa band is nearly the molecular weight of curcin 2. This finding suggests that the protein of 32 kDa should be related to curcin 2. The presence of this protein molecular marker under stresses may provide an experimental foundation to study the stress proteins in J. curcas.

  1. Late-assembly of human ribosomal protein S20 in the cytoplasm is essential for the functioning of the small subunit ribosome

    International Nuclear Information System (INIS)

    Using immuno-fluorescent probing and Western blotting analysis, we reveal the exclusive cytoplasm nature of the small subunit ribosomal protein S20. To illustrate the importance of the cellular compartmentation of S20 to the function of small subunit 40S, we created a nuclear resident S20NLS mutant gene and examined polysome profile of cells that had been transfected with the S20NLS gene. As a result, we observed the formation of recombinant 40S carried S20NLS but this recombinant 40S was never found in the polysome, suggesting such a recombinant 40S was translation incompetent. Moreover, by the tactic of the energy depletion and restoration, we were able to restrain the nuclear-resided S20NLS in the cytoplasm. Yet, along a progressive energy restoration, we observed the presence of recombinant 40S subunits carrying the S20NLS in the polysome. This proves that S20 needs to be cytoplasmic in order to make a functional 40S subunit. Furthermore, it also implies that the assembly order of ribosomal protein in eukaryote is orderly regulated. - Highlights: • The step of S20 assembled on 40S is happened in the cytoplasm. • A small subunit assembled with a nuclear S20NLS is translational incompetence. • Using energy depletion and recovery to manipulate the cellular compartment of S20NLS. • Cytoplasm-retained S20NLS is crucial for creating a functional small subunit

  2. La Autoantigen Induces Ribosome Binding Protein 1 (RRBP1) Expression through Internal Ribosome Entry Site (IRES)-Mediated Translation during Cellular Stress Condition.

    Science.gov (United States)

    Gao, Wenqing; Li, Qi; Zhu, Ruiyu; Jin, Jian

    2016-01-01

    The function of ribosome binding protein 1 (RRBP1) is regulating the transportation and secretion of some intracellular proteins in mammalian cells. Transcription of RRBP1 is induced by various cytokines. However, few studies focused on the process of RRPB1 mRNA translation. The RRBP1 mRNA has a long 5' untranslated region that potentially formed a stable secondary structure. In this study, we show that the 5' UTR of RRBP1 mRNA contains an internal ribosome entry site (IRES). Moreover, the RRBP1 expression is induced by chemotherapeutic drug paclitaxel or adriamycin in human hepatocellular carcinoma cells and accompanied with the increased expression of La autoantigen (La), which binds to RRBP1 IRES element and facilitates translation initiation. Interestingly, we found IRES-mediated RRBP1 translation is also activated during serum-starvation condition which can induce cytoplasmic localization of La. After mapping the entire RRBP1 5' UTR, we determine the core IRES activity is located between nt-237 and -58. Furthermore, two apical GARR loops within the functional RRBP1 IRES elements may be important for La binding. These results strongly suggest an important role for IRES-dependent translation of RRBP1 mRNA in hepatocellular carcinoma cells during cellular stress conditions. PMID:27447629

  3. A Ribosomal Protein AgRPS3aE from Halophilic Aspergillus glaucus Confers Salt Tolerance in Heterologous Organisms

    Directory of Open Access Journals (Sweden)

    Xilong Liang

    2015-01-01

    Full Text Available High salt in soils is one of the abiotic stresses that significantly reduces crop yield, although saline lands are considered potential resources arable for agriculture. Currently, genetic engineering for enhancing salt tolerance is being tested as an efficient and viable strategy for crop improvement. We previously characterized a large subunit of the ribosomal protein RPL44, which is involved in osmotic stress in the extremely halophilic fungus Aspergillus glaucus. Here, we screened another ribosomal protein (AgRPS3aE that also produced high-salt tolerance in yeast. Bioinformatics analysis indicated that AgRPS3aE encodes a 29.2 kDa small subunit of a ribosomal protein belonging to the RPS3Ae family in eukaryotes. To further confirm its protective function against salinity, we expressed AgRPS3aE in three heterologous systems, the filamentous fungus Magnaporthe oryzae and two model plants Arabidopsis and tobacco. Overexpression of AgRPS3aE in all tested transformants significantly alleviated stress symptoms compared with controls, suggesting that AgRPS3aE functions not only in fungi but also in plants. Considering that ribosomal proteins are housekeeping components in organisms from prokaryotes to eukaryotes, we propose that AgRPS3aE is one of the optimal genes for improving high-salt tolerance in crops.

  4. Ribosomes: Ribozymes that Survived Evolution Pressures but Is Paralyzed by Tiny Antibiotics

    Science.gov (United States)

    Yonath, Ada

    An impressive number of crystal structures of ribosomes, the universal cellular machines that translate the genetic code into proteins, emerged during the last decade. The determination of ribosome high resolution structure, which was widely considered formidable, led to novel insights into the ribosomal function, namely, fidelity, catalytic mechanism, and polymerize activities. They also led to suggestions concerning its origin and shed light on the action, selectivity and synergism of ribosomal antibiotics; illuminated mechanisms acquiring bacterial resistance and provided structural information for drug improvement and design. These studies required the pioneering and implementation of advanced technologies, which directly influenced the remarkable increase of the number of structures deposited in the Protein Data Bank.

  5. Comparative proteomic analysis of the silkworm middle silk gland reveals the importance of ribosome biogenesis in silk protein production.

    Science.gov (United States)

    Li, Jian-ying; Ye, Lu-peng; Che, Jia-qian; Song, Jia; You, Zheng-ying; Yun, Ki-chan; Wang, Shao-hua; Zhong, Bo-xiong

    2015-08-01

    The silkworm middle silk gland (MSG) is the sericin synthesis and secretion unique sub-organ. The molecular mechanisms of regulating MSG protein synthesis are largely unknown. Here, we performed shotgun proteomic analysis on the three MSG subsections: the anterior (MSG-A), middle (MSG-M), and posterior (MSG-P) regions. The results showed that more strongly expressed proteins in the MSG-A were involved in multiple processes, such as silk gland development and silk protein protection. The proteins that were highly expressed in the MSG-M were enriched in the ribosome pathway. MSG-P proteins with stronger expression were mainly involved in the oxidative phosphorylation and citrate cycle pathways. These results suggest that the MSG-M is the most active region in the sericin synthesis. Furthermore, comparing the proteome of the MSG with the posterior silk gland (PSG) revealed that the specific and highly expressed proteins in the MSG were primarily involved in the ribosome and aminoacyl-tRNA biosynthesis pathways. These results indicate that silk protein synthesis is much more active as a result of the enhancement of translation-related pathways in the MSG. These results also suggest that enhancing ribosome biogenesis is important to the efficient synthesis of silk proteins. PMID:26051239

  6. Similarity between the association factor of ribosomal subunits and the protein Stm1p from Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Heriberto Correia

    2004-11-01

    Full Text Available A ribosome association factor (AF was isolated from the yeast Sacchharomyces cerevisiae. Partial amino acid sequence of AF was determined from its fragment of 25 kDa isolated by treating AF with 2-(2-nitrophenylsulfenyl-3-methyl-3'-Bromoindolenine (BNPS-skatole. This sequence has a 86% identity to the product of the single-copy S. cerevisiae STM1 gene that is apparently involved in several events like binding to quadruplex and triplex nucleic acids and participating in apoptosis, stability of telomere structures, cell cycle, and ribosomal function. Here we show that AF and Stm1p share some characteristics: both bind to quadruplex and Pu triplex DNA, associates ribosomal subunits, and are thermostable. These observations suggest that these polypeptides belong to a family of proteins that may have roles in the translation process.

  7. Endogenous ribosomal protein L29 (RPL29: a newly identified regulator of angiogenesis in mice

    Directory of Open Access Journals (Sweden)

    Dylan T. Jones

    2013-01-01

    Cellular ribosomal protein L29 (RPL29 is known to be important in protein synthesis, but its function during angiogenesis has never been described before. We have shown previously that mice lacking β3-integrins support enhanced tumour angiogenesis and, therefore, deletion of endothelial αvβ3 can provide a method for discovery of novel regulators of tumour angiogenesis. Here, we describe significant upregulation of RPL29 in β3-null endothelial cells at both the mRNA and protein level. Ex vivo, we show that VEGF-stimulated microvessel sprouting was reduced significantly in Rpl29-heterozygous and Rpl29-null aortic ring assays compared with wild-type controls. Moreover, we provide in vivo evidence that RPL29 can regulate tumour angiogenesis. Tumour blood vessel density in subcutaneously grown Lewis lung carcinomas was reduced significantly in Rpl29-mutant mice. Additionally, depletion of Rpl29 using RNA interference inhibited VEGF-induced aortic ring sprouting, suggesting that anti-RPL29 strategies might have anti-angiogenic potential. Overall, our results identify that loss or depletion of RPL29 can reduce angiogenesis in vivo and ex vivo.

  8. γ-irradiated ribosomes from Micrococcus radiodurans in a cell-free protein synthesizing system

    International Nuclear Information System (INIS)

    γ-irradiation inactivation of isolated ribosomes of Micrococcus radiodurans was studied by examining poly U directed synthesis of polyphenylalanine. Ribosomes of M. radiodurans did not show significant γ-radiation sensitivity up to a dose of approx. 11.6 k Gy. Cells of M. radiodurans take up more magnesium than E. coli cells under the same conditions. The magnesium content of ribosomes of M. radiodurans was 18% higher than that of E.coli ribosomes. A possible relation between Mg2+-content and γ-resistance is discussed. (orig.)

  9. Bacterial Protein N-Glycosylation: New Perspectives and Applications*

    OpenAIRE

    Nothaft, Harald; Szymanski, Christine M.

    2013-01-01

    Protein glycosylation is widespread throughout all three domains of life. Bacterial protein N-glycosylation and its application to engineering recombinant glycoproteins continue to be actively studied. Here, we focus on advances made in the last 2 years, including the characterization of novel bacterial N-glycosylation pathways, examination of pathway enzymes and evolution, biological roles of protein modification in the native host, and exploitation of the N-glycosylation pathways to create ...

  10. Selection of IgE-binding aptameric green fluorescent protein (Ap-GFP) by the ribosome display (RD) platform

    International Nuclear Information System (INIS)

    GFP-Cκ fusion protein was previously shown selectable on ribosome display platform with solid phase antibodies against GFP determinant [Y.-M. Yang, T.J. Barankiewicz, M. He, M. Taussig, S.-S. Chen, Selection of antigenic markers on a GFP-Cκ fusion scaffold with high sensitivity by eukaryotic ribosome display, Biochem. Biophys. Res. Commun. 359 (2007) 251-257]. Herein, we show that members of aptameric peptide library constructed within the site 6 and site 8/9 loops of GFP of the ribosome display construct are selectable upon binding to the solid phase IgE antigen. An input of 1.0 μg of the dual site aptameric GFP library exhibiting a diversity of 7.5 x 1011 was transcribed, translated and incubated with solid phase IgE. RT-PCR products were amplified from mRNA of the aptamer-ribosome-mRNA (ARM) complex captured on the solid phase IgE. Clones of aptameric GFP were prepared from RT-PCR product of ARM complex following repetitive selection. Recombinant aptameric GFP proteins from the selected clones bind IgE coated on the 96-well plate, and the binding was abrogated by incubation with soluble human IgE but not human IgG. Selected aptameric GFP proteins also exhibit binding to three different sources of human IgE (IgE PS, BED, and JW8) but not irrelevant proteins. These observations indicate that appropriately selected aptameric GFP on a solid phase ligand by ribosome display may serve as an affinity reagent for blocking reactivity of a biological ligand

  11. The Cytotoxicity of Elderberry Ribosome-Inactivating Proteins Is Not Solely Determined by Their Protein Translation Inhibition Activity.

    Directory of Open Access Journals (Sweden)

    Chenjing Shang

    Full Text Available Although the protein translation inhibition activity of ribosome inactivating proteins (RIPs is well documented, little is known about the contribution of the lectin chain to the biological activity of these proteins. In this study, we compared the in vitro and intracellular activity of several S. nigra (elderberry RIPs and non-RIP lectins. Our data demonstrate that RIPs from elderberry are much more toxic to HeLa cells than to primary fibroblasts. Differences in the cytotoxicity between the elderberry proteins correlated with differences in glycan specificity of their lectin domain, cellular uptake efficiency and intracellular destination. Despite the fact that the bulk of the RIPs accumulated in the lysosomes and partly in the Golgi apparatus, we could demonstrate effective inhibition of protein synthesis in cellula. As we also observed cytotoxicity for non-RIP lectins, it is clear that the lectin chain triggers additional pathways heralding cell death. Our data suggest that one of these pathways involves the induction of autophagy.

  12. Structure of the JmjC domain-containing protein NO66 complexed with ribosomal protein Rpl8

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Chengliang [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Chinese Academy of Sciences, Hefei, Anhui 230026, People’s Republic of (China); Zhang, Qiongdi [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Hang, Tianrong [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Chinese Academy of Sciences, Hefei, Anhui 230026, People’s Republic of (China); Tao, Yue [Shanghai Children’s Medical Center, 1678 Dongfang Road, Pudong, Shanghai 200120, People’s Republic of (China); Ma, Xukai [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Wu, Minhao; Zhang, Xuan, E-mail: xuanzbin@ustc.edu.cn; Zang, Jianye, E-mail: xuanzbin@ustc.edu.cn [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Chinese Academy of Sciences, Hefei, Anhui 230026, People’s Republic of (China)

    2015-08-28

    The structure of the complex of NO66 and Rpl8 was solved in the native state and NO66 recognizes the consensus motif NHXH . Tetramerization is required for efficient substrate binding and catalysis by NO66. The JmjC domain-containing proteins belong to a large family of oxygenases possessing distinct substrate specificities which are involved in the regulation of different biological processes, such as gene transcription, RNA processing and translation. Nucleolar protein 66 (NO66) is a JmjC domain-containing protein which has been reported to be a histone demethylase and a ribosome protein 8 (Rpl8) hydroxylase. The present biochemical study confirmed the hydroxylase activity of NO66 and showed that oligomerization is required for NO66 to efficiently catalyze the hydroxylation of Rpl8. The structures of NO66{sup 176–C} complexed with Rpl8{sup 204–224} in a tetrameric form and of the mutant protein M2 in a dimeric form were solved. Based on the results of structural and biochemical analyses, the consensus sequence motif NHXH recognized by NO66 was confirmed. Several potential substrates of NO66 were found by a BLAST search according to the consensus sequence motif. When binding to substrate, the relative positions of each subunit in the NO66 tetramer shift. Oligomerization may facilitate the motion of each subunit in the NO66 tetramer and affect the catalytic activity.

  13. Structure of the JmjC domain-containing protein NO66 complexed with ribosomal protein Rpl8

    International Nuclear Information System (INIS)

    The structure of the complex of NO66 and Rpl8 was solved in the native state and NO66 recognizes the consensus motif NHXH . Tetramerization is required for efficient substrate binding and catalysis by NO66. The JmjC domain-containing proteins belong to a large family of oxygenases possessing distinct substrate specificities which are involved in the regulation of different biological processes, such as gene transcription, RNA processing and translation. Nucleolar protein 66 (NO66) is a JmjC domain-containing protein which has been reported to be a histone demethylase and a ribosome protein 8 (Rpl8) hydroxylase. The present biochemical study confirmed the hydroxylase activity of NO66 and showed that oligomerization is required for NO66 to efficiently catalyze the hydroxylation of Rpl8. The structures of NO66176–C complexed with Rpl8204–224 in a tetrameric form and of the mutant protein M2 in a dimeric form were solved. Based on the results of structural and biochemical analyses, the consensus sequence motif NHXH recognized by NO66 was confirmed. Several potential substrates of NO66 were found by a BLAST search according to the consensus sequence motif. When binding to substrate, the relative positions of each subunit in the NO66 tetramer shift. Oligomerization may facilitate the motion of each subunit in the NO66 tetramer and affect the catalytic activity

  14. Molecular ecology of tetracycline resistance: development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins.

    Science.gov (United States)

    Aminov, R I; Garrigues-Jeanjean, N; Mackie, R I

    2001-01-01

    Phylogenetic analysis of tetracycline resistance genes encoding the ribosomal protection proteins (RPPs) revealed the monophyletic origin of these genes. The most deeply branching class, exemplified by tet and otrA, consisted of genes from the antibiotic-producing organisms Streptomyces rimosus and Streptomyces lividans. With a high degree of confidence, the corresponding genes of the other seven classes (Tet M, Tet S, Tet O, Tet W, Tet Q, Tet T, and TetB P) formed phylogenetically distinct separate clusters. Based on this phylogenetic analysis, a set of PCR primers for detection, retrieval, and sequence analysis of the corresponding gene fragments from a variety of bacterial and environmental sources was developed and characterized. A pair of degenerate primers targeted all tetracycline resistance genes encoding RPPs except otrA and tet, and seven other primer pairs were designed to target the specific classes. The primers were used to detect the circulation of these genes in the rumina of cows, in swine feed and feces, and in swine fecal streptococci. Classes Tet O and Tet W were found in the intestinal contents of both animals, while Tet M was confined to pigs and Tet Q was confined to the rumen. The tet(O) and tet(W) genes circulating in the microbiota of the rumen and the gastrointestinal tract of pigs were identical despite the differences in animal hosts and antibiotic use regimens. Swine fecal streptococci uniformly possessed the tet(O) gene, and 22% of them also carried tet(M). This population could be considered one of the main reservoirs of these two resistance genes in the pig gastrointestinal tract. All classes of RPPs except Tet T and TetB P were found in the commercial components of swine feed. This is the first demonstration of the applicability of molecular ecology techniques to estimation of the gene pool and the flux of antibiotic resistance genes in production animals. PMID:11133424

  15. Cloning, purification, crystallization and preliminary X-ray analysis of the Burkholderia pseudomallei L1 ribosomal protein

    International Nuclear Information System (INIS)

    The L1 ribosomal protein from B. pseudomallei has been overexpressed, purified and crystallized in a form suitable for X-ray analysis. The gene encoding the L1 ribosomal protein from Burkholderia pseudomallei strain D286 has been cloned into the pETBLUE-1 vector system, overexpressed in Escherichia coli and purified. Crystals of the native protein were grown by the hanging-drop vapour-diffusion technique using PEG 3350 as a precipitant and diffracted to beyond 1.65 Å resolution. The crystals belonged to space group P21212, with unit-cell parameters a = 53.6, b = 127.1, c = 31.8 Å and with a single molecule in the asymmetric unit

  16. Purification and partial characterization of single-chain ribosome-inactivating proteins from the seeds of Phytolacca dioica L.

    Science.gov (United States)

    Parente, A; De Luca, P; Bolognesi, A; Barbieri, L; Battelli, M G; Abbondanza, A; Sande, M J; Gigliano, G S; Tazzari, P L; Stirpe, F

    1993-10-19

    Three ribosome-inactivating proteins (RIPs) similar to those already known (Stirpe et al. (1992) Bio/Technology 10, 405-412) were purified from the seeds of Phytolacca dioica. These proteins, called Phytolacca dioica RIPs (PD-S1, PD-S2 and PD-S3 RIPs), are glycoproteins, with M(r) approx. 30,000, inhibit protein synthesis by a rabbit reticulocyte lysate and phenylalanine polymerization by isolated ribosomes, and depurinate rat liver rRNA in an apparently identical manner as the A-chain of ricin and other RIPs (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). Part of the purified rat liver ribosomes appeared resistant to the action of PD-S RIPs. The most abundant protein, PD-S2 RIP, gave a weak or nil cross-reaction with sera against various other RIPs, including a pokeweed antiviral protein from the roots of Phytolacca americana. PD-S2 RIP was linked to a monoclonal antibody (Ber-H2) against the CD30 human lymphocyte antigen and the resulting immunotoxin was selectively toxic to the CD30 + Hodgkin's lymphoma-derived L540 cell line. PMID:8218414

  17. Convergent evolution among immunoglobulin G-binding bacterial proteins.

    OpenAIRE

    Frick, I M; Wikström, M.; Forsén, S.; Drakenberg, T; Gomi, H.; Sjöbring, U; Björck, L

    1992-01-01

    Protein G, a bacterial cell-wall protein with high affinity for the constant region of IgG (IgGFc) antibodies, contains homologous repeats responsible for the interaction with IgGFc. A synthetic peptide corresponding to an 11-amino acid-long sequence in the COOH-terminal region of the repeats was found to bind to IgGFc and block the interaction with protein G. Moreover, two other IgGFc-binding bacterial proteins (proteins A and H), which do not contain any sequences homologous to the peptide,...

  18. Identification, characterization and structure analysis of a type I ribosome-inactivating protein from Sapium sebiferum (Euphorbiaceae)

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Ying [Key Laboratory of Ion Beam Bioengineering and Bioenergy Forest Research Center of State Forestry Administration, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, Anhui (China); School of Life Sciences, University of Science and Technology of China, Hefei 230027, Anhui (China); College of Food and Bioengineering, Henan University of Science and Technology, Luoyang 471023, Henan (China); Mao, Yingji [Key Laboratory of Ion Beam Bioengineering and Bioenergy Forest Research Center of State Forestry Administration, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, Anhui (China); School of Life Sciences, University of Science and Technology of China, Hefei 230027, Anhui (China); Jin, Shan; Hou, Jinyan; Du, Hua [Key Laboratory of Ion Beam Bioengineering and Bioenergy Forest Research Center of State Forestry Administration, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, Anhui (China); Yang, Minglei, E-mail: yml888@mail.ustc.edu.cn [Key Laboratory of Ion Beam Bioengineering and Bioenergy Forest Research Center of State Forestry Administration, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, Anhui (China); Wu, Lifang, E-mail: lfwu@ipp.ac.cn [Key Laboratory of Ion Beam Bioengineering and Bioenergy Forest Research Center of State Forestry Administration, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, Anhui (China); School of Life Sciences, University of Science and Technology of China, Hefei 230027, Anhui (China)

    2015-08-07

    Ribosome-inactivating proteins (RIPs) are N-glycosidases (EC3.2.2.22) that universally inactivate the ribosome, thereby inhibiting protein biosynthesis. In this study, a novel type I RIPs named SEBIN was identified in Sapium sebiferum. Nuclear acid depurine experiment showed that SEBIN had rRNA N-Glycosidase activity. Further experiment indicated that SEBIN significantly inhibited Caenorhabditis elegans development as well as resulted in worm cell apoptosis. This is the first report to evaluate RIPs toxicity using C. elegans. We proposed that SEBIN may impaire C. elegans reproduction in a DNA-damage manner besides traditional protein synthesis inhibition approach. The predicted 3D structure was modeled using threading and ab initio modeling, and the r-RNA binding residue of SEBIN was identified through the protein-ligand docking approach. It showed the amino acid residues, Glu195, Asn81, Ala82, Tyr83, Glu164, Ser163, Ile159 and Arg167, played critical roles in catalytic process. Our results provided the theoretical foundation of structure–function relationships between enzymatic properties, toxicity and structural characterization of SEBIN. - Graphical abstract: Superposition of main chains of ricin (cyan) and SEBIN (brown), and adenine binding site residues of SEBIN. - Highlights: • A Ribosome-inactivating proteins gene (SEBIN) was isolated from Sapium sebiferum. • SEBIN had DNase activity besides widely reported ribosome inactivation via N-glycosidases activity. • SEBIN significantly inhibited Caenorhabditis elegans development in vivo. • SEBIN may impaire C. elegans reproduction in a DNA-damage manner with the aid of mutant strains hus-1 and clk-2. • The possible active sites between SEBIN and the adenine of rRNA were predicted.

  19. Identification, characterization and structure analysis of a type I ribosome-inactivating protein from Sapium sebiferum (Euphorbiaceae)

    International Nuclear Information System (INIS)

    Ribosome-inactivating proteins (RIPs) are N-glycosidases (EC3.2.2.22) that universally inactivate the ribosome, thereby inhibiting protein biosynthesis. In this study, a novel type I RIPs named SEBIN was identified in Sapium sebiferum. Nuclear acid depurine experiment showed that SEBIN had rRNA N-Glycosidase activity. Further experiment indicated that SEBIN significantly inhibited Caenorhabditis elegans development as well as resulted in worm cell apoptosis. This is the first report to evaluate RIPs toxicity using C. elegans. We proposed that SEBIN may impaire C. elegans reproduction in a DNA-damage manner besides traditional protein synthesis inhibition approach. The predicted 3D structure was modeled using threading and ab initio modeling, and the r-RNA binding residue of SEBIN was identified through the protein-ligand docking approach. It showed the amino acid residues, Glu195, Asn81, Ala82, Tyr83, Glu164, Ser163, Ile159 and Arg167, played critical roles in catalytic process. Our results provided the theoretical foundation of structure–function relationships between enzymatic properties, toxicity and structural characterization of SEBIN. - Graphical abstract: Superposition of main chains of ricin (cyan) and SEBIN (brown), and adenine binding site residues of SEBIN. - Highlights: • A Ribosome-inactivating proteins gene (SEBIN) was isolated from Sapium sebiferum. • SEBIN had DNase activity besides widely reported ribosome inactivation via N-glycosidases activity. • SEBIN significantly inhibited Caenorhabditis elegans development in vivo. • SEBIN may impaire C. elegans reproduction in a DNA-damage manner with the aid of mutant strains hus-1 and clk-2. • The possible active sites between SEBIN and the adenine of rRNA were predicted

  20. Sumoylation of Rap1 mediates the recruitment of TFIID to promote transcription of ribosomal protein genes.

    Science.gov (United States)

    Chymkowitch, Pierre; Nguéa, Aurélie P; Aanes, Håvard; Koehler, Christian J; Thiede, Bernd; Lorenz, Susanne; Meza-Zepeda, Leonardo A; Klungland, Arne; Enserink, Jorrit M

    2015-06-01

    Transcription factors are abundant Sumo targets, yet the global distribution of Sumo along the chromatin and its physiological relevance in transcription are poorly understood. Using Saccharomyces cerevisiae, we determined the genome-wide localization of Sumo along the chromatin. We discovered that Sumo-enriched genes are almost exclusively involved in translation, such as tRNA genes and ribosomal protein genes (RPGs). Genome-wide expression analysis showed that Sumo positively regulates their transcription. We also discovered that the Sumo consensus motif at RPG promoters is identical to the DNA binding motif of the transcription factor Rap1. We demonstrate that Rap1 is a molecular target of Sumo and that sumoylation of Rap1 is important for cell viability. Furthermore, Rap1 sumoylation promotes recruitment of the basal transcription machinery, and sumoylation of Rap1 cooperates with the target of rapamycin kinase complex 1 (TORC1) pathway to promote RPG transcription. Strikingly, our data reveal that sumoylation of Rap1 functions in a homeostatic feedback loop that sustains RPG transcription during translational stress. Taken together, Sumo regulates the cellular translational capacity by promoting transcription of tRNA genes and RPGs. PMID:25800674

  1. Mutation in ribosomal protein S5 leads to spectinomycin resistance in Neisseria gonorrhoeae.

    Science.gov (United States)

    Ilina, Elena N; Malakhova, Maya V; Bodoev, Ivan N; Oparina, Nina Y; Filimonova, Alla V; Govorun, Vadim M

    2013-01-01

    Spectinomycin remains a useful reserve option for therapy of gonorrhea. The emergence of multidrug-resistant Neisseria gonorrhoeae strains with decreased susceptibility to cefixime and to ceftriaxone makes it the only medicine still effective for treatment of gonorrhea infection in analogous cases. However, adoption of spectinomycin as a routinely used drug of choice was soon followed by reports of spectinomycin resistance. The main molecular mechanism of spectinomycin resistance in N. gonorrhoeae was C1192T substitution in 16S rRNA genes. Here we reported a Thr-24→Pro mutation in ribosomal protein S5 (RPS5) found in spectinomycin resistant clinical N. gonorrhoeae strain, which carried no changes in 16S rRNA. In a series of experiments, the transfer of rpsE gene allele encoding the mutant RPS5 to the recipient N. gonorrhoeae strains was analyzed. The relatively high rate of transformation [ca. 10(-5) colony-forming units (CFUs)] indicates the possibility of spread of spectinonycin resistance within gonococcal population due to the horizontal gene transfer (HGT). PMID:23847609

  2. Integrative analyses shed new light on human ribosomal protein gene regulation.

    Science.gov (United States)

    Li, Xin; Zheng, Yiyu; Hu, Haiyan; Li, Xiaoman

    2016-01-01

    Ribosomal protein genes (RPGs) are important house-keeping genes that are well-known for their coordinated expression. Previous studies on RPGs are largely limited to their promoter regions. Recent high-throughput studies provide an unprecedented opportunity to study how human RPGs are transcriptionally modulated and how such transcriptional regulation may contribute to the coordinate gene expression in various tissues and cell types. By analyzing the DNase I hypersensitive sites under 349 experimental conditions, we predicted 217 RPG regulatory regions in the human genome. More than 86.6% of these computationally predicted regulatory regions were partially corroborated by independent experimental measurements. Motif analyses on these predicted regulatory regions identified 31 DNA motifs, including 57.1% of experimentally validated motifs in literature that regulate RPGs. Interestingly, we observed that the majority of the predicted motifs were shared by the predicted distal and proximal regulatory regions of the same RPGs, a likely general mechanism for enhancer-promoter interactions. We also found that RPGs may be differently regulated in different cells, indicating that condition-specific RPG regulatory regions still need to be discovered and investigated. Our study advances the understanding of how RPGs are coordinately modulated, which sheds light to the general principles of gene transcriptional regulation in mammals. PMID:27346035

  3. Ribosome Protein L4 is essential for Epstein-Barr Virus Nuclear Antigen 1 function.

    Science.gov (United States)

    Shen, Chih-Lung; Liu, Cheng-Der; You, Ren-In; Ching, Yung-Hao; Liang, Jun; Ke, Liangru; Chen, Ya-Lin; Chen, Hong-Chi; Hsu, Hao-Jen; Liou, Je-Wen; Kieff, Elliott; Peng, Chih-Wen

    2016-02-23

    Epstein-Barr Virus (EBV) Nuclear Antigen 1 (EBNA1)-mediated origin of plasmid replication (oriP) DNA episome maintenance is essential for EBV-mediated tumorigenesis. We have now found that EBNA1 binds to Ribosome Protein L4 (RPL4). RPL4 shRNA knockdown decreased EBNA1 activation of an oriP luciferase reporter, EBNA1 DNA binding in lymphoblastoid cell lines, and EBV genome number per lymphoblastoid cell line. EBV infection increased RPL4 expression and redistributed RPL4 to cell nuclei. RPL4 and Nucleolin (NCL) were a scaffold for an EBNA1-induced oriP complex. The RPL4 N terminus cooperated with NCL-K429 to support EBNA1 and oriP-mediated episome binding and maintenance, whereas the NCL C-terminal K380 and K393 induced oriP DNA H3K4me2 modification and promoted EBNA1 activation of oriP-dependent transcription. These observations provide new insights into the mechanisms by which EBV uses NCL and RPL4 to establish persistent B-lymphoblastoid cell infection. PMID:26858444

  4. Expression and localization of VCX/Y proteins and their possible involvement in regulation of ribosome assembly during spermatogenesis

    Institute of Scientific and Technical Information of China (English)

    SHENG WEI ZOU; JIAN CHAO ZHANG; XIAO DONG ZHANG; SHI YING MIAO; SHU DONG ZONG; QI SHENG; LIN FANG WANG

    2003-01-01

    Variable Charge X/Y (VCX/Y) is a human testis-specific gene family that localized on X and Y chromo-somes. In this study, VCY protein was expressed in E. coli in the form of glutathione-S-transferase (GST)fusion protein. With the purified fusion protein as antigen, the anti-GST-VCY antibody was generated andthe localization of VCY protein in human testis was determined by immunohistochemistry. In the testisseminiferous epithelium, VCY proteins were highly expressed in nuclei of germ cells. Using propidium io-dide staining and green fluorescent protein (GFP) tag technologies, VCY and VCX-8r proteins were mainlylocalized in the nucleoli of COS7 cells. In addition, the colocalization for VCY and VCX-8r in COS7 cellswas also observed. With VCY cDNA as bait, a cDNA fragment of acidic ribosomal protein PO was obtainedusing yeast two-hybrid system. All the information above indicates that VCX/Y protein family might beinvolved in the regulation of ribosome assembly during spermatogenesis.

  5. Anti-Human Endoglin (hCD105 Immunotoxin—Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1

    Directory of Open Access Journals (Sweden)

    Begoña Barriuso

    2016-06-01

    Full Text Available Endoglin (CD105 is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT—containing recombinant musarmin 1 (single chain ribosome-inactivating proteins linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio propionate (SPDP. The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10−10 to 10−9 M.

  6. A Ribosomal Protein AgRPS3aE from Halophilic Aspergillus glaucus Confers Salt Tolerance in Heterologous Organisms

    OpenAIRE

    Xilong Liang; Yiling Liu; Lixia Xie; Xiaodan Liu; Yi Wei; Xiaoyang Zhou; Shihong Zhang

    2015-01-01

    High salt in soils is one of the abiotic stresses that significantly reduces crop yield, although saline lands are considered potential resources arable for agriculture. Currently, genetic engineering for enhancing salt tolerance is being tested as an efficient and viable strategy for crop improvement. We previously characterized a large subunit of the ribosomal protein RPL44, which is involved in osmotic stress in the extremely halophilic fungus Aspergillus glaucus. Here, we screened another...

  7. Ribosomal proteins L11 and L10.(L12)4 and the antibiotic thiostrepton interact with overlapping regions of the 23 S rRNA backbone in the ribosomal GTPase centre

    DEFF Research Database (Denmark)

    Rosendahl, G; Douthwaite, S

    1993-01-01

    23 S rRNA. Within the ribosome, L11 also interacts with this rRNA region, although the protection effects are subtly different and extend to nucleotide 1098. The pentameric r-protein complex L10.(L12)4 binds to an adjacent site on the rRNA, protecting riboses at positions 1043, 1046 to 1049, 1053 to...... by L10.(L12)4 and other proteins within the ribosome. The antibiotics thiostrepton and micrococcin inhibit the catalytic functions of this region by slotting in between the accessible loops and interacting with nucleotides there....

  8. Exploring the diversity of protein modifications: special bacterial phosphorylation systems

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Grangeasse, Christophe; Turgay, Kürşad

    2016-01-01

    physiology, and regulatory networks. Investigating these unusual bacterial kinase and phosphatases is not only important to understand their role in bacterial physiology but will help to generally understand the full potential and evolution of protein phosphorylation for signal transduction, protein...... has been most thoroughly investigated. Unlike in eukarya, a large diversity of enzyme families has been shown to phosphorylate and dephosphorylate proteins on various amino acids with different chemical properties in bacteria. In this review, after a brief overview of the known bacterial...... phosphorylation systems, we focus on more recently discovered and less widely known kinases and phosphatases. Namely, we describe in detail tyrosine- and arginine-phosphorylation together with some examples of unusual serine-phosphorylation systems and discuss their potential role and function in bacterial...

  9. Diversity and recombination of dispersed ribosomal DNA and protein coding genes in microsporidia.

    Directory of Open Access Journals (Sweden)

    Joseph Edward Ironside

    Full Text Available Microsporidian strains are usually classified on the basis of their ribosomal DNA (rDNA sequences. Although rDNA occurs as multiple copies, in most non-microsporidian species copies within a genome occur as tandem arrays and are homogenised by concerted evolution. In contrast, microsporidian rDNA units are dispersed throughout the genome in some species, and on this basis are predicted to undergo reduced concerted evolution. Furthermore many microsporidian species appear to be asexual and should therefore exhibit reduced genetic diversity due to a lack of recombination. Here, DNA sequences are compared between microsporidia with different life cycles in order to determine the effects of concerted evolution and sexual reproduction upon the diversity of rDNA and protein coding genes. Comparisons of cloned rDNA sequences between microsporidia of the genus Nosema with different life cycles provide evidence of intragenomic variability coupled with strong purifying selection. This suggests a birth and death process of evolution. However, some concerted evolution is suggested by clustering of rDNA sequences within species. Variability of protein-coding sequences indicates that considerable intergenomic variation also occurs between microsporidian cells within a single host. Patterns of variation in microsporidian DNA sequences indicate that additional diversity is generated by intragenomic and/or intergenomic recombination between sequence variants. The discovery of intragenomic variability coupled with strong purifying selection in microsporidian rRNA sequences supports the hypothesis that concerted evolution is reduced when copies of a gene are dispersed rather than repeated tandemly. The presence of intragenomic variability also renders the use of rDNA sequences for barcoding microsporidia questionable. Evidence of recombination in the single-copy genes of putatively asexual microsporidia suggests that these species may undergo cryptic sexual

  10. Phenotypically Dormant and Immature Leukaemia Cells Display Increased Ribosomal Protein S6 Phosphorylation.

    Directory of Open Access Journals (Sweden)

    Monica Pallis

    Full Text Available Mechanistic/mammalian target of rapamycin (mTOR activity drives a number of key metabolic processes including growth and protein synthesis. Inhibition of the mTOR pathway promotes cellular dormancy. Since cells from patients with acute myeloid leukaemia (AML can be phenotypically dormant (quiescent, we examined biomarkers of their mTOR pathway activity concurrently with Ki-67 and CD71 (indicators of cycling cells by quantitative flow cytometry. Using antibodies to phosphorylated epitopes of mTOR (S2448 and its downstream targets ribosomal protein S6 (rpS6, S235/236 and 4E-BP1 (T36/45, we documented that these phosphorylations were negligible in lymphocytes, but evident in dormant as well as proliferating subsets of both mobilised normal stem cell harvest CD34+ cells and AML blasts. Although mTOR phosphorylation in AML blasts was lower than that of the normal CD34+ cells, p-4E-BP1 was 2.6-fold higher and p-rpS6 was 22-fold higher. Moreover, in contrast to 4E-BP1, rpS6 phosphorylation was higher in dormant than proliferating AML blasts, and was also higher in the immature CD34+CD38- blast subset. Data from the Cancer Genome Atlas show that rpS6 expression is associated with that of respiratory chain enzymes in AML. We conclude that phenotypic quiescence markers do not necessarily predict metabolic dormancy and that elevated rpS6 ser235/236 phosphorylation is characteristic of AML.

  11. Phenotypically Dormant and Immature Leukaemia Cells Display Increased Ribosomal Protein S6 Phosphorylation.

    Science.gov (United States)

    Pallis, Monica; Harvey, Tamsin; Russell, Nigel

    2016-01-01

    Mechanistic/mammalian target of rapamycin (mTOR) activity drives a number of key metabolic processes including growth and protein synthesis. Inhibition of the mTOR pathway promotes cellular dormancy. Since cells from patients with acute myeloid leukaemia (AML) can be phenotypically dormant (quiescent), we examined biomarkers of their mTOR pathway activity concurrently with Ki-67 and CD71 (indicators of cycling cells) by quantitative flow cytometry. Using antibodies to phosphorylated epitopes of mTOR (S2448) and its downstream targets ribosomal protein S6 (rpS6, S235/236) and 4E-BP1 (T36/45), we documented that these phosphorylations were negligible in lymphocytes, but evident in dormant as well as proliferating subsets of both mobilised normal stem cell harvest CD34+ cells and AML blasts. Although mTOR phosphorylation in AML blasts was lower than that of the normal CD34+ cells, p-4E-BP1 was 2.6-fold higher and p-rpS6 was 22-fold higher. Moreover, in contrast to 4E-BP1, rpS6 phosphorylation was higher in dormant than proliferating AML blasts, and was also higher in the immature CD34+CD38- blast subset. Data from the Cancer Genome Atlas show that rpS6 expression is associated with that of respiratory chain enzymes in AML. We conclude that phenotypic quiescence markers do not necessarily predict metabolic dormancy and that elevated rpS6 ser235/236 phosphorylation is characteristic of AML. PMID:26985829

  12. cDNA sequence analysis of ribosomal protein S13 gene in Plutella xylostella (Lepidoptera: Plutellidae)

    Institute of Scientific and Technical Information of China (English)

    SHAO-LIWANG; CHENG-FASHENG; CHUAN-LINGQIAO; MIYATATADASHI

    2005-01-01

    Ribosomal protein S 13 gene has been cloned and analyzed in many organisms,but there are few documents relating to insects. In this communication, the full-length cDNA sequence of ribosomal protein S 13 gene in the diamondback moth, Plutella xylostella(Lepidoptera: Plutellidae), was determined by using PCR amplification technique. The features of the ribosomal protein S 13 gene sequence were analyzed and the deduced amino acids sequence was compared with those from other insects. The results of multi-alignment of the amino acid sequences between the diamondback moth and other insect species revealed that this gene sequence is highly conserved in insects. Based on maximum likelihood method, a phylogenetic tree was constructed from 10 different species using PHYLIP software. It showed that nematode is one separate lineage and the five insect speciesbe long to another lineage, whereas those species higher than insects form the third one. The pattern of this phylogenetic tree evidently represented the evolution of different species.

  13. GUN1 Controls Accumulation of the Plastid Ribosomal Protein S1 at the Protein Level and Interacts with Proteins Involved in Plastid Protein Homeostasis.

    Science.gov (United States)

    Tadini, Luca; Pesaresi, Paolo; Kleine, Tatjana; Rossi, Fabio; Guljamow, Arthur; Sommer, Frederik; Mühlhaus, Timo; Schroda, Michael; Masiero, Simona; Pribil, Mathias; Rothbart, Maxi; Hedtke, Boris; Grimm, Bernhard; Leister, Dario

    2016-03-01

    Developmental or metabolic changes in chloroplasts can have profound effects on the rest of the plant cell. Such intracellular responses are associated with signals that originate in chloroplasts and convey information on their physiological status to the nucleus, which leads to large-scale changes in gene expression (retrograde signaling). A screen designed to identify components of retrograde signaling resulted in the discovery of the so-called genomes uncoupled (gun) mutants. Genetic evidence suggests that the chloroplast protein GUN1 integrates signals derived from perturbations in plastid redox state, plastid gene expression, and tetrapyrrole biosynthesis (TPB) in Arabidopsis (Arabidopsis thaliana) seedlings, exerting biogenic control of chloroplast functions. However, the molecular mechanism by which GUN1 integrates retrograde signaling in the chloroplast is unclear. Here we show that GUN1 also operates in adult plants, contributing to operational control of chloroplasts. The gun1 mutation genetically interacts with mutations of genes for the chloroplast ribosomal proteins S1 (PRPS1) and L11. Analysis of gun1 prps1 lines indicates that GUN1 controls PRPS1 accumulation at the protein level. The GUN1 protein physically interacts with proteins involved in chloroplast protein homeostasis based on coimmunoprecipitation experiments. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments suggest that GUN1 might transiently interact with several TPB enzymes, including Mg-chelatase subunit D (CHLD) and two other TPB enzymes known to activate retrograde signaling. Moreover, the association of PRPS1 and CHLD with protein complexes is modulated by GUN1. These findings allow us to speculate that retrograde signaling might involve GUN1-dependent formation of protein complexes. PMID:26823545

  14. BLANNOTATOR: enhanced homology-based function prediction of bacterial proteins

    Directory of Open Access Journals (Sweden)

    Kankainen Matti

    2012-02-01

    Full Text Available Abstract Background Automated function prediction has played a central role in determining the biological functions of bacterial proteins. Typically, protein function annotation relies on homology, and function is inferred from other proteins with similar sequences. This approach has become popular in bacterial genomics because it is one of the few methods that is practical for large datasets and because it does not require additional functional genomics experiments. However, the existing solutions produce erroneous predictions in many cases, especially when query sequences have low levels of identity with the annotated source protein. This problem has created a pressing need for improvements in homology-based annotation. Results We present an automated method for the functional annotation of bacterial protein sequences. Based on sequence similarity searches, BLANNOTATOR accurately annotates query sequences with one-line summary descriptions of protein function. It groups sequences identified by BLAST into subsets according to their annotation and bases its prediction on a set of sequences with consistent functional information. We show the results of BLANNOTATOR's performance in sets of bacterial proteins with known functions. We simulated the annotation process for 3090 SWISS-PROT proteins using a database in its state preceding the functional characterisation of the query protein. For this dataset, our method outperformed the five others that we tested, and the improved performance was maintained even in the absence of highly related sequence hits. We further demonstrate the value of our tool by analysing the putative proteome of Lactobacillus crispatus strain ST1. Conclusions BLANNOTATOR is an accurate method for bacterial protein function prediction. It is practical for genome-scale data and does not require pre-existing sequence clustering; thus, this method suits the needs of bacterial genome and metagenome researchers. The method and a

  15. Discrimination of Burkholderia mallei/pseudomallei from Burkholderia thailandensis by sequence comparison of a fragment of the ribosomal protein S21 (rpsU) gene

    OpenAIRE

    Frickmann, H.; Chantratita, N.; Gauthier, Y. P.; Neubauer, H.; Hagen, R. M.

    2012-01-01

    Discrimination of Burkholderia (B.) pseudomallei and B. mallei from environmental B. thailandensis is challenging. We describe a discrimination method based on sequence comparison of the ribosomal protein S21 (rpsU) gene.

  16. Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies

    KAUST Repository

    Wang, Yong

    2009-10-09

    Bacterial 16S ribosomal DNA (rDNA) amplicons have been widely used in the classification of uncultured bacteria inhabiting environmental niches. Primers targeting conservative regions of the rDNAs are used to generate amplicons of variant regions that are informative in taxonomic assignment. One problem is that the percentage coverage and application scope of the primers used in previous studies are largely unknown. In this study, conservative fragments of available rDNA sequences were first mined and then used to search for candidate primers within the fragments by measuring the coverage rate defined as the percentage of bacterial sequences containing the target. Thirty predicted primers with a high coverage rate (>90%) were identified, which were basically located in the same conservative regions as known primers in previous reports, whereas 30% of the known primers were associated with a coverage rate of <90%. The application scope of the primers was also examined by calculating the percentages of failed detections in bacterial phyla. Primers A519-539, E969- 983, E1063-1081, U515 and E517, are highly recommended because of their high coverage in almost all phyla. As expected, the three predominant phyla, Firmicutes, Gemmatimonadetes and Proteobacteria, are best covered by the predicted primers. The primers recommended in this report shall facilitate a comprehensive and reliable survey of bacterial diversity in metagenomic studies. © 2009 Wang, Qian.

  17. Effect of ultraviolet irradiation on 30-S ribosomal subunits. Identification of the RNA region crosslinked to protein S7

    International Nuclear Information System (INIS)

    The effects of ultraviolet irradiation on Escherichia coli 30-S ribosomal subunits were studied. At the doses of radiation used in this work (0-4.5 x 105 quanta/30-S subunit), only protein S7 was found to be significantly crosslinked to the 16-S RNA. In conditions where 25% of the protein was covalently crosslinked, the ability of the irradiated 30-S subunits to reassociate with 50-S subunits and their activity in polyphenylalanine synthesis decreased strongly. Similar results were obtained by irradiation with a germicide lamp (254 nm) or with a monochromatic ultraviolet light at 248 nm. No additional proteins were crosslinked to the 16-S RNA by irradiating 30-S subunits depleted in protein S1 or 70-S ribosomes. The covalent complex of 16-S RNA and protein S7 was isolated and digested by T1 ribonuclease. The oligonucleotide remaining attached to the crosslinked protein was characterised as A-C-C-U-C-G (position 1261-1266, see the sequence published by Carbon et al. (1979) Eur. J. Biochem. 160, 399-410). Analysis of this fragment suggests that protein S7 was linked to the cytosine at position 1265 in the RNA sequence. (orig.)

  18. Inhibition of protein synthesis occurring on tetracycline-resistant, TetM-protected ribosomes by a novel class of tetracyclines, the glycylcyclines.

    OpenAIRE

    Rasmussen, B A; Gluzman, Y; Tally, F P

    1994-01-01

    One of the two major mechanisms of tetracycline resistance is ribosomal protection. Of this resistance type, tet(M) is the best characterized. Although the mechanism of tet(M) resistance has not yet been fully elucidated, it has been demonstrated that ribosomes isolated from a tet(M) strain are resistant to inhibition of protein synthesis by tetracycline. A new generation of tetracycline compounds, the glycylcyclines, that are able to inhibit protein synthesis occurring on tetracycline-resist...

  19. Comparative effects of whey protein versus L-leucine on skeletal muscle protein synthesis and markers of ribosome biogenesis following resistance exercise.

    Science.gov (United States)

    Mobley, C Brooks; Fox, Carlton D; Thompson, Richard M; Healy, James C; Santucci, Vincent; Kephart, Wesley C; McCloskey, Anna E; Kim, Mike; Pascoe, David D; Martin, Jeffrey S; Moon, Jordan R; Young, Kaelin C; Roberts, Michael D

    2016-03-01

    We compared immediate post-exercise whey protein (WP, 500 mg) versus L-leucine (LEU, 54 mg) feedings on skeletal muscle protein synthesis (MPS) mechanisms and ribosome biogenesis markers 3 h following unilateral plantarflexor resistance exercise in male, Wistar rats (~250 g). Additionally, in vitro experiments were performed on differentiated C2C12 myotubes to compare nutrient (i.e., WP, LEU) and 'exercise-like' treatments (i.e., caffeine, hydrogen peroxide, and AICAR) on ribosome biogenesis markers. LEU and WP significantly increased phosphorylated-rpS6 (Ser235/236) in the exercised (EX) leg 2.4-fold (P  0.05). Compared to the non-EX leg, MPS levels increased 32 % and 52 % in the EX leg of CTL (P  0.05). Several genes associated with ribosome biogenesis robustly increased in the EX versus non-EX legs of all treatments; specifically, c-Myc mRNA, Nop56 mRNA, Bop1 mRNA, Ncl mRNA, Npm1 mRNA, Fb1 mRNA, and Xpo-5 mRNA. However, only LEU significantly increased 45S pre-rRNA levels in the EX leg (63 %, P < 0.001). In vitro findings confirmed that 'exercise-like' treatments similarly altered markers of ribosome biogenesis, but only LEU increased 47S pre-rRNA levels (P < 0.01). Collectively, our data suggests that resistance exercise, as well as 'exercise-like' signals in vitro, acutely increase the expression of genes associated with ribosome biogenesis independent of nutrient provision. Moreover, while EX with or without WP appears superior for enhancing translational efficiency (i.e., increasing MPS per unit of RNA), LEU administration (or co-administration) may further enhance ribosome biogenesis over prolonged periods with resistance exercise. PMID:26507545

  20. Bacterial S-layer protein coupling to lipids

    DEFF Research Database (Denmark)

    Weygand, M.; Wetzer, B.; Pum, D.; Sleytr, U.B.; Cuvillier, N.; Kjær, K.; Howes, P.B.; Lösche, M.

    1999-01-01

    The coupling of bacterial surface (S)-layer proteins to lipid membranes is studied in molecular detail for proteins from Bacillus sphaericus CCM2177 and B. coagulans E38-66 recrystallized at dipalmitoylphosphatidylethanolamine (DPPE) monolayers on aqueous buffer. A comparison of the monolayer str...

  1. Crystallization and preliminary crystallographic studies of L30e, a ribosomal protein from Methanocaldococcus jannaschii (MJ1044)

    International Nuclear Information System (INIS)

    The ribosomal protein (L30e) from M. jannaschii was cloned from the gene MJ1044, expressed, purified and crystallized. The crystal belongs to the primitive tetragonal space group P43 and diffracted to 1.9 Å resolution. In view of the biological significance of understanding the ribosomal machinery of both prokaryotes and eukaryotes, the L30e ribosomal protein from Methanocaldococcus jannaschii was cloned, overexpressed, purified and crystallized using the microbatch-under-oil method with the crystallization conditions 40% PEG 400, 0.1 M MES pH 6.0 and 5% PEG 3000 at 291 K. A diffraction-quality crystal (0.20 × 0.20 × 0.35 mm) was obtained that belonged to the primitive tetragonal space group P43, with unit-cell parameters a = 46.1, b = 46.1, c = 98.5 Å, and diffracted to a resolution of 1.9 Å. Preliminary calculations reveal that the asymmetric unit contains two monomers with a Matthews coefficient (VM) of 2.16 Å3 Da−1

  2. Characteristic differences between the promoters of intron-containing and intronless ribosomal protein genes in yeast

    Directory of Open Access Journals (Sweden)

    Vingron Martin

    2008-10-01

    Full Text Available Abstract Background More than two thirds of the highly expressed ribosomal protein (RP genes in Saccharomyces cerevisiae contain introns, which is in sharp contrast to the genome-wide five percent intron-containing genes. It is well established that introns carry regulatory sequences and that the transcription of RP genes is extensively and coordinately regulated. Here we test the hypotheses that introns are innately associated with heavily transcribed genes and that introns of RP genes contribute regulatory TF binding sequences. Moreover, we investigate whether promoter features are significantly different between intron-containing and intronless RP genes. Results We find that directly measured transcription rates tend to be lower for intron-containing compared to intronless RP genes. We do not observe any specifically enriched sequence motifs in the introns of RP genes other than those of the branch point and the two splice sites. Comparing the promoters of intron-containing and intronless RP genes, we detect differences in number and position of Rap1-binding and IFHL motifs. Moreover, the analysis of the length distribution and the folding free energies suggest that, at least in a sub-population of RP genes, the 5' untranslated sequences are optimized for regulatory function. Conclusion Our results argue against the direct involvement of introns in the regulation of transcription of highly expressed genes. Moreover, systematic differences in motif distributions suggest that RP transcription factors may act differently on intron-containing and intronless gene promoters. Thus, our findings contribute to the decoding of the RP promoter architecture and may fuel the discussion on the evolution of introns.

  3. Protein quality control in the bacterial periplasm.

    Science.gov (United States)

    Merdanovic, Melisa; Clausen, Tim; Kaiser, Markus; Huber, Robert; Ehrmann, Michael

    2011-01-01

    Protein quality control involves sensing and treatment of defective or incomplete protein structures. Misfolded or mislocalized proteins trigger dedicated signal transduction cascades that upregulate the production of protein quality-control factors. Corresponding proteases and chaperones either degrade or repair damaged proteins, thereby reducing the level of aggregation-prone molecules. Because the periplasm of gram-negative bacteria is particularly exposed to environmental changes and respective protein-folding stresses connected with the presence of detergents, low or high osmolarity of the medium, elevated temperatures, and the host's immune response, fine-tuned protein quality control systems are essential for survival under these unfavorable conditions. This review discusses recent advances in the identification and characterization of the key cellular factors and the emerging general principles of the underlying molecular mechanisms. PMID:21639788

  4. The divergently transcribed genes encoding yeast ribosomal proteins L46 and S24 are activated by shared RPG-boxes.

    OpenAIRE

    Kraakman, L.S.; Mager, W H; Maurer, K T; Nieuwint, R T; Planta, R J

    1989-01-01

    Transcription of the majority of the ribosomal protein (rp) genes in yeast is activated through common cis-acting elements, designated RPG-boxes. These elements have been shown to act as specific binding sites for the protein factor TUF/RAP1/GRF1 in vitro. Two such elements occur in the intergenic region separating the divergently transcribed genes encoding L46 and S24. To investigate whether the two RPG-boxes mediate transcription activation of both the L46 and S24 gene, two experimental str...

  5. Nutritional and growth control of ribosomal protein mRNA and rRNA in Neurospora crassa.

    OpenAIRE

    Cujec, T P; Tyler, B M

    1996-01-01

    The effects of changing growth rates on the levels of 40S pre-rRNA and two r-protein mRNAs were examined to gain insight into the coordinate transcriptional regulation of ribosomal genes in the ascomycete fungus Neurospora crassa. Growth rates were varied either by altering carbon nutritional conditions, or by subjecting the isolates to inositol-limiting conditions. During carbon up- or down-shifts, r-protein mRNA levels were stoichiometrically coordinated. Changes in 40S pre-rRNA levels para...

  6. Cloning and expression of a cDNA encoding ribosomal protein S4 from Rice (Oryza sativa)

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A cDNA clone, pS4, has been isolated from a cDNA library prepared from rice anthers of about 1.0 mm in length. DNA sequence analysis and database search show that the cDNA encodes a protein which is highly homologous to eukaryotic 80S ribosomal protein subunit 4 (S4). Northern hybridization indicates that this gene expresses in all tissues analyzed although the expression level varies and it cannot be induced by mechanical wounding in leaves. Southern blot analysis demonstrates that this rice S4 gene is from a multigene family.

  7. Demodex-associated bacterial proteins induce neutrophil activation.

    LENUS (Irish Health Repository)

    2012-02-01

    Background: Patients with rosacea demonstrate a higher density of Demodex mites in their skin than controls. A bacterium isolated from a Demodex mite from a patient with papulopustular rosacea (PPR) was previously shown to provoke an immune response in patients with PPR or ocular rosacea thus suggesting a possible role for bacterial proteins in the etiology of this condition. Objectives: To examine the response of neutrophils to proteins derived from a bacterium isolated from a Demodex mite. Methods: Bacterial cells were lysed and proteins were partially purified by AKTA-FPLC. Isolated neutrophils were exposed to bacterial proteins and monitored for alterations in migration, degranulation and cytokine production. Results: Neutrophils exposed to proteins from Bacillus cells demonstrated increased levels of migration and elevated release of MMP-9, an enzyme known to degrade collagen and cathelicidin, an antimicrobial peptide. In addition neutrophils exposed to the bacterial proteins demonstrated elevated rates of Il-8 and TNF-alpha production. Conclusions: Proteins produced by a bacterium isolated from a Demodex mite have the ability to increase the migration, degranulation and cytokine production abilities of neutrophils. These results suggest that bacteria may play a role in the inflammatory erythema associated with rosacea.

  8. Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases

    DEFF Research Database (Denmark)

    Issinger, O G; Beier, H; Speichermann, N; Flokerzi, V; Hofmann, F

    1980-01-01

    Phosphorylation of eukaryotic ribosomal proteins in vitro by essentially homogeneous preparations of cyclic AMP-dependent protein kinase catalytic subunit and cyclic GMP-dependent protein kinase was compared. Each protein kinase was added at a concentration of 30nM. Ribosomal proteins were...... cyclic AMP-dependent enzyme. Between 0.1 and 0.2 mol of phosphate was incorporated/mol of these phosphorylated proteins. With the exception of protein S7, the same proteins were also major substrates for the cyclic GMP-dependent protein kinase. Time courses of the phosphorylation of individual proteins...... from the small and large ribosomal subunits in the presence of either protein kinase suggested four types of phosphorylation reactions: (1) proteins S2, S10 and L5 were preferably phosphorylated by the cyclic GMP-dependent protein kinase; (2) proteins S3 and L6 were phosphorylated at very similar rates...

  9. Prolonged inhibition of bacterial protein synthesis abolishes Salmonella invasion.

    OpenAIRE

    MacBeth, K J; Lee, C. A.

    1993-01-01

    We have found that prolonged inhibition of bacterial protein synthesis abolishes the ability of Salmonella typhimurium to enter HEp-2 cells. Our results suggest that an essential invasion factor has a functional half-life that is seen as a gradual loss of invasiveness in the absence of protein synthesis. Therefore, Salmonella invasiveness appears to be a transient phenotype that is lost unless protein synthesis is maintained. This finding may explain why salmonellae grown to stationary phase ...

  10. A novel full-length gene of human ribosomal protein L14.22 related to human glioma

    Institute of Scientific and Technical Information of China (English)

    QI Zhen-yu; HUI Guo-zhen; LI Yao; ZHOU Zong-xiang; GU Shao-hua; XIE Yi

    2006-01-01

    Background This study was undertaken to obtain differentially expressed genes related to human glioma by cDNA microarray and the characterization of a novel full-length gene. Methods Total RNA was extracted from human glioma and normal brain tissues, and mRNA was used as a probe. The results of hybridization procedure were scanned with the computer system. The gene named 507E08clone was subsequently analyzed by northern blot, bioinformatic approach, and protein expression.Results Fifteen differentially expressed genes were obtained from human glioma by hybridization and scanning for four times. Northern blot analysis confirmed that the 507E08 clone was low expressed in human brain tissue and over expressed in human glioma tissues. The analysis of BLASTn and BLASTx showed that the 507E08clone was a novel full-length gene, which codes 203 amino acid of protein and is called human ribosomal protein 14.22 gene. The nucleotide sequence had been submitted to the GenBankTM with the accession number of AF329277. After expression in E. Coli., protein yielded a major band of apparent molecular mass 22 kDa on an SDS-PAGE gel.Conclusions cDNA microarray technology can be successfully used to identify differentially expressed genes.The novel full-length gene of human ribosomal protein 14.22 may be correlated with the development of human glioma.

  11. Ribosome Assembly as Antimicrobial Target.

    Science.gov (United States)

    Nikolay, Rainer; Schmidt, Sabine; Schlömer, Renate; Deuerling, Elke; Nierhaus, Knud H

    2016-01-01

    Many antibiotics target the ribosome and interfere with its translation cycle. Since translation is the source of all cellular proteins including ribosomal proteins, protein synthesis and ribosome assembly are interdependent. As a consequence, the activity of translation inhibitors might indirectly cause defective ribosome assembly. Due to the difficulty in distinguishing between direct and indirect effects, and because assembly is probably a target in its own right, concepts are needed to identify small molecules that directly inhibit ribosome assembly. Here, we summarize the basic facts of ribosome targeting antibiotics. Furthermore, we present an in vivo screening strategy that focuses on ribosome assembly by a direct fluorescence based read-out that aims to identify and characterize small molecules acting as primary assembly inhibitors. PMID:27240412

  12. Slow formation of stable complexes during coincubation of minimal rRNA and ribosomal protein S4.

    Science.gov (United States)

    Mayerle, Megan; Bellur, Deepti L; Woodson, Sarah A

    2011-09-23

    Ribosomal protein S4 binds and stabilizes a five-helix junction or five-way junction (5WJ) in the 5' domain of 16S ribosomal RNA (rRNA) and is one of two proteins responsible for nucleating 30S ribosome assembly. Upon binding, both protein S4 and 5WJ reorganize their structures. We show that labile S4 complexes rearrange into stable complexes within a few minutes at 42 °C, with longer coincubation leading to an increased population of stable complexes. In contrast, prefolding the rRNA has a smaller effect on stable S4 binding. Experiments with minimal rRNA fragments show that this structural change depends only on 16S residues within the S4 binding site. SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemical probing experiments showed that S4 strongly stabilizes 5WJ and the helix (H) 18 pseudoknot, which become tightly folded within the first minute of S4 binding. However, a kink in H16 that makes specific contacts with the S4 N-terminal extension, as well as a right-angle motif between H3, H4, and H18, requires a minute or more to become fully structured. Surprisingly, S4 structurally reorganizes the 530-loop and increases the flexibility of H3, which is proposed to undergo a conformational switch during 30S assembly. These elements of the S4 binding site may require other 30S proteins to reach a stable conformation. PMID:21821049

  13. Ribosomal protein genes are highly enriched among genes with allele-specific expression in the interspecific F1 hybrid catfish.

    Science.gov (United States)

    Chen, Ailu; Wang, Ruijia; Liu, Shikai; Peatman, Eric; Sun, Luyang; Bao, Lisui; Jiang, Chen; Li, Chao; Li, Yun; Zeng, Qifan; Liu, Zhanjiang

    2016-06-01

    Interspecific hybrids provide a rich source for the analysis of allele-specific expression (ASE). In this work, we analyzed ASE in F1 hybrid catfish using RNA-Seq datasets. While the vast majority of genes were expressed with both alleles, 7-8 % SNPs exhibited significant differences in allele ratios of expression. Of the 66,251 and 177,841 SNPs identified from the datasets of the liver and gill, 5420 (8.2 %) and 13,390 (7.5 %) SNPs were identified as significant ASE-SNPs, respectively. With these SNPs, a total of 1519 and 3075 ASE-genes were identified. Gene Ontology analysis revealed that genes encoding cytoplasmic ribosomal proteins (RP) were highly enriched among ASE genes. Parent-of-origin was determined for 27 and 30 ASE RP genes in the liver and gill, respectively. The results indicated that genes from both channel catfish and blue catfish were involved in ASE. However, each RP gene appeared to be almost exclusively expressed from only one parent, indicating that ribosomes in the hybrid catfish were in the "hybrid" form. Overall representation of RP transcripts among the transcriptome appeared lower in the F1 hybrid catfish than in channel catfish or blue catfish, suggesting that the "hybrid" ribosomes may work more efficiently for translation in the F1 hybrid catfish. PMID:26747053

  14. The essential nucleolar yeast protein Nop8p controls the exosome function during 60S ribosomal subunit maturation.

    Directory of Open Access Journals (Sweden)

    Marcia C T Santos

    Full Text Available The yeast nucleolar protein Nop8p has previously been shown to interact with Nip7p and to be required for 60S ribosomal subunit formation. Although depletion of Nop8p in yeast cells leads to premature degradation of rRNAs, the biochemical mechanism responsible for this phenotype is still not known. In this work, we show that the Nop8p amino-terminal region mediates interaction with the 5.8S rRNA, while its carboxyl-terminal portion interacts with Nip7p and can partially complement the growth defect of the conditional mutant strain Δnop8/GAL::NOP8. Interestingly, Nop8p mediates association of Nip7p to pre-ribosomal particles. Nop8p also interacts with the exosome subunit Rrp6p and inhibits the complex activity in vitro, suggesting that the decrease in 60S ribosomal subunit levels detected upon depletion of Nop8p may result from degradation of pre-rRNAs by the exosome. These results strongly indicate that Nop8p may control the exosome function during pre-rRNA processing.

  15. YbxF, a protein associated with exponential-phase ribosomes in Bacillus subtilis

    Czech Academy of Sciences Publication Activity Database

    Sojka, Luděk; Fučík, Vladimír; Krásný, Libor; Barvík, I.; Jonák, Jiří

    2007-01-01

    Roč. 189, č. 13 (2007), s. 4809-4814. ISSN 0021-9193 R&D Projects: GA AV ČR IAA5052206 Institutional research plan: CEZ:AV0Z50520514 Keywords : ybxF * ymxC * ribosomes * Bacillus subtilis * GFP * growth phase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.013, year: 2007

  16. Slow formation of stable complexes during coincubation of a minimal rRNA and ribosomal protein S4

    Science.gov (United States)

    Mayerle, Megan; Bellur, Deepti L.; Woodson, Sarah A.

    2011-01-01

    Ribosomal protein S4 binds and stabilizes a five-helix junction in the 5’ domain of the 16S rRNA, and is one of two proteins responsible for nucleating 30S ribosome assembly. Upon binding, both protein S4 and the five-helix junction reorganize their structures. We show that labile S4 complexes rearrange to stable complexes within a few minutes at 42°C, with longer coincubation leading to an increased population of stable complexes. In contrast, prefolding the rRNA has a smaller effect on stable S4 binding. Experiments with minimal rRNA fragments show this structural change depends only on 16S residues within the S4 binding site. SHAPE chemical-probing experiments showed that S4 strongly stabilizes the five-helix junction and helix 18 pseudoknot, which become tightly folded within the first minute of S4 binding. However, a kink in helix 16 that makes specific contacts with the S4 N-terminal extension, and a right angle motif between helices 3, 4 and 18, require a minute or more to become fully structured. Surprisingly, S4 structurally reorganizes the 530-loop and increases the flexibility of helix 3, which is proposed to undergo a conformational switch during 30S assembly. These elements of the S4 binding site may require other 30S proteins to reach a stable conformation. PMID:21821049

  17. HrpA, a new ribosome-associated protein which appears in heat-stressed Mycobacterium bovis bacillus Calmette-Guérin.

    OpenAIRE

    Ohara, N; Naito, M.; Miyazaki, C.; Matsumoto, S.; Tabira, Y; Yamada, T.

    1997-01-01

    A novel 18-kDa heat shock protein, HrpA, has been identified from Mycobacterium bovis BCG. HrpA was rapidly synthesized in membrane and ribosome fractions but not in the cytoplasmic fraction under heat shock stress. HrpA bound tightly to 70S ribosomes, mainly in 30S subunits. HrpA might be involved in the initiation step of translation at high temperature.

  18. The Chaotic Structure of Bacterial Virulence Protein Sequences

    Directory of Open Access Journals (Sweden)

    Sevdanur Genc

    2015-01-01

    Full Text Available Bacterial virulence proteins, which have been class ified on structure of virulence, causes several diseases. For instance, Adhesins play an important role in th e host cells. They are inserted DNA sequences for a variety of virulence properties. Several important methods conducted for the prediction of bacterial virulence proteins for finding new drugs or vaccines. In this study, we propose a method for feature sele ction about classification of bacterial virulence protein. The features are constituted dir ectly from the amino acid sequence of a given protein. Amino acids form proteins, which are criti cal to life, and have many important functions in living cells. They occurring with diff erent physicochemical properties by a vector of 20 numerical values, and collected in AAIndex datab ases of known 544 indices. For all that, this approach have two steps. Firstly , the amino acid sequence of a given protein analysed with Lyapunov Exponents that they have a chaotic structure in accordance wi th the chaos theory. After that, if the results show chara cterization over the complete distribution in the phase space from the point of deterministic sys tem, it means related protein will show a chaotic structure. Empirical results revealed that generated feature v ectors give the best performance with chaotic structure of physicochemical features of amino acid s with Adhesins and non-Adhesins data sets.

  19. Identification of ligands for bacterial sensor proteins.

    Science.gov (United States)

    Fernández, Matilde; Morel, Bertrand; Corral-Lugo, Andrés; Rico-Jiménez, Miriam; Martín-Mora, David; López-Farfán, Diana; Reyes-Darias, José Antonio; Matilla, Miguel A; Ortega, Álvaro; Krell, Tino

    2016-02-01

    Bacteria have evolved a variety of different signal transduction mechanisms. However, the cognate signal molecule for the very large amount of corresponding sensor proteins is unknown and their functional annotation represents a major bottleneck in the field of signal transduction. The knowledge of the signal molecule is an essential prerequisite to understand the signalling mechanisms. Recently, the identification of signal molecules by the high-throughput protein screening of commercially available ligand collections using differential scanning fluorimetry has shown promise to resolve this bottleneck. Based on the analysis of a significant number of different ligand binding domains (LBDs) in our laboratory, we identified two issues that need to be taken into account in the experimental design. Since a number of LBDs require the dimeric state for ligand recognition, it has to be assured that the protein analysed is indeed in the dimeric form. A number of other examples demonstrate that purified LBDs can contain bound ligand which prevents further binding. In such cases, the apo-form can be generated by denaturation and subsequent refolding. We are convinced that this approach will accelerate the functional annotation of sensor proteins which will help to understand regulatory circuits in bacteria. PMID:26511375

  20. RNA:protein ratio of the unicellular organism as a characteristic of phosphorous and nitrogen stoichiometry and of the cellular requirement of ribosomes for protein synthesis

    OpenAIRE

    Sams Carl E; Greenwood Duncan J; Karpinets Tatiana V; Ammons John T

    2006-01-01

    Abstract Background Mean phosphorous:nitrogen (P:N) ratios and relationships of P:N ratios with the growth rate of organisms indicate a surprising similarity among and within microbial species, plants, and insect herbivores. To reveal the cellular mechanisms underling this similarity, the macromolecular composition of seven microorganisms and the effect of specific growth rate (SGR) on RNA:protein ratio, the number of ribosomes, and peptide elongation rate (PER) were analyzed under different ...

  1. A novel protein-RNA binding assay: functional interactions of the foot-and-mouth disease virus internal ribosome entry site with cellular proteins.

    OpenAIRE

    Stassinopoulos, I A; Belsham, G J

    2001-01-01

    Translation initiation on foot-and-mouth disease virus (FMDV) RNA occurs by a cap-independent mechanism directed by a highly structured element (approximately 435 nt) termed an internal ribosome entry site (IRES). A functional assay to identify proteins that bind to the FMDV IRES and are necessary for FMDV IRES-mediated translation initiation has been developed. In vitro-transcribed polyadenylated RNAs corresponding to the whole or part of the FMDV IRES were immobilized on oligo-dT Dynabeads ...

  2. Ribosome recycling induces optimal translation rate at low ribosomal availability

    OpenAIRE

    Marshall, E.; Stansfield, I; Romano, M. C.

    2014-01-01

    During eukaryotic cellular protein synthesis, ribosomal translation is made more efficient through interaction between the two ends of the messenger RNA (mRNA). Ribosomes reaching the 3′ end of the mRNA can thus recycle and begin translation again on the same mRNA, the so-called ‘closed-loop’ model. Using a driven diffusion lattice model of translation, we study the effects of ribosome recycling on the dynamics of ribosome flow and density on the mRNA. We show that ribosome recycling induces ...

  3. Quinacrine impairs enterovirus 71 RNA replication by preventing binding of polypyrimidine-tract binding protein with internal ribosome entry sites.

    Directory of Open Access Journals (Sweden)

    Jianmin Wang

    Full Text Available Since the 1980s, epidemics of enterovirus 71 (EV71 and other enteroviruses have occurred in Asian countries and regions, causing a wide range of human diseases. No effective therapy is available for the treatment of these infections. Internal ribosome entry sites (IRESs are indispensable for the initiation of translation in enteroviruses. Several cellular factors, as well as the ribosome, are recruited to the conserved IRES during this process. Quinacrine intercalates into the RNA architecture and inhibits RNA transcription and protein synthesis, and a recent study showed that quinacrine inhibited encephalomyocarditis virus and poliovirus IRES-mediated translation in vitro without disrupting internal cellular IRES. Here, we report that quinacrine was highly active against EV71, protecting cells from EV71 infection. Replication of viral RNA, expression of viral capsid protein, and production of virus were all strongly inhibited by quinacrine. Interaction of the polypyrimidine tract-binding protein (PTB with the conserved IRES was prevented by quinacrine. Coxsackieviruses and echovirus were also inhibited by quinacrine in cultured cells. These results indicate that quinacrine may serve as a potential protective agent for use in the treatment of patients with chronic enterovirus infection.

  4. Bacterial Microcompartment Organelles: Protein Shell Structure and Evolution

    OpenAIRE

    Yeates, Todd O; Crowley, Christopher S.; Tanaka, Shiho

    2010-01-01

    Some bacteria contain organelles or microcompartments consisting of a large virion-like protein shell encapsulating sequentially acting enzymes. These organized microcompartments serve to enhance or protect key metabolic pathways inside the cell. The variety of bacterial microcompartments provide diverse metabolic functions, ranging from CO2 fixation to the degradation of small organic molecules. Yet they share an evolutionarily related shell, which is defined by a conserved protein domain th...

  5. Surface display of proteins by Gram-negative bacterial autotransporters

    Directory of Open Access Journals (Sweden)

    Mourez Michael

    2006-06-01

    Full Text Available Abstract Expressing proteins of interest as fusions to proteins of the bacterial envelope is a powerful technique with many biotechnological and medical applications. Autotransporters have recently emerged as a good tool for bacterial surface display. These proteins are composed of an N-terminal signal peptide, followed by a passenger domain and a translocator domain that mediates the outer membrane translocation of the passenger. The natural passenger domain of autotransporters can be replaced by heterologous proteins that become displayed at the bacterial surface by the translocator domain. The simplicity and versatility of this system has made it very attractive and it has been used to display functional enzymes, vaccine antigens as well as polypeptides libraries. The recent advances in the study of the translocation mechanism of autotransporters have raised several controversial issues with implications for their use as display systems. These issues include the requirement for the displayed polypeptides to remain in a translocation-competent state in the periplasm, the requirement for specific signal sequences and "autochaperone" domains, and the influence of the genetic background of the expression host strain. It is therefore important to better understand the mechanism of translocation of autotransporters in order to employ them to their full potential. This review will focus on the recent advances in the study of the translocation mechanism of autotransporters and describe practical considerations regarding their use for bacterial surface display.

  6. Primary structure of dihydrofolate reductase and mitochondrial ribosomal protein L36 genes from the basidiomycete Coprinus cinereus.

    Science.gov (United States)

    Aimi, Tadanori; Fukuhara, Shoji; Ishiguro, Maki; Kitamoto, Yutaka; Morinaga, Tsutomu

    2004-08-01

    We amplified and sequenced the dihydrofolate reductase (DHFR) gene of the basidiomycete Coprinus cinereus. Downstream of the DHFR coding region, a mitochondrial (mt) ribosomal protein L36 (RPL36) gene was discovered in the opposite orientation to DHFR gene. Putative polyadenylation signals of the two genes overlapped, both containing the 8-bp palindrome 5'-aatatatt-3'. The finding that C. cinereus DHFR gene is closely clustered with a mt protein gene strongly suggests that C. cinereus DHFR is closely related to mt function and evolution. The amino acid sequence of C. cinereus DHFR is most homologous to eukaryotic proteins such as Cryptococcus neoformans and Pneumocystis carinii DHFRs. However, the sequence of C. cinereus mt RPL36 closely resembles RPL36 of bacteria and cyanobacteria such as Synechocystis sp. and Escherichia coli. This result strongly supports the serial endosymbiotic theory of the development of ancestral eukaryotes, and suggests that C. cinereus mt RPL36 gene originated from the ancestral eubacterial genome. PMID:15620217

  7. Bacterial characterization using protein profiling in a microchip separations platform.

    Science.gov (United States)

    Pizarro, Shelly A; Lane, Pamela; Lane, Todd W; Cruz, Evelyn; Haroldsen, Brent; VanderNoot, Victoria A

    2007-12-01

    A rapid microanalytical protein-based approach to bacterial characterization is presented. Chip gel electrophoresis (CGE) coupled with LIF detection was used to analyze lysates from different bacterial cell lines to obtain signature profiles of the soluble protein composition. The study includes Escherichia coli, Bacillus subtilis, and Bacillus anthracis (Delta Sterne strain) vegetative cells as well as endospores formed from the latter two species as model organisms to demonstrate the method. A unified protein preparation protocol was developed for both cell types to streamline the benchtop process and aid future automation. Cells and spores were lysed and proteins solubilized using a combination of thermal and chemical lysis methods. Reducing agents, necessary to solubilize spore proteins, were eliminated using a small-scale rapid size-exclusion chromatography step to eliminate interference with down-stream protein labeling. This approach was found to be compatible with nonspore cells (i.e., vegetative cells) as well, not adversely impacting the protein signatures. Data are presented demonstrating distinct CGE protein signatures for our model organisms, suggesting the potential for discrimination of organisms on the basis of empirical protein patterns. The goal of this work is to develop a fast and field-portable method for characterizing bacteria via their proteomes. PMID:18008300

  8. Diversity and Recombination of Dispersed Ribosomal DNA and Protein Coding Genes in Microsporidia

    OpenAIRE

    Ironside, Joseph Edward

    2013-01-01

    Microsporidian strains are usually classified on the basis of their ribosomal DNA (rDNA) sequences. Although rDNA occurs as multiple copies, in most non-microsporidian species copies within a genome occur as tandem arrays and are homogenised by concerted evolution. In contrast, microsporidian rDNA units are dispersed throughout the genome in some species, and on this basis are predicted to undergo reduced concerted evolution. Furthermore many microsporidian species appear to be asexual and sh...

  9. The Interaction Pattern between a Homology Model of 40S Ribosomal S9 Protein of Rhizoctonia solani and 1-Hydroxyphenaize by Docking Study

    Directory of Open Access Journals (Sweden)

    Seema Dharni

    2014-01-01

    Full Text Available 1-Hydroxyphenazine (1-OH-PHZ, a natural product from Pseudomonas aeruginosa strain SD12, was earlier reported to have potent antifungal activity against Rhizoctonia solani. In the present work, the antifungal activity of 1-OH-PHZ on 40S ribosomal S9 protein was validated by molecular docking approach. 1-OH-PHZ showed interaction with two polar contacts with residues, Arg69 and Phe19, which inhibits the synthesis of fungal protein. Our study reveals that 1-OH-PHZ can be a potent inhibitor of 40S ribosomal S9 protein of R. solani that may be a promising approach for the management of fungal diseases.

  10. Prolonged inhibition of bacterial protein synthesis abolishes Salmonella invasion.

    Science.gov (United States)

    MacBeth, K J; Lee, C A

    1993-01-01

    We have found that prolonged inhibition of bacterial protein synthesis abolishes the ability of Salmonella typhimurium to enter HEp-2 cells. Our results suggest that an essential invasion factor has a functional half-life that is seen as a gradual loss of invasiveness in the absence of protein synthesis. Therefore, Salmonella invasiveness appears to be a transient phenotype that is lost unless protein synthesis is maintained. This finding may explain why salmonellae grown to stationary phase lose their ability to enter cultured cells. In addition, a short-lived capacity to enter cells may be important during infection so that bacterial invasiveness is limited to certain times and host sites during pathogenesis. PMID:8454361

  11. Ribosomal protein L11 is related to brain maturation during the adult phase in Apis cerana cerana (Hymenoptera, Apidae)

    Science.gov (United States)

    Meng, Fei; Lu, Wenjing; Yu, Feifei; Kang, Mingjiang; Guo, Xingqi; Xu, Baohua

    2012-05-01

    Ribosomal proteins (RPs) play pivotal roles in developmental regulation. The loss or mutation of ribosomal protein L11 ( RPL11) induces various developmental defects. However, few RPs have been functionally characterized in Apis cerana cerana. In this study, we isolated a single copy gene, AccRPL11, and characterized its connection to brain maturation. AccRPL11 expression was highly concentrated in the adult brain and was significantly induced by abiotic stresses such as pesticides and heavy metals. Immunofluorescence assays demonstrated that AccRPL11 was localized to the medulla, lobula and surrounding tissues of esophagus in the brain. The post-transcriptional knockdown of AccRPL11 gene expression resulted in a severe decrease in adult brain than in other tissues. The expression levels of other brain development-related genes, p38, ERK2, CacyBP and CREB, were also reduced. Immunofluorescence signal attenuation was also observed in AccRPL11-rich regions of the brain in ds AccRPL11-injected honeybees. Taken together, these results suggest that AccRPL11 may be functional in brain maturation in honeybee adults.

  12. cDNA Cloning, Expression and Characterization of an Allergenic 60s Ribosomal Protein of Almond (Prunus dulcis

    Directory of Open Access Journals (Sweden)

    Abolhassani Mohsen

    2009-06-01

    Full Text Available Tree nuts, including almond (prunus dulcis are a source of food allergens often associated with life-threatening allergic reactions in susceptible individuals. Although the proteins in almonds have been biochemically characterized, relatively little has been reported regarding the identity of the allergens involved in almond sensitivity. The present study was undertaken to identify the allergens of the almond by cDNA library approach. cDNA library of almond seeds was constructed in Uni-Zap XR lamda vector and expressed in E. coli XL-1 blue. Plaques were immunoscreened with pooled sera of allergic patients. The cDNA clone reacting significantly with specific IgE antibodies was selected and subcloned and subsequently expressed in E. coli. The amino acids deducted from PCR product of clone showed homology to 60s acidic ribosomal protein of almond. The expressed protein was 11,450 Dalton without leader sequence. Immunoreactivity of the recombinant 60s ribosomal protein (r60sRP was evaluated with dot blot analysis using pooled and individual sera of allergic patients. The data showed that r60sRP and almond extract (as positive control possess the ability to bind the IgE antibodies. The results showed that expressed protein is an almond allergen.Whether this r60sRP represents a major allergen of almond needs to be further studied which requires a large number of sera from the almond atopic patients and also need to determine the IgE-reactive frequencies of each individual allergen.

  13. Structural Aspects of Bacterial Outer Membrane Protein Assembly.

    Science.gov (United States)

    Calmettes, Charles; Judd, Andrew; Moraes, Trevor F

    2015-01-01

    The outer membrane of Gram-negative bacteria is predominantly populated by β-Barrel proteins and lipid anchored proteins that serve a variety of biological functions. The proper folding and assembly of these proteins is essential for bacterial viability and often plays a critical role in virulence and pathogenesis. The β-barrel assembly machinery (Bam) complex is responsible for the proper assembly of β-barrels into the outer membrane of Gram-negative bacteria, whereas the localization of lipoproteins (Lol) system is required for proper targeting of lipoproteins to the outer membrane. PMID:26621472

  14. Incorporation of single dinitrophenyl-modified proteins in to the 30S subunit of Escherichia coli ribosomes by total reconstitution for localization by immune electron microscopy

    International Nuclear Information System (INIS)

    The ribosome is a structurally defined organelle whose function is central to the existence of all organisms. It is the unique site of protein biosynthesis in all cells. A detailed understanding of ribosome structure is essential in understanding the process of translation. This thesis represents a new approach to the systematic localization of individual proteins contained in the small subunit of Escherichia coli ribosomes using immunoelectron microscopy. All 30S proteins were purified using high performance liquid chromatography (HPLC) and eight isolated proteins (S12,S21,S14,S19,S18,S17,S16 and S13) were derivatized with 2,4-[3,5-3H]dinitrofluorobenzene (DNFB). The extent of modification of these proteins was estimated by both radioactivity and integrated peak areas, using dual wavelength monitoring at 214nm to detect protein and 360nm (to detect dinitrophenyl groups). Each dinitrophenylated protein was introduced in place of the corresponding unmodified protein into totally reconstituted 30S subunits. Antibodies raised against the DNP-hapten bound effectively to such reconstituted subunits and did not cause dissociation of the modified protein from the subunit. Electron microscopy of the immune complexes was used to localize the modified protein on the subunit surface. Incorporation of any of the DNP-modified proteins, with the exception of DNP-S18, does not interfere with the functionality of the ribosome as measure by the binding of Phe-tRNAPhe or the synthesis of poly(Phe) in a poly(U)-dependent manner. Results show that unmodified protein competes with DNP-protein and that DNP-protein can function, as its native counterpart, in stimulating uptake of specific proteins during reconstitution. This data provides evidence that each DNP-protein occupies the same position in 30S subunits as does the corresponding unmodified protein

  15. Protein Factor Requirements of the Apaf-1 Internal Ribosome Entry Segment: Roles of Polypyrimidine Tract Binding Protein and upstream of N-ras

    OpenAIRE

    Mitchell, Sally A.; Brown, Emma C; Coldwell, Mark J; Jackson, Richard J.; Willis, Anne E.

    2001-01-01

    It has been reported previously that the 5′ untranslated region of the mRNA encoding Apaf-1 (apoptotic protease-activating factor 1) has an internal ribosome entry site (IRES), whose activity varies widely among different cell types. Here it is shown that the Apaf-1 IRES is active in rabbit reticulocyte lysates, provided that the system is supplemented with polypyrimidine tract binding protein (PTB) and upstream of N-ras (unr), two cellular RNA binding proteins previously identified to be req...

  16. A ribosomal protein gene cluster is encoded in the mitochondrial DNA of Dictyostelium discoideum: UGA termination codons and similarity of gene order to Acanthamoeba castellanii.

    Science.gov (United States)

    Iwamoto, M; Pi, M; Kurihara, M; Morio, T; Tanaka, Y

    1998-04-01

    We sequenced a region of about 14.5 kb downstream from the ribosomal protein L11 gene (rpl11) in the mitochondrial DNA (54+/-2 kb) of the cellular slime mold Dictyostelium discoideum. Sequence analysis revealed that eleven ribosomal protein genes and six open reading frames (ORFs) formed a cluster arranged in the order: rpl11-orf189-rps12-rps7-rpl2-rps19-+ ++orf425-orf1740-rpl16-rpl14-orf188- rps14-rps8-rpl6-rps13-orf127-orf796. This order was very similar to that of homologous genes in Acanthamoeba castellanii mitochondrial DNA. The N-terminal region of ORF425 and the C-terminal region of ORF1740 had partial similarities to the S3 ribosomal protein of other organisms. The termination codons of rpl16 and orf188 were UGA, which has not hitherto been found in genes encoded in D. discoideum mitochondrial DNA. PMID:9560439

  17. Recombinant Expression Screening of P. aeruginosa Bacterial Inner Membrane Proteins

    Directory of Open Access Journals (Sweden)

    Jeffery Constance J

    2010-11-01

    Full Text Available Abstract Background Transmembrane proteins (TM proteins make up 25% of all proteins and play key roles in many diseases and normal physiological processes. However, much less is known about their structures and molecular mechanisms than for soluble proteins. Problems in expression, solubilization, purification, and crystallization cause bottlenecks in the characterization of TM proteins. This project addressed the need for improved methods for obtaining sufficient amounts of TM proteins for determining their structures and molecular mechanisms. Results Plasmid clones were obtained that encode eighty-seven transmembrane proteins with varying physical characteristics, for example, the number of predicted transmembrane helices, molecular weight, and grand average hydrophobicity (GRAVY. All the target proteins were from P. aeruginosa, a gram negative bacterial opportunistic pathogen that causes serious lung infections in people with cystic fibrosis. The relative expression levels of the transmembrane proteins were measured under several culture growth conditions. The use of E. coli strains, a T7 promoter, and a 6-histidine C-terminal affinity tag resulted in the expression of 61 out of 87 test proteins (70%. In this study, proteins with a higher grand average hydrophobicity and more transmembrane helices were expressed less well than less hydrophobic proteins with fewer transmembrane helices. Conclusions In this study, factors related to overall hydrophobicity and the number of predicted transmembrane helices correlated with the relative expression levels of the target proteins. Identifying physical characteristics that correlate with protein expression might aid in selecting the "low hanging fruit", or proteins that can be expressed to sufficient levels using an E. coli expression system. The use of other expression strategies or host species might be needed for sufficient levels of expression of transmembrane proteins with other physical

  18. Mice deficient in ribosomal protein S6 phosphorylation suffer from muscle weakness that reflects a growth defect and energy deficit.

    Directory of Open Access Journals (Sweden)

    Igor Ruvinsky

    Full Text Available BACKGROUND: Mice, whose ribosomal protein S6 cannot be phosphorylated due to replacement of all five phosphorylatable serine residues by alanines (rpS6(P-/-, are viable and fertile. However, phenotypic characterization of these mice and embryo fibroblasts derived from them, has established the role of these modifications in the regulation of the size of several cell types, as well as pancreatic beta-cell function and glucose homeostasis. A relatively passive behavior of these mice has raised the possibility that they suffer from muscle weakness, which has, indeed, been confirmed by a variety of physical performance tests. METHODOLOGY/PRINCIPAL FINDINGS: A large variety of experimental methodologies, including morphometric measurements of histological preparations, high throughput proteomic analysis, positron emission tomography (PET and numerous biochemical assays, were used in an attempt to establish the mechanism underlying the relative weakness of rpS6(P-/- muscles. Collectively, these experiments have demonstrated that the physical inferiority appears to result from two defects: a a decrease in total muscle mass that reflects impaired growth, rather than aberrant differentiation of myofibers, as well as a diminished abundance of contractile proteins; and b a reduced content of ATP and phosphocreatine, two readily available energy sources. The abundance of three mitochondrial proteins has been shown to diminish in the knockin mouse. However, the apparent energy deficiency in this genotype does not result from a lower mitochondrial mass or compromised activity of enzymes of the oxidative phosphorylation, nor does it reflect a decline in insulin-dependent glucose uptake, or diminution in storage of glycogen or triacylglycerol (TG in the muscle. CONCLUSIONS/SIGNIFICANCE: This study establishes rpS6 phosphorylation as a determinant of muscle strength through its role in regulation of myofiber growth and energy content. Interestingly, a similar

  19. Predicted class-I aminoacyl tRNA synthetase-like proteins in non-ribosomal peptide synthesis

    Directory of Open Access Journals (Sweden)

    Iyer Lakshminarayan M

    2010-08-01

    Full Text Available Abstract Background Recent studies point to a great diversity of non-ribosomal peptide synthesis systems with major roles in amino acid and co-factor biosynthesis, secondary metabolism, and post-translational modifications of proteins by peptide tags. The least studied of these systems are those utilizing tRNAs or aminoacyl-tRNA synthetases (AAtRS in non-ribosomal peptide ligation. Results Here we describe novel examples of AAtRS related proteins that are likely to be involved in the synthesis of widely distributed peptide-derived metabolites. Using sensitive sequence profile methods we show that the cyclodipeptide synthases (CDPSs are members of the HUP class of Rossmannoid domains and are likely to be highly derived versions of the class-I AAtRS catalytic domains. We also identify the first eukaryotic CDPSs in fungi and in animals; they might be involved in immune response in the latter organisms. We also identify a paralogous version of the methionyl-tRNA synthetase, which is widespread in bacteria, and present evidence using contextual information that it might function independently of protein synthesis as a peptide ligase in the formation of a peptide- derived secondary metabolite. This metabolite is likely to be heavily modified through multiple reactions catalyzed by a metal-binding cupin domain and a lysine N6 monooxygenase that are strictly associated with this paralogous methionyl-tRNA synthetase (MtRS. We further identify an analogous system wherein the MtRS has been replaced by more typical peptide ligases with the ATP-grasp or modular condensation-domains. Conclusions The prevalence of these predicted biosynthetic pathways in phylogenetically distant, pathogenic or symbiotic bacteria suggests that metabolites synthesized by them might participate in interactions with the host. More generally, these findings point to a complete spectrum of recruitment of AAtRS to various non-ribosomal biosynthetic pathways, ranging from the

  20. Inactivation of indispensable bacterial proteins by early proteins of bacteriophages: implication in antibacterial drug discovery.

    Science.gov (United States)

    Sau, S; Chattoraj, P; Ganguly, T; Chanda, P K; Mandal, N C

    2008-06-01

    Bacteriophages utilize host bacterial cellular machineries for their own reproduction and completion of life cycles. The early proteins that phage synthesize immediately after the entry of their genomes into bacterial cells participate in inhibiting host macromolecular biosynthesis, initiating phage-specific replication and synthesizing late proteins. Inhibition of synthesis of host macromolecules that eventually leads to cell death is generally performed by the physical and/or chemical modification of indispensable host proteins by early proteins. Interestingly, most modified bacterial proteins were shown to take part actively in phage-specific transcription and replication. Research on phages in last nine decades has demonstrated such lethal early proteins that interact with or chemically modify indispensable host proteins. Among the host proteins inhibited by lethal phage proteins, several are not inhibited by any chemical inhibitor available today. Under the context of widespread dissemination of antibiotic-resistant strains of pathogenic bacteria in recent years, the information of lethal phage proteins and cognate host proteins could be extremely invaluable as they may lead to the identification of novel antibacterial compounds. In this review, we summarize the current knowledge about some early phage proteins, their cognate host proteins and their mechanism of action and also describe how the above interacting proteins had been exploited in antibacterial drug discovery. PMID:18537683

  1. Bacterial protein meal in diets for pigs and minks

    DEFF Research Database (Denmark)

    Hellwing, Anne Louise Frydendahl; Tauson, Anne-Helene; Skrede, Anders;

    2007-01-01

    The effect of increasing the dietary content of bacterial protein meal (BPM) on protein turnover rate, and on nucleic acid and creatinine metabolism in growing minks and pigs was investigated in two experiments. In each experiment, 16 animals were allocated to four experimental diets. The diets...... containing no BPM served as controls, i.e. for minks diet M1, for pigs P1; the experimental diets contained increasing levels of BPM to replace fish meal (minks) or soybean meal (pigs), so that up to 17% (P2), 20% (M2), 35% (P3), 40% (M3), 52% (P4), and 60% (M4) of digestible N was BPM derived. Protein...... turnover rate was measured by means of the end-product method using [15N]glycine as tracer and urinary nitrogen as end-product. In minks, protein flux, synthesis, and breakdown increased significantly with increasing dietary BPM. In pigs, diet had no observed effect on protein turnover rate. The intake...

  2. Mitochondrial Ribosomal Protein L12 Is Required for POLRMT Stability and Exists as Two Forms Generated by Alternative Proteolysis during Import.

    Science.gov (United States)

    Nouws, Jessica; Goswami, Arvind V; Bestwick, Megan; McCann, Beverly Jo; Surovtseva, Yulia V; Shadel, Gerald S

    2016-01-01

    To translate the 13 mtDNA-encoded mRNAs involved in oxidative phosphorylation (OXPHOS), mammalian mitochondria contain a dedicated set of ribosomes comprising rRNAs encoded by the mitochondrial genome and mitochondrial ribosomal proteins (MRPs) that are encoded by nuclear genes and imported into the matrix. In addition to their role in the ribosome, several MRPs have auxiliary functions or have been implicated in other cellular processes like cell cycle regulation and apoptosis. For example, we have shown that human MRPL12 binds and activates mitochondrial RNA polymerase (POLRMT), and hence has distinct functions in the ribosome and mtDNA transcription. Here we provide concrete evidence that there are two mature forms of mammalian MRPL12 that are generated by a two-step cleavage during import, involving efficient cleavage by mitochondrial processing protease and a second inefficient or regulated cleavage by mitochondrial intermediate protease. We also show that knock-down of MRPL12 by RNAi results in instability of POLRMT, but not other primary mitochondrial transcription components, and a corresponding decrease in mitochondrial transcription rates. Knock-down of MRPL10, the binding partner of MRPL12 in the ribosome, results in selective degradation of the mature long form of MRPL12, but has no effect on POLRMT. We propose that the two forms of MRPL12 are involved in homeostatic regulation of mitochondrial transcription and ribosome biogenesis that likely contribute to cell cycle, growth regulation, and longevity pathways to which MRPL12 has been linked. PMID:26586915

  3. Bacterial protein translocation requires only one copy of the SecY complex in vivo

    OpenAIRE

    Park, Eunyong; Rapoport, Tom A.

    2012-01-01

    The transport of proteins across the plasma membrane in bacteria requires a channel formed from the SecY complex, which cooperates with either a translating ribosome in cotranslational translocation or the SecA ATPase in post-translational translocation. Whether translocation requires oligomers of the SecY complex is an important but controversial issue: it determines channel size, how the permeation of small molecules is prevented, and how the channel interacts with the ribosome and SecA. He...

  4. TORC1 and TORC2 work together to regulate ribosomal protein S6 phosphorylation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Yerlikaya, Seda; Meusburger, Madeleine; Kumari, Romika; Huber, Alexandre; Anrather, Dorothea; Costanzo, Michael; Boone, Charles; Ammerer, Gustav; Baranov, Pavel V; Loewith, Robbie

    2016-01-15

    Nutrient-sensitive phosphorylation of the S6 protein of the 40S subunit of the eukaryote ribosome is highly conserved. However, despite four decades of research, the functional consequences of this modification remain unknown. Revisiting this enigma in Saccharomyces cerevisiae, we found that the regulation of Rps6 phosphorylation on Ser-232 and Ser-233 is mediated by both TOR complex 1 (TORC1) and TORC2. TORC1 regulates phosphorylation of both sites via the poorly characterized AGC-family kinase Ypk3 and the PP1 phosphatase Glc7, whereas TORC2 regulates phosphorylation of only the N-terminal phosphosite via Ypk1. Cells expressing a nonphosphorylatable variant of Rps6 display a reduced growth rate and a 40S biogenesis defect, but these phenotypes are not observed in cells in which Rps6 kinase activity is compromised. Furthermore, using polysome profiling and ribosome profiling, we failed to uncover a role of Rps6 phosphorylation in either global translation or translation of individual mRNAs. Taking the results together, this work depicts the signaling cascades orchestrating Rps6 phosphorylation in budding yeast, challenges the notion that Rps6 phosphorylation plays a role in translation, and demonstrates that observations made with Rps6 knock-ins must be interpreted cautiously. PMID:26582391

  5. Function, structure, and mechanism in bacterial photosensory LOV proteins

    OpenAIRE

    Herrou, Julien; Crosson, Sean

    2011-01-01

    LOV domains are protein photosensors conserved in bacteria, archaea, plants and fungi that detect blue light via a flavin cofactor. In the bacterial kingdom, LOV domains are present in both chemotrophic and phototrophic species, where they are found N-terminally of signaling and regulatory domains such as sensor histidine kinases, diguanylate cyclases/phosphodiesterases, DNA-binding domains, and σ factor regulators. In this review, we describe the current state of knowledge on the function of...

  6. Crystallization of the two-domain N-terminal fragment of the archaeal ribosomal protein L10(P0) in complex with a specific fragment of 23S rRNA

    International Nuclear Information System (INIS)

    Lateral L12-stalk (P1-stalk in Archaea, P1/P2-stalk in eukaryotes) is an obligatory morphological element of large ribosomal subunits in all organisms studied. This stalk is composed of the complex of ribosomal proteins L10(P0) and L12(P1) and interacts with 23S rRNA through the protein L10(P0). L12(P1)-stalk is involved in the formation of GTPase center of the ribosome and plays an important role in the ribosome interaction with translation factors. High mobility of this stalk puts obstacles in determination of its structure within the intact ribosome. Crystals of a two-domain N-terminal fragment of ribosomal protein L10(P0) from the archaeon Methanococcus jannaschii in complex with a specific fragment of rRNA from the same organism have been obtained. The crystals diffract X-rays at 3.2 Å resolution.

  7. Silencing of ribosomal protein S9 elicits a multitude of cellular responses inhibiting the growth of cancer cells subsequent to p53 activation.

    Directory of Open Access Journals (Sweden)

    Mikael S Lindström

    Full Text Available BACKGROUND: Disruption of the nucleolus often leads to activation of the p53 tumor suppressor pathway through inhibition of MDM2 that is mediated by a limited set of ribosomal proteins including RPL11 and RPL5. The effects of ribosomal protein loss in cultured mammalian cells have not been thoroughly investigated. Here we characterize the cellular stress response caused by depletion of ribosomal protein S9 (RPS9. METHODOLOGY/PRINCIPAL FINDINGS: Depletion of RPS9 impaired production of 18S ribosomal RNA and induced p53 activity. It promoted p53-dependent morphological differentiation of U343MGa Cl2:6 glioma cells as evidenced by intensified expression of glial fibrillary acidic protein and profound changes in cell shape. U2OS osteosarcoma cells displayed a limited senescence response with increased expression of DNA damage response markers, whereas HeLa cervical carcinoma cells underwent cell death by apoptosis. Knockdown of RPL11 impaired p53-dependent phenotypes in the different RPS9 depleted cell cultures. Importantly, knockdown of RPS9 or RPL11 also markedly inhibited cell proliferation through p53-independent mechanisms. RPL11 binding to MDM2 was retained despite decreased levels of RPL11 protein following nucleolar stress. In these settings, RPL11 was critical for maintaining p53 protein stability but was not strictly required for p53 protein synthesis. CONCLUSIONS: p53 plays an important role in the initial restriction of cell proliferation that occurs in response to decreased level of RPS9. Our results do not exclude the possibility that other nucleolar stress sensing molecules act upstream or in parallel to RPL11 to activate p53. Inhibiting the expression of certain ribosomal proteins, such as RPS9, could be one efficient way to reinitiate differentiation processes or to induce senescence or apoptosis in rapidly proliferating tumor cells.

  8. Structure of the GTP Form of Elongation Factor 4 (EF4) Bound to the Ribosome.

    Science.gov (United States)

    Kumar, Veerendra; Ero, Rya; Ahmed, Tofayel; Goh, Kwok Jian; Zhan, Yin; Bhushan, Shashi; Gao, Yong-Gui

    2016-06-17

    Elongation factor 4 (EF4) is a member of the family of ribosome-dependent translational GTPase factors, along with elongation factor G and BPI-inducible protein A. Although EF4 is highly conserved in bacterial, mitochondrial, and chloroplast genomes, its exact biological function remains controversial. Here we present the cryo-EM reconstitution of the GTP form of EF4 bound to the ribosome with P and E site tRNAs at 3.8-Å resolution. Interestingly, our structure reveals an unrotated ribosome rather than a clockwise-rotated ribosome, as observed in the presence of EF4-GDP and P site tRNA. In addition, we also observed a counterclockwise-rotated form of the above complex at 5.7-Å resolution. Taken together, our results shed light on the interactions formed between EF4, the ribosome, and the P site tRNA and illuminate the GTPase activation mechanism at previously unresolved detail. PMID:27137929

  9. Chemiluminescence enzyme immunoassay using ProteinA-bacterial magnetite complex

    Science.gov (United States)

    Matsunaga, Tadashi; Sato, Rika; Kamiya, Shinji; Tanaka, Tsuyosi; Takeyama, Haruko

    1999-04-01

    Bacterial magnetic particles (BMPs) which have ProteinA expressed on their surface were constructed using magA which is a key gene in BMP biosynthesis in the magnetic bacterium Magnetospirillum sp. AMB-1. Homogenous chemiluminescence enzyme immunoassay using antibody bound ProteinA-BMP complexes was developed for detection of human IgG. A good correlation between the luminescence yield and the concentration of human IgG was obtained in the range of 1-10 3 ng/ml.

  10. Cooperative assembly of proteins in the ribosomal GTPase centre demonstrated by their interactions with mutant 23S rRNAs

    DEFF Research Database (Denmark)

    Rosendahl, G; Douthwaite, S

    1995-01-01

    within a hairpin loop (1068-1072), where the protein probably makes nucleotide contacts. Some of these mutations also interfere with binding of the r-protein complex L10.(L12)4 to an adjacent site on the rRNA. When added together to rRNA, proteins L10.(L12)4 and L11 bind cooperatively to overcome the......The ribosomal protein L11 binds to the region of 23S rRNA associated with the GTPase-dependent steps of protein synthesis. Nucleotides 1054-1107 within this region of the Escherichia coli 23S rRNA gene were mutagenized with bisulphite. Twenty point mutations (G-->A and C-->T transitions) and...... numerous multiple mutations were generated. Expression of mutant 23S rRNAs in vivo shows that all the mutations detectably alter the phenotype, with effects ranging from a slight growth rate reduction to lack of viability. Temperature sensitivity is conferred by 1071G-->A and 1092C-->U substitutions. These...

  11. Single protein omission reconstitution studies of tetracycline binding to the 30S subunit of Escherichia coli ribosomes

    International Nuclear Information System (INIS)

    In previous work the authors showed that on photolysis of Escherichia coli ribosomes in the presence of [3H]tetracycline (TC) the major protein labeled is S7, and they presented strong evidence that such labeling takes place from a high-affinity site related to the inhibitory action of TC. In this work they use single protein omission reconstitution (SPORE) experiments to identify those proteins that are important for high-affinity TC binding to the 30S subunit, as measured by both cosedimentation and filter binding assays. With respect to both sedimentation coefficients and relative Phe-tRNAPhe binding, the properties of the SPORE particles they obtain parallel very closely those measured earlier, with the exception of the SPORE particle lacking S13. A total of five proteins, S3, S7, S8, S14, and S19, are shown to be important for TC binding, with the largest effects seen on omission of proteins S7 and S14. Determination of the protein compositions of the corresponding SPORE particles demonstrates that the observed effects are, for the most part, directly attributable to the omission of the given protein rather than reflecting an indirect effect of omitting one protein on the uptake of another. A large body of evidence supports the notion that four of these proteins, S3, S7, S14, and S19, are included, along with 16S rRNA bases 920-1,396, in one of the major domains of the 30S subunit. The results support the conclusion that the structure of this domain is important for the binding of TC and that, within this domain, TC binds directly to S7

  12. Ribosome recycling induces optimal translation rate at low ribosomal availability.

    Science.gov (United States)

    Marshall, E; Stansfield, I; Romano, M C

    2014-09-01

    During eukaryotic cellular protein synthesis, ribosomal translation is made more efficient through interaction between the two ends of the messenger RNA (mRNA). Ribosomes reaching the 3' end of the mRNA can thus recycle and begin translation again on the same mRNA, the so-called 'closed-loop' model. Using a driven diffusion lattice model of translation, we study the effects of ribosome recycling on the dynamics of ribosome flow and density on the mRNA. We show that ribosome recycling induces a substantial increase in ribosome current. Furthermore, for sufficiently large values of the recycling rate, the lattice does not transition directly from low to high ribosome density, as seen in lattice models without recycling. Instead, a maximal current phase becomes accessible for much lower values of the initiation rate, and multiple phase transitions occur over a wide region of the phase plane. Crucially, we show that in the presence of ribosome recycling, mRNAs can exhibit a peak in protein production at low values of the initiation rate, beyond which translation rate decreases. This has important implications for translation of certain mRNAs, suggesting that there is an optimal concentration of ribosomes at which protein synthesis is maximal, and beyond which translational efficiency is impaired. PMID:25008084

  13. Near-infrared fluorescent proteins engineered from bacterial phytochromes.

    Science.gov (United States)

    Shcherbakova, Daria M; Baloban, Mikhail; Verkhusha, Vladislav V

    2015-08-01

    Near-infrared fluorescent proteins (NIR FPs), photoactivatable NIR FPs and NIR reporters of protein-protein interactions developed from bacterial phytochrome photoreceptors (BphPs) have advanced non-invasive deep-tissue imaging. Here we provide a brief guide to the BphP-derived NIR probes with an emphasis on their in vivo applications. We describe phenotypes of NIR FPs and their photochemical and intracellular properties. We discuss NIR FP applications for imaging of various cell types, tissues and animal models in basic and translational research. In this discussion, we focus on NIR FPs that efficiently incorporate endogenous biliverdin chromophore and therefore can be used as straightforward as GFP-like proteins. We also overview a usage of NIR FPs in different imaging platforms, from planar epifluorescence to tomographic and photoacoustic technologies. PMID:26115447

  14. Prokaryote phylogeny based on ribosomal proteins and aminoacyl tRNA synthetases by using the compositional distance approach

    Institute of Scientific and Technical Information of China (English)

    WEI; Haibin; QI; Ji; HAO; Bailin

    2004-01-01

    In order to show that the newly developed K-string composition distance method,based on counting oligopeptide frequencies,for inferring phylogenetic relations of prokaryotes works equally well without requiring the whole proteome data,we used all ribosomal proteins and the set of aminoacyl tRNA synthetases for each species.The latter group has been known to yield inconsistent trees if used individually.Our trees are obtained without making any sequence alignment.Altogether 16 Archaea,105 Bacteria and 2 Eucarya are represented on the tree.Most of the lower branchings agree well with the latest,2003,Outline of the second edition of the Bergey's Manual of Systematic Bacteriology and the trees also suggest some relationships among higher taxa.

  15. Structural insights into ribosome translocation.

    Science.gov (United States)

    Ling, Clarence; Ermolenko, Dmitri N

    2016-09-01

    During protein synthesis, tRNA and mRNA are translocated from the A to P to E sites of the ribosome thus enabling the ribosome to translate one codon of mRNA after the other. Ribosome translocation along mRNA is induced by the universally conserved ribosome GTPase, elongation factor G (EF-G) in bacteria and elongation factor 2 (EF-2) in eukaryotes. Recent structural and single-molecule studies revealed that tRNA and mRNA translocation within the ribosome is accompanied by cyclic forward and reverse rotations between the large and small ribosomal subunits parallel to the plane of the intersubunit interface. In addition, during ribosome translocation, the 'head' domain of small ribosomal subunit undergoes forward- and back-swiveling motions relative to the rest of the small ribosomal subunit around the axis that is orthogonal to the axis of intersubunit rotation. tRNA/mRNA translocation is also coupled to the docking of domain IV of EF-G into the A site of the small ribosomal subunit that converts the thermally driven motions of the ribosome and tRNA into the forward translocation of tRNA/mRNA inside the ribosome. Despite recent and enormous progress made in the understanding of the molecular mechanism of ribosome translocation, the sequence of structural rearrangements of the ribosome, EF-G and tRNA during translocation is still not fully established and awaits further investigation. WIREs RNA 2016, 7:620-636. doi: 10.1002/wrna.1354 For further resources related to this article, please visit the WIREs website. PMID:27117863

  16. Involvement of the N Terminus of Ribosomal Protein L11 in Regulation of the RelA Protein of Escherichia coli†

    OpenAIRE

    Yang, Xiaoming; Ishiguro, Edward E.

    2001-01-01

    Amino acid-deprived rplK (previously known as relC) mutants of Escherichia coli cannot activate (p)ppGpp synthetase I (RelA) and consequently exhibit relaxed phenotypes. The rplK gene encodes ribosomal protein L11, suggesting that L11 is involved in regulating the activity of RelA. To investigate the role of L11 in the stringent response, a derivative of rplK encoding L11 lacking the N-terminal 36 amino acids (designated ′L11) was constructed. Bacteria overexpressing ′L11 exhibited a relaxed ...

  17. Chaperoning ribosome assembly

    OpenAIRE

    Karbstein, Katrin

    2010-01-01

    Chaperones help proteins fold in all cellular compartments, and many associate directly with ribosomes, capturing nascent chains to assist their folding and prevent aggregation. In this issue, new data from Koplin et al. (2010. J. Cell Biol. doi: 10.1083/jcb.200910074) and Albanèse et al. (2010. J. Cell Biol. doi: 10.1083/jcb.201001054) suggest that in addition to promoting protein folding, the chaperones ribosome-associated complex (RAC), nascent chain–associated complex (NAC), and Jjj1 also...

  18. The Structure of Aquifex aeolicus Ribosomal Protein S8 Reveals a Unique Subdomain That Contributes to Extremely-Tight Association With 16S rRNA

    OpenAIRE

    Menichelli, Elena; Edgcomb, Stephen P.; Recht, Michael I.; Williamson, James R.

    2011-01-01

    The assembly of ribonucleoprotein complexes occurs in a broad range of conditions, but the principles that promote assembly and allow function at high temperature are poorly understood. The ribosomal protein S8 from the hyperthemophilic bacterium Aquifex aeolicus (AS8) is unique in that there is a 41 residue insertion in the consensus S8 sequence. In addition, AS8 exhibits an unusually-high affinity for the 16S ribosomal RNA (rRNA), characterized by a picomolar dissociation constant that is a...

  19. Quinupristin-Dalfopristin Resistance in Streptococcus pneumoniae: Novel L22 Ribosomal Protein Mutation in Two Clinical Isolates from the SENTRY Antimicrobial Surveillance Program

    OpenAIRE

    Ronald N Jones; Farrell, David J.; Morrissey, Ian

    2003-01-01

    Resistance to quinupristin-dalfopristin (Q/D) among gram-positive cocci has been very uncommon. Two clinical isolates among 8,837 (0.02%) Streptococcus pneumoniae isolates were discovered in 2001 to 2002 with Q/D MICs of 4 μg/ml. Each had a 5-amino-acid tandem duplication (RTAHI) in the L22 ribosomal protein gene (rplV) preventing synergistic ribosomal binding of the streptogramin combination. Similar gene duplication has been reported in Q/D-resistant Staphylococcus aureus.

  20. Atomic resolution structure of cucurmosin, a novel type 1 ribosome-inactivating protein from the sarcocarp of Cucurbita moschata

    Energy Technology Data Exchange (ETDEWEB)

    Hou, Xiaomin; Meehan, Edward J.; Xie, Jieming; Huang, Mingdong; Chen, Minghuang; Chen, Liqing (UAH); (Fujian); (Chinese Aca. Sci.)

    2008-10-27

    A novel type 1 ribosome-inactivating protein (RIP) designated cucurmosin was isolated from the sarcocarp of Cucurbita moschata (pumpkin). Besides rRNA N-glycosidase activity, cucurmosin exhibits strong cytotoxicities to three cancer cell lines of both human and murine origins, but low toxicity to normal cells. Plant genomic DNA extracted from the tender leaves was amplified by PCR between primers based on the N-terminal sequence and X-ray sequence of the C-terminal. The complete mature protein sequence was obtained from N-terminal protein sequencing and partial DNA sequencing, confirmed by high resolution crystal structure analysis. The crystal structure of cucurmosin has been determined at 1.04 {angstrom}, a resolution that has never been achieved before for any RIP. The structure contains two domains: a large N-terminal domain composed of seven {alpha}-helices and eight {beta}-strands, and a smaller C-terminal domain consisting of three {alpha}-helices and two {beta}-strands. The high resolution structure established a glycosylation pattern of GlcNAc{sub 2}Man3Xyl. Asn225 was identified as a glycosylation site. Residues Tyr70, Tyr109, Glu158 and Arg161 define the active site of cucurmosin as an RNA N-glycosidase. The structural basis of cytotoxicity difference between cucurmosin and trichosanthin is discussed.

  1. BDP-30, a systemic resistance inducer from Boerhaavia diffusa L., suppresses TMV infection, and displays homology with ribosome- inactivating proteins

    Indian Academy of Sciences (India)

    Shalini Srivastava; H N Verma; Aparana Srivastava; Vivek Prasad

    2015-03-01

    Root extract of Boerhaavia diffusa L. induced systemic resistance in tobacco against Tobacco mosaic virus. A 30 kDa protein was isolated as the active component, called BDP-30 on the basis of the molecular weight and source plant. BDP-30, a glycoprotein, was found to be temperature and protease resistant. It was basic, possessing a pI greater than 9.0. In-gel proteolytic digestion of BDP-30 generated two peptides that possessed the amino acid sequence KLYDIPPLR and KVTLPYSGNYER by LC/MS/MS. Both peptides shared absolute sequence identity with trichosanthin, a ribosome-inactivating protein from Trichosanthes kirilowii, and a 78% and 100% homology respectively with an RIP from Bryonia dioica, bryodin. Further, effort was made to look at the fate of TMV in induced resistant Nicotiana tabacum cv. Xanthi, a systemic host of the virus, at specified days after inoculation in control and treated plants. TMV coat protein (CP) was detected by immunoblot 7 days post inoculation up to 21 days in the control set, but not in treated resistant plants. TMV RNA was detected by RT-PCR using TMV-CP specific primers. Resistant tobacco did not show presence of TMV RNA up to 21 days of inoculation. This suggests that BDP-30 may be suppressing TMV replication.

  2. Role of horizontal gene transfer as a control on the coevolution of ribosomal proteins and the genetic code

    Energy Technology Data Exchange (ETDEWEB)

    Woese, Carl R.; Goldenfeld, Nigel; Luthey-Schulten, Zaida

    2011-03-31

    Our main goal is to develop the conceptual and computational tools necessary to understand the evolution of the universal processes of translation and replication and to identify events of horizontal gene transfer that occurred within the components. We will attempt to uncover the major evolutionary transitions that accompanied the development of protein synthesis by the ribosome and associated components of the translation apparatus. Our project goes beyond standard genomic approaches to explore homologs that are represented at both the structure and sequence level. Accordingly, use of structural phylogenetic analysis allows us to probe further back into deep evolutionary time than competing approaches, permitting greater resolution of primitive folds and structures. Specifically, our work focuses on the elements of translation, ranging from the emergence of the canonical genetic code to the evolution of specific protein folds, mediated by the predominance of horizontal gene transfer in early life. A unique element of this study is the explicit accounting for the impact of phenotype selection on translation, through a coevolutionary control mechanism. Our work contributes to DOE mission objectives through: (1) sophisticated computer simulation of protein dynamics and evolution, and the further refinement of techniques for structural phylogeny, which complement sequence information, leading to improved annotation of genomic databases; (2) development of evolutionary approaches to exploring cellular function and machinery in an integrated way; and (3) documentation of the phenotype interaction with translation over evolutionary time, reflecting the system response to changing selection pressures through horizontal gene transfer.

  3. 20-Hydroxyecdysone stimulates nuclear accumulation of BmNep1, a nuclear ribosome biogenesis-related protein in the silkworm, Bombyx mori.

    Science.gov (United States)

    Ji, M-M; Liu, A-Q; Sima, Y-H; Xu, S-Q

    2016-10-01

    The pathway of communication between endocrine hormones and ribosome biogenesis critical for physiological adaptation is largely unknown. Nucleolar essential protein 1 (Nep1) is an essential gene for ribosome biogenesis and is functionally conserved in many in vertebrate and invertebrate species. In this study, we cloned Bombyx mori Nep1 (BmNep1) due to its high expression in silk glands of silkworms on day 3 of the fifth instar. We found that BmNep1 mRNA and protein levels were upregulated in silk glands during fourth-instar ecdysis and larval-pupal metamorphosis. By immunoprecipitation with the anti-BmNep1 antibody and liquid chromatography-tandem mass spectrometry analyses, it was shown that BmNep1 probably interacts with proteins related to ribosome structure formation. Immunohistochemistry, biochemical fractionation and immunocytochemistry revealed that BmNep1 is localized to the nuclei in Bombyx cells. Using BmN cells originally derived from ovaries, we demonstrated that 20-hydroxyecdysone (20E) induced BmNep1 expression and stimulated nuclear accumulation of BmNep1. Under physiological conditions, BmNep1 was also upregulated in ovaries during larval-pupal metamorphosis. Overall, our results indicate that the endocrine hormone 20E facilitates nuclear accumulation of BmNep1, which is involved in nuclear ribosome biogenesis in Bombyx. PMID:27329527

  4. A Novel Method for Simultaneous Production of Two Ribosome-Inactivating Proteins, α-MMC and MAP30, from Momordica charantia L

    OpenAIRE

    Yao Meng; Sen Lin; Shuangfeng Liu; Xiang Fan; Gangrui Li; Yanfa Meng

    2014-01-01

    Alpha-momorcharin (α-MMC) and momordica anti-HIV protein (MAP30) from Momordica charantia L. have been confirmed to possess anti-tumor and anti-virus activities. Traditional purification methods of these two ribosome-inactivating proteins (RIPs) were separate which was time consuming and cost effective as well as low efficient. In order to obtain sufficient samples for researches, a strategy combining ion-exchange and gel filtration chromatography was developed and optimized in this study. Us...

  5. Phosphorylation of ribosomal protein S6 in avian sarcoma virus-transformed chicken embryo fibroblasts.

    OpenAIRE

    Decker, S.

    1981-01-01

    Protein phosphorylation was examined in whole cell extracts from normal and avian sarcoma virus-transformed chicken embryo fibroblasts. The addition of serum or epidermal growth factor to serum-starved normal cells resulted in increased 32P labeling of a Mr 30,000 protein. In extracts from cells transformed by a temperature-sensitive mutant of Schmidt-Ruppin virus, subgroup A, and grown at the permissive temperature, the protein was phosphorylated regardless of serum starvation. This Mr 30,00...

  6. Double subgenomic alphaviruses expressing multiple fluorescent proteins using a Rhopalosiphum padi virus internal ribosome entry site element.

    Directory of Open Access Journals (Sweden)

    Michael R Wiley

    Full Text Available Double subgenomic Sindbis virus (dsSINV vectors are widely used for the expression of proteins, peptides, and RNA sequences. These recombinant RNA viruses permit high level expression of a heterologous sequence in a wide range of animals, tissues, and cells. However, the alphavirus genome structure and replication strategy is not readily amenable to the expression of more than one heterologous sequence. The Rhopalosiphum padi virus (RhPV genome contains two internal ribosome entry site (IRES elements that mediate cap-independent translation of the virus nonstructural and structural proteins. Most IRES elements that have been characterized function only in mammalian cells but previous work has shown that the IRES element present in the 5' untranslated region (UTR of the RhPV genome functions efficiently in mammalian, insect, and plant systems. To determine if the 5' RhPV IRES element could be used to express more than one heterologous sequence from a dsSINV vector, RhPV 5' IRES sequences were placed between genes for two different fluorescent marker proteins in the dsSINV, TE/3'2J/mcs. While mammalian and insect cells infected with recombinant viruses containing the RhPV sequences expressed both fluorescent marker proteins, only single marker proteins were routinely observed in cells infected with dsSINV vectors in which the RhPV IRES had been replaced by a luciferase fragment, an antisense RhPV IRES, or no intergenic sequence. Thus, we report development of a versatile tool for the expression of multiple sequences in diverse cell types.

  7. Adjustable under-expression of yeast mating pathway proteins in Saccharomyces cerevisiae using a programmed ribosomal frameshift.

    Science.gov (United States)

    Choi, Min-Yeon; Park, Sang-Hyun

    2016-06-01

    Experimental research in molecular biology frequently relies on the promotion or suppression of gene expression, an important tool in the study of its functions. Although yeast is among the most studied model systems with the ease of maintenance and manipulation, current experimental methods are mostly limited to gene deletion, suppression or overexpression of genes. Therefore, the ability to reduce protein expressions and then observing the effects would promote a better understanding of the exact functions and their interactions. Reducing protein expression is mainly limited by the difficulties associated with controlling the reduction level, and in some cases, the initial endogenous abundance is too low. For the under-expression to be useful as an experimental tool, repeatability and stability of reduced expression is important. We found that cis-elements in programmed -1 ribosomal frameshifting (-1RFS) of beet western yellow virus (BWYV) could be utilized to reduced protein expression in Saccharomyces cerevisiae. The two main advantages of using -1RFS are adjustable reduction rates and ease of use. To demonstrate the utility of this under-expression system, examples of reduced protein abundance were shown using yeast mating pathway components. The abundance of MAP kinase Fus3 was reduced to approximately 28-75 % of the wild-type value. Other MAP kinase mating pathway components, including Ste5, Ste11, and Ste7, were also under-expressed to verify that the -1RFS system works with different proteins. Furthermore, reduced Fus3 abundance altered the overall signal transduction outcome of the mating pathway, demonstrating the potential for further studies of signal transduction adjustment via under-expression. PMID:26837218

  8. Invasion of protein coding genes by green algal ribosomal group I introns.

    Science.gov (United States)

    McManus, Hilary A; Lewis, Louise A; Fučíková, Karolina; Haugen, Peik

    2012-01-01

    The spread of group I introns depends on their association with intron-encoded homing endonucleases. Introns that encode functional homing endonuclease genes (HEGs) are highly invasive, whereas introns that only encode the group I ribozyme responsible for self-splicing are generally stably inherited (i.e., vertical inheritance). A number of recent case studies have provided new knowledge on the evolution of group I introns, however, there are still large gaps in understanding of their distribution on the tree of life, and how they have spread into new hosts and genic sites. During a larger phylogenetic survey of chlorophyceaen green algae, we found that 23 isolates contain at least one group I intron in the rbcL chloroplast gene. Structural analyses show that the introns belong to one of two intron lineages, group IA2 intron-HEG (GIY-YIG family) elements inserted after position 462 in the rbcL gene, and group IA1 introns inserted after position 699. The latter intron type sometimes encodes HNH homing endonucleases. The distribution of introns was analyzed on an exon phylogeny and patterns were recovered that are consistent with vertical inheritance and possible horizontal transfer. The rbcL 462 introns are thus far reported only within the Volvocales, Hydrodictyaceae and Bracteacoccus, and closely related isolates of algae differ in the presence of rbcL introns. Phylogenetic analysis of the intron conserved regions indicates that the rbcL699 and rbcL462 introns have distinct evolutionary origins. The rbcL699 introns were likely derived from ribosomal RNA L2449 introns, whereas the rbcL462 introns form a close relationship with psbA introns. PMID:22056605

  9. Sepsis and development impede muscle protein synthesis in neonatal pigs by different ribosomal mechanisms

    Science.gov (United States)

    In muscle, sepsis reduces protein synthesis (MPS) by restraining translation in neonates and adults. Even though protein accretion decreases with development as neonatal MPS rapidly declines by maturation, the changes imposed by development on the sepsis-associated decrease in MPS have not been desc...

  10. Purification and properties of a new ribosome-inactivating protein with RNA N-glycosidase activity suitable for immunotoxin preparation from the seeds of Momordica cochinchinensis.

    Science.gov (United States)

    Bolognesi, A; Barbieri, L; Carnicelli, D; Abbondanza, A; Cenini, P; Falasca, A I; Dinota, A; Stirpe, F

    1989-12-01

    A ribosome-inactivating protein similar to those already known (Stirpe and Barbieri (1986) FEBS Lett. 195, 1-8) was purified from the seeds of Momordica cochinchinensis. This protein, for which the name of momorcochin-S is proposed, is a glycoprotein, has an Mr of approx. 30,000, and an alkaline isoelectric point and can be considered as an iso-form of the previously purified momorcochin from the roots of M. cochinchinensis. Momorcochin-S inhibits protein synthesis by a rabbit-reticulocyte lysate and phenylalanine polymerization by isolated ribosomes, and alters rRNA in a similar manner as the A-chain of ricin and related toxins (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). Momorcochin-S was linked to a monoclonal antibody (8A) against human plasma cells, and the resulting immunotoxin was selectively toxic to target cells. PMID:2597699

  11. Interaction of ribosomal protein L22 with casein kinase 2α: a novel mechanism for understanding the biology of non-small cell lung cancer.

    Science.gov (United States)

    Yang, Mingxia; Sun, Haibo; He, Ji; Wang, Hong; Yu, Xiaowei; Ma, Lei; Zhu, Changliang

    2014-07-01

    Dysfunction of ribosomal proteins (RPs) may play an important role in molecular tumorigenesis, such as lung cancer, acting in extraribosomal functions. Many protein-protein interaction studies and genetic screens have confirmed the extraribosomal capacity of RPs. As reported, ribosomal protein L22 (RPL22) dysfunction could increase cancer risk. In the present study, we examined RPL22-protein complexes in lung cancer cells. Tandem affinity purification (TAP) was used to screen the RPL22-protein complexes, and GST pull-down experiments and confocal microscopy were used to assess the protein-protein interaction. The experiment of kinase assay was used to study the function of the RPL22-protein complexes. The results showed that several differentially expressed proteins were isolated and identified by LC-MS/MS, which revealed that one of the protein complexes included casein kinase 2α (CK2α). RPL22 and CK2α interact in vitro. RPL22 also inhibited CK2α substrate phosphorylation in vitro. This is the first report of the RPL22-CK2α relationship in lung cancer. Dysregulated CK2 may impact cell proliferation and apoptosis, key features of cancer cell biology. Our results indicate that RPL22 may be a candidate anticancer agent due to its CK2α-binding and -inhibitory functions in human lung cancer. PMID:24840952

  12. p53 and the ribosomal protein L5 participate in high molecular mass complex formation with protein kinase CK2 in murine teratocarcinoma cell line F9 after serum stimulation and cisplatin treatment

    DEFF Research Database (Denmark)

    Guerra, B; Issinger, O G

    evidence by immunoprecipitation for an association of protein kinase CK2 holoenzyme (alpha2beta2), p53, and the ribosomal protein L5. The results suggest complexes between the CK2 holoenzyme and p53 but also p53/CKbeta complexes. Furthermore we provide evidence for the existence of high molecular mass...

  13. Bacterial protein meal in diets for growing pigs

    DEFF Research Database (Denmark)

    Hellwing, Anne Louise Frydendahl; Tauson, Anne-Helene; Kjos, N.P.;

    2007-01-01

    This experiment investigated the effects of increasing the dietary content of bacterial protein meal (BPM) on the protein and energy metabolism of pigs from weaning to a live weight of 80 kg. FOur litters with four castrated male pigs in each litter were used. The litters were divided into two...... blocks according to age. One pig from each litter was fed one of the four experimental diets. Soya-bean meal was replaced with BPM on the basis of digestible protein, and the BPM contents in the four diets were 0% (BP0), 5% (BP5), 10% (BP10) and 15% (BP15), corresponding to 0%, 17%, 35% and 52...... by inclusion level of BPM. Retention of energy was 620 (BP0), 696 (BP5), 613 (BP10) and 664 kJ/kg0.75 per day (BP15), the differences among diets being non-significant. The N-free respiratory quotient was similar on all diets. It was concluded that the overall protein and energy metabolism in growing pigs were...

  14. Holo- And Apo- Structures of Bacterial Periplasmic Heme Binding Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ho, W.W.; Li, H.; Eakanunkul, S.; Tong, Y.; Wilks, A.; Guo, M.; Poulos, T.L.

    2009-06-01

    An essential component of heme transport in Gram-negative bacterial pathogens is the periplasmic protein that shuttles heme between outer and inner membranes. We have solved the first crystal structures of two such proteins, ShuT from Shigella dysenteriae and PhuT from Pseudomonas aeruginosa. Both share a common architecture typical of Class III periplasmic binding proteins. The heme binds in a narrow cleft between the N- and C-terminal binding domains and is coordinated by a Tyr residue. A comparison of the heme-free (apo) and -bound (holo) structures indicates little change in structure other than minor alterations in the heme pocket and movement of the Tyr heme ligand from an 'in' position where it can coordinate the heme iron to an 'out' orientation where it points away from the heme pocket. The detailed architecture of the heme pocket is quite different in ShuT and PhuT. Although Arg{sup 228} in PhuT H-bonds with a heme propionate, in ShuT a peptide loop partially takes up the space occupied by Arg{sup 228}, and there is no Lys or Arg H-bonding with the heme propionates. A comparison of PhuT/ShuT with the vitamin B{sub 12}-binding protein BtuF and the hydroxamic-type siderophore-binding protein FhuD, the only two other structurally characterized Class III periplasmic binding proteins, demonstrates that PhuT/ShuT more closely resembles BtuF, which reflects the closer similarity in ligands, heme and B{sub 12}, compared with ligands for FhuD, a peptide siderophore.

  15. Ribosomal Protein Genes S23 and L35 from Amphioxus Branchiostoma belcheri tsingtauense: Identification and Copy Number

    Institute of Scientific and Technical Information of China (English)

    Xian LI; Shi-Cui ZHANG; Zhen-Hui LIU; Hong-Yan LI

    2005-01-01

    The complete cDNA and deduced amino acid sequences of the ribosomal proteins S23 (AmphiS23) and L35 (AmphiL35) from amphioxus Branchiostoma belcheri tsingtauense were identified in this study. AmphiS23 cDNA is 546 bp long and encodes a protein of 143 amino acids. It has a predicted molecular mass of 15,851 Da and a pI of 10.7. AmphiL35 cDNA comprises 473 bp, and codes for a protein of 123 amino acids with a predicted molecular mass of 14,543 Da and a pI of 10.8. AmphiS23 shares more than 83% identity with its homologues in the vertebrates and more than 84% identity with those in the invertebrates. AmphiL35 is more than 63% identical to its counterparts in the vertebrates and more than 52% identical to those in the invertebrates, Southern blot analysis demonstrated the existence of 1-2 copies of the S23 gene and 2-3 copies of the L35 gene in the genome of amphioxus B. belcheri tsingtauense. This is in sharp contrast to the presence of 6-13 copies of the S23 gene and 15-17 copies of the L35 gene in the rat genome. It is clear that the housekeeping genes like S23 and L35 underwent a large-scale duplication in the vertebrate lineage, reinforcing the gene/genome duplication hypothesis.

  16. Proteomic and biochemical analyses reveal the activation of unfolded protein response, ERK-1/2 and ribosomal protein S6 signaling in experimental autoimmune myocarditis rat model

    Directory of Open Access Journals (Sweden)

    Kim Chan

    2011-10-01

    Full Text Available Abstract Background To investigate the molecular and cellular pathogenesis underlying myocarditis, we used an experimental autoimmune myocarditis (EAM-induced heart failure rat model that represents T cell mediated postinflammatory heart disorders. Results By performing unbiased 2-dimensional electrophoresis of protein extracts from control rat heart tissues and EAM rat heart tissues, followed by nano-HPLC-ESI-QIT-MS, 67 proteins were identified from 71 spots that exhibited significantly altered expression levels. The majority of up-regulated proteins were confidently associated with unfolded protein responses (UPR, while the majority of down-regulated proteins were involved with the generation of precursor metabolites and energy metabolism in mitochondria. Although there was no difference in AKT signaling between EAM rat heart tissues and control rat heart tissues, the amounts and activities of extracellular signal-regulated kinase (ERK-1/2 and ribosomal protein S6 (rpS6 were significantly increased. By comparing our data with the previously reported myocardial proteome of the Coxsackie viruses of group B (CVB-mediated myocarditis model, we found that UPR-related proteins were commonly up-regulated in two murine myocarditis models. Even though only two out of 29 down-regulated proteins in EAM rat heart tissues were also dysregulated in CVB-infected rat heart tissues, other proteins known to be involved with the generation of precursor metabolites and energy metabolism in mitochondria were also dysregulated in CVB-mediated myocarditis rat heart tissues, suggesting that impairment of mitochondrial functions may be a common underlying mechanism of the two murine myocarditis models. Conclusions UPR, ERK-1/2 and S6RP signaling were activated in both EAM- and CVB-induced myocarditis murine models. Thus, the conserved components of signaling pathways in two murine models of acute myocarditis could be targets for developing new therapeutic drugs or

  17. Antifungal activity of the ribosome-inactivating protein BE27 from sugar beet (Beta vulgaris L.) against the green mould Penicillium digitatum.

    Science.gov (United States)

    Citores, Lucía; Iglesias, Rosario; Gay, Carolina; Ferreras, José Miguel

    2016-02-01

    The ribosome-inactivating protein BE27 from sugar beet (Beta vulgaris L.) leaves is an apoplastic protein induced by signalling compounds, such as hydrogen peroxide and salicylic acid, which has been reported to be involved in defence against viruses. Here, we report that, at a concentration much lower than that present in the apoplast, BE27 displays antifungal activity against the green mould Penicillium digitatum, a necrotrophic fungus that colonizes wounds and grows in the inter- and intracellular spaces of the tissues of several edible plants. BE27 is able to enter into the cytosol and kill fungal cells, thus arresting the growth of the fungus. The mechanism of action seems to involve ribosomal RNA (rRNA) N-glycosylase activity on the sarcin-ricin loop of the major rRNA which inactivates irreversibly the fungal ribosomes, thus inhibiting protein synthesis. We compared the C-terminus of the BE27 structure with antifungal plant defensins and hypothesize that a structural motif composed of an α-helix and a β-hairpin, similar to the γ-core motif of defensins, might contribute to the specific interaction with the fungal plasma membranes, allowing the protein to enter into the cell. PMID:25976013

  18. Bacterial Hydrolysis of Protein and Methylated Protein and Its Implications for Studies of Protein Degradation in Aquatic Systems

    OpenAIRE

    Keil, Richard G.; Kirchman, David L.

    1992-01-01

    Ribulose 1,5-bisphosphate carboxylase was radiolabelled by in vitro translation, resulting in uniformly labelled ribulose 1,5-bisphosphate carboxylase, and also by reductive methylation. We investigated the degradation of the two forms of radiolabelled protein by natural bacterial populations. Although total hydrolysis of uniformly labelled protein and methylated protein was nearly equal, percent assimilation, respiration, and release as low-molecular-weight material were different. Radioacti...

  19. Ribosomal protein s6-ps240 is expressed in lesional skin from patients with autoimmune skin blistering diseases

    Directory of Open Access Journals (Sweden)

    Ana Maria Abreu Velez

    2013-01-01

    Full Text Available Background: The in situ signaling transduction within skin biopsies from patients affected by autoimmune skin blistering diseases is not well-characterized. Aim : In autoimmune skin blistering diseases, autoantibodies seem to trigger several intracellular signaling pathways and we investigated the presence of the phosphorylated form of ribosomal protein S6-pS240 within autoimmune skin blistering diseases biopsies. Materials and Methods: We utilized immunohistochemistry to evaluate the presence of S6-pS240 in lesional skin biopsies of patients affected by autoimmune skin blistering diseases including patients with an endemic and nonendemic pemphigus foliaceus (non EPF, with bullous pemphigoid (BP, pemphigus vulgaris (PV, dermatitis herpetiformis (DH, and the respective controls. Results: Most autoimmune bullous skin diseases biopsies stained positive for S6-pS240 around lesional blisters, including adjacent areas of the epidermis; and within upper dermal inflammatory infiltrates, and/or mesenchymal-endothelial cell junctions within the dermis. Conclusions: We document that S6-pS240 is expressed in lesional areas of skin biopsies from patients with autoimmune skin blistering diseases, as well as on eccrine glands and piloerector muscles. Thus, the role of this molecule in autoimmune skin blistering diseases warrants further study.

  20. Preparation and Primary Application of Monoclonal Antibodies against a Novel Ribosome-inactivating Protein Moschatin from Pumpkin Seeds

    Institute of Scientific and Technical Information of China (English)

    Heng-ChuanXIA; Wei-GuoHU; Xin-XiuYANG; FengLI; Zu-ChuanZHANG

    2004-01-01

    Plant ribosome-inactivating proteins (RIPs) have multiple biological functions, and have beenwidely used in the studies on biomedical and agronomic applications. Moschatin is a novel single-chain RIPrecently purified from pumpkin seeds, and it has been successfully applied to construct the immunotoxin thatcan selectively kill the cultured human melanoma cells. Six stable strains of hybridomas (2H8, 4A8, 5B6,6F8, 4H10 and 6C2) that can secrete high specific monoclonal antibodies against Moschatin have beensuccessfully prepared using hybridoma technique. The isotypes of these monoclonal antibodies areIgG1, IgG1, IgG1, IgG1, IgG2a and IgM. Their affinity constants were determined to be 1.42×108, 2.71×108,8.72×07, 2.06×l08, 1.36×108 and 1.51×108 M-1 in a sequent order, measured by non-competitive ELISA.The monoclonal antibody 4A8 has been used to detect Moschatin in Western blot. An immunoaffinity gel,which consisted ofa monoclonal antibody 4H 10 and Sepharose 4B, was prepared and used to purify Moschatinfrom pumpkin seeds crude extract.

  1. Preparation and Primary Application of Monoclonal Antibodies against a Novel Ribosome-inactivating Protein Moschatin from Pumpkin Seeds

    Institute of Scientific and Technical Information of China (English)

    Heng-Chuan XIA; Wei-Guo HU; Xin-Xiu YANG; Feng LI; Zu-Chuan ZHANG

    2004-01-01

    Plant ribosome-inactivating proteins (RIPs) have multiple biological functions, and have beenwidely used in the studies on biomedical and agronomic applications. Moschatin is a novel single-chain RIPrecently purified from pumpkin seeds, and it has been successfully applied to construct the immunotoxin thatcan selectively kill the cultured human melanoma cells. Six stable strains of hybridomas (2H8, 4A8, 5B6,6F8, 4H 10 and 6C2) that can secrete high specific monoclonal antibodies against Moschatin have beensuccessfully prepared using hybridoma technique. The isotypes of these monoclonal antibodies areIgG1, IgG1, IgG1, IgG1, IgG2a and IgM. Their affinity constants were determined to be 1.42×108, 2.71×108,8.72×107, 2.06×108, 1.36×108 and 1.51×108M-1 in a sequent order, measured by non-competitive ELISA.The monoclonal antibody 4A8 has been used to detect Moschatin in Western blot. An immunoaffinity gel,which consisted ofa monoclonal antibody 4H10 and Sepharose 4B, was prepared and used to purify Moschatinfrom pumpkin seeds crude extract.

  2. An ancient spliceosomal intron in the ribosomal protein L7a gene (Rpl7a of Giardia lamblia

    Directory of Open Access Journals (Sweden)

    Gray Michael W

    2005-08-01

    Full Text Available Abstract Background Only one spliceosomal-type intron has previously been identified in the unicellular eukaryotic parasite, Giardia lamblia (a diplomonad. This intron is only 35 nucleotides in length and is unusual in possessing a non-canonical 5' intron boundary sequence, CT, instead of GT. Results We have identified a second spliceosomal-type intron in G. lamblia, in the ribosomal protein L7a gene (Rpl7a, that possesses a canonical GT 5' intron boundary sequence. A comparison of the two known Giardia intron sequences revealed extensive nucleotide identity at both the 5' and 3' intron boundaries, similar to the conserved sequence motifs recently identified at the boundaries of spliceosomal-type introns in Trichomonas vaginalis (a parabasalid. Based on these observations, we searched the partial G. lamblia genome sequence for these conserved features and identified a third spliceosomal intron, in an unassigned open reading frame. Our comprehensive analysis of the Rpl7a intron in other eukaryotic taxa demonstrates that it is evolutionarily conserved and is an ancient eukaryotic intron. Conclusion An analysis of the phylogenetic distribution and properties of the Rpl7a intron suggests its utility as a phylogenetic marker to evaluate particular eukaryotic groupings. Additionally, analysis of the G. lamblia introns has provided further insight into some of the conserved and unique features possessed by the recently identified spliceosomal introns in related organisms such as T. vaginalis and Carpediemonas membranifera.

  3. Influence of intron length on interaction characters between post-spliced intron and its CDS in ribosomal protein genes

    Science.gov (United States)

    Zhao, Xiaoqing; Li, Hong; Bao, Tonglaga; Ying, Zhiqiang

    2012-09-01

    Many experiment evidences showed that sequence structures of introns and intron loss/gain can influence gene expression, but current mechanisms did not refer to the functions of post-spliced introns directly. We propose that postspliced introns play their functions in gene expression by interacting with their mRNA sequences and the interaction is characterized by the matched segments between introns and their CDS. In this study, we investigated the interaction characters with length series by improved Smith-Waterman local alignment software for the ribosomal protein genes in C. elegans and D. melanogaster. Our results showed that RF values of five intron groups are significantly high in the central non-conserved region and very low in 5'-end and 3'-end splicing region. It is interesting that the number of the optimal matched regions gradually increases with intron length. Distributions of the optimal matched regions are different for five intron groups. Our study revealed that there are more interaction regions between longer introns and their CDS than shorter, and it provides a positive pattern for regulating the gene expression.

  4. The Listeria monocytogenes hibernation-promoting factor is required for the formation of 100S ribosomes, optimal fitness, and pathogenesis.

    Science.gov (United States)

    Kline, Benjamin C; McKay, Susannah L; Tang, William W; Portnoy, Daniel A

    2015-02-01

    During exposure to certain stresses, bacteria dimerize pairs of 70S ribosomes into translationally silent 100S particles in a process called ribosome hibernation. Although the biological roles of ribosome hibernation are not completely understood, this process appears to represent a conserved and adaptive response that contributes to optimal survival during stress and post-exponential-phase growth. Hibernating ribosomes are formed by the activity of one or more highly conserved proteins; gammaproteobacteria produce two relevant proteins, ribosome modulation factor (RMF) and hibernation promoting factor (HPF), while most Gram-positive bacteria produce a single, longer HPF protein. Here, we report the formation of 100S ribosomes by an HPF homolog in Listeria monocytogenes. L. monocytogenes 100S ribosomes were observed by sucrose density gradient centrifugation of bacterial extracts during mid-logarithmic phase, peaked at the transition to stationary phase, and persisted at lower levels during post-exponential-phase growth. 100S ribosomes were undetectable in bacteria carrying an hpf::Himar1 transposon insertion, indicating that HPF is required for ribosome hibernation in L. monocytogenes. Additionally, epitope-tagged HPF cosedimented with 100S ribosomes, supporting its previously described direct role in 100S formation. We examined hpf mRNA by quantitative PCR (qPCR) and identified several conditions that upregulated its expression, including carbon starvation, heat shock, and exposure to high concentrations of salt or ethanol. Survival of HPF-deficient bacteria was impaired under certain conditions both in vitro and during animal infection, providing evidence for the biological relevance of 100S ribosome formation. PMID:25422304

  5. Neisseria gonorrhoeae Strain with High-Level Resistance to Spectinomycin Due to a Novel Resistance Mechanism (Mutated Ribosomal Protein S5) Verified in Norway

    OpenAIRE

    Unemo, Magnus; Golparian, Daniel; Skogen, Vegard; Olsen, Anne Olaug; Moi, Harald; Syversen, Gaute; Hjelmevoll, Stig Ove

    2013-01-01

    Gonorrhea may become untreatable, and new treatment options are essential. Verified resistance to spectinomycin is exceedingly rare. However, we describe a high-level spectinomycin-resistant (MIC, >1,024 μg/ml) Neisseria gonorrhoeae strain from Norway with a novel resistance mechanism. The resistance determinant was a deletion of codon 27 (valine) and a K28E alteration in the ribosomal protein 5S. The traditional spectinomycin resistance gene (16S rRNA) was wild type. Despite this exceedingly...

  6. Revealing pancrustacean relationships: Phylogenetic analysis of ribosomal protein genes places Collembola (springtails) in a monophyletic Hexapoda and reinforces the discrepancy between mitochondrial and nuclear DNA markers.

    OpenAIRE

    Mariën J; Roelofs D; Timmermans MJTN; Straalen NM van

    2008-01-01

    Abstract Background In recent years, several new hypotheses on phylogenetic relations among arthropods have been proposed on the basis of DNA sequences. One of the challenged hypotheses is the monophyly of hexapods. This discussion originated from analyses based on mitochondrial DNA datasets that, due to an unusual positioning of Collembola, suggested that the hexapod body plan evolved at least twice. Here, we re-evaluate the position of Collembola using ribosomal protein gene sequences. Resu...

  7. Conserved codon composition of ribosomal protein coding genes in Escherichia coli, Mycobacterium tuberculosis and Saccharomyces cerevisiae: lessons from supervised machine learning in functional genomics

    OpenAIRE

    Lin, Kui; Kuang, Yuyu; Joseph, Jeremiah S.; Kolatkar, Prasanna R

    2002-01-01

    Genomics projects have resulted in a flood of sequence data. Functional annotation currently relies almost exclusively on inter-species sequence comparison and is restricted in cases of limited data from related species and widely divergent sequences with no known homologs. Here, we demonstrate that codon composition, a fusion of codon usage bias and amino acid composition signals, can accurately discriminate, in the absence of sequence homology information, cytoplasmic ribosomal protein gene...

  8. Antibodies to ribosomal P proteins of Trypanosoma cruzi in Chagas disease possess functional autoreactivity with heart tissue and differ from anti-P autoantibodies in lupus.

    Science.gov (United States)

    Kaplan, D; Ferrari, I; Bergami, P L; Mahler, E; Levitus, G; Chiale, P; Hoebeke, J; Van Regenmortel, M H; Levin, M J

    1997-09-16

    Anti-P antibodies present in sera from patients with chronic Chagas heart disease (cChHD) recognize peptide R13, EEEDDDMGFGLFD, which encompasses the C-terminal region of the Trypanosoma cruzi ribosomal P1 and P2 proteins. This peptide shares homology with the C-terminal region (peptide H13 EESDDDMGFGLFD) of the human ribosomal P proteins, which is in turn the target of anti-P autoantibodies in systemic lupus erythematosus (SLE), and with the acidic epitope, AESDE, of the second extracellular loop of the beta1-adrenergic receptor. Anti-P antibodies from chagasic patients showed a marked preference for recombinant parasite ribosomal P proteins and peptides, whereas anti-P autoantibodies from SLE reacted with human and parasite ribosomal P proteins and peptides to the same extent. A semi-quantitative estimation of the binding of cChHD anti-P antibodies to R13 and H13 using biosensor technology indicated that the average affinity constant was about 5 times higher for R13 than for H13. Competitive enzyme immunoassays demonstrated that cChHD anti-P antibodies bind to the acidic portions of peptide H13, as well as to peptide H26R, encompassing the second extracellular loop of the beta1 adrenoreceptor. Anti-P antibodies isolated from cChHD patients exert a positive chronotropic effect in vitro on cardiomyocytes from neonatal rats, which resembles closely that of anti-beta1 receptor antibodies isolated from the same patient. In contrast, SLE anti-P autoantibodies have no functional effect. Our results suggest that the adrenergic-stimulating activity of anti-P antibodies may be implicated in the induction of functional myocardial impairments observed in cChHD. PMID:9294205

  9. The quantitative assessment of the role played by basic amino acid clusters in the nuclear uptake of human ribosomal protein L7

    Energy Technology Data Exchange (ETDEWEB)

    Tai, Lin-Ru [Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan, ROC (China); Chou, Chang-Wei [Institute of Clinical Dentistry Science, National Yang-Ming University, Taipei, Taiwan, ROC (China); Lee, I-Fang; Kirby, Ralph [Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan, ROC (China); Lin, Alan, E-mail: alin@ym.edu.tw [Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan, ROC (China); Institute of Clinical Dentistry Science, National Yang-Ming University, Taipei, Taiwan, ROC (China)

    2013-02-15

    In this study, we used a multiple copy (EGFP){sub 3} reporter system to establish a numeric nuclear index system to assess the degree of nuclear import. The system was first validated by a FRAP assay, and then was applied to evaluate the essential and multifaceted nature of basic amino acid clusters during the nuclear import of ribosomal protein L7. The results indicate that the sequence context of the basic cluster determines the degree of nuclear import, and that the number of basic residues in the cluster is irrelevant; rather the position of the pertinent basic residues is crucial. Moreover, it also found that the type of carrier protein used by basic cluster has a great impact on the degree of nuclear import. In case of L7, importin β2 or importin β3 are preferentially used by clusters with a high import efficiency, notwithstanding that other importins are also used by clusters with a weaker level of nuclear import. Such a preferential usage of multiple basic clusters and importins to gain nuclear entry would seem to be a common practice among ribosomal proteins in order to ensure their full participation in high rate ribosome synthesis. - Highlights: ► We introduce a numeric index system that represents the degree of nuclear import. ► The rate of nuclear import is dictated by the sequence context of the basic cluster. ► Importin β2 and β3 were mainly responsible for the N4 mediated nuclear import.

  10. Conserved codon composition of ribosomal protein coding genes in Escherichia coli, Mycobacterium tuberculosis and Saccharomyces cerevisiae: lessons from supervised machine learning in functional genomics.

    Science.gov (United States)

    Lin, Kui; Kuang, Yuyu; Joseph, Jeremiah S; Kolatkar, Prasanna R

    2002-06-01

    Genomics projects have resulted in a flood of sequence data. Functional annotation currently relies almost exclusively on inter-species sequence comparison and is restricted in cases of limited data from related species and widely divergent sequences with no known homologs. Here, we demonstrate that codon composition, a fusion of codon usage bias and amino acid composition signals, can accurately discriminate, in the absence of sequence homology information, cytoplasmic ribosomal protein genes from all other genes of known function in Saccharomyces cerevisiae, Escherichia coli and Mycobacterium tuberculosis using an implementation of support vector machines, SVM(light). Analysis of these codon composition signals is instructive in determining features that confer individuality to ribosomal protein genes. Each of the sets of positively charged, negatively charged and small hydrophobic residues, as well as codon bias, contribute to their distinctive codon composition profile. The representation of all these signals is sensitively detected, combined and augmented by the SVMs to perform an accurate classification. Of special mention is an obvious outlier, yeast gene RPL22B, highly homologous to RPL22A but employing very different codon usage, perhaps indicating a non-ribosomal function. Finally, we propose that codon composition be used in combination with other attributes in gene/protein classification by supervised machine learning algorithms. PMID:12034849

  11. Extremophilic 50S Ribosomal RNA-Binding Protein L35Ae as a Basis for Engineering of an Alternative Protein Scaffold.

    Directory of Open Access Journals (Sweden)

    Anna V Lomonosova

    Full Text Available Due to their remarkably high structural stability, proteins from extremophiles are particularly useful in numerous biological applications. Their utility as alternative protein scaffolds could be especially valuable in small antibody mimetic engineering. These artificial binding proteins occupy a specific niche between antibodies and low molecular weight substances, paving the way for development of innovative approaches in therapeutics, diagnostics, and reagent use. Here, the 50S ribosomal RNA-binding protein L35Ae from the extremophilic archaea Pyrococcus horikoshii has been probed for its potential to serve as a backbone in alternative scaffold engineering. The recombinant wild type L35Ae has a native-like secondary structure, extreme thermal stability (mid-transition temperature of 90°C and a moderate resistance to the denaturation by guanidine hydrochloride (half-transition at 2.6 M. Chemical crosslinking and dynamic light scattering data revealed that the wild type L35Ae protein has a propensity for multimerization and aggregation correlating with its non-specific binding to a model cell surface of HEK293 cells, as evidenced by flow cytometry. To suppress these negative features, a 10-amino acid mutant (called L35Ae 10X was designed, which lacks the interaction with HEK293 cells, is less susceptible to aggregation, and maintains native-like secondary structure and thermal stability. However, L35Ae 10X also shows lowered resistance to guanidine hydrochloride (half-transition at 2.0M and is more prone to oligomerization. This investigation of an extremophile protein's scaffolding potential demonstrates that lowered resistance to charged chemical denaturants and increased propensity to multimerization may limit the utility of extremophile proteins as alternative scaffolds.

  12. AMPLIFICATION OF RIBOSOMAL RNA SEQUENCES

    Science.gov (United States)

    This book chapter offers an overview of the use of ribosomal RNA sequences. A history of the technology traces the evolution of techniques to measure bacterial phylogenetic relationships and recent advances in obtaining rRNA sequence information. The manual also describes procedu...

  13. Initiation factor 2 stabilizes the ribosome in a semirotated conformation.

    Science.gov (United States)

    Ling, Clarence; Ermolenko, Dmitri N

    2015-12-29

    Intersubunit rotation and movement of the L1 stalk, a mobile domain of the large ribosomal subunit, have been shown to accompany the elongation cycle of translation. The initiation phase of protein synthesis is crucial for translational control of gene expression; however, in contrast to elongation, little is known about the conformational rearrangements of the ribosome during initiation. Bacterial initiation factors (IFs) 1, 2, and 3 mediate the binding of initiator tRNA and mRNA to the small ribosomal subunit to form the initiation complex, which subsequently associates with the large subunit by a poorly understood mechanism. Here, we use single-molecule FRET to monitor intersubunit rotation and the inward/outward movement of the L1 stalk of the large ribosomal subunit during the subunit-joining step of translation initiation. We show that, on subunit association, the ribosome adopts a distinct conformation in which the ribosomal subunits are in a semirotated orientation and the L1 stalk is positioned in a half-closed state. The formation of the semirotated intermediate requires the presence of an aminoacylated initiator, fMet-tRNA(fMet), and IF2 in the GTP-bound state. GTP hydrolysis by IF2 induces opening of the L1 stalk and the transition to the nonrotated conformation of the ribosome. Our results suggest that positioning subunits in a semirotated orientation facilitates subunit association and support a model in which L1 stalk movement is coupled to intersubunit rotation and/or IF2 binding. PMID:26668356

  14. Ribosome-inactivating proteins from the seeds of Saponaria officinalis L. (soapwort), of Agrostemma githago L. (corn cockle) and of Asparagus officinalis L. (asparagus), and from the latex of Hura crepitans L. (sandbox tree).

    Science.gov (United States)

    Stirpe, F; Gasperi-Campani, A; Barbieri, L; Falasca, A; Abbondanza, A; Stevens, W A

    1983-12-15

    Ribosome-inactivating proteins, similar to those already known [Barbieri & Stirpe (1982) Cancer Surveys 1, 489-520] were purified from the seeds of Saponaria officinalis (two proteins), of Agrostemma githago (three proteins), and of Asparagus officinalis (three proteins), and from the latex of Hura crepitans (one protein). The yield ranged from 8 to 400 mg/100 g of starting material. All proteins have an Mr of approx. 30000 and an alkaline isoelectric point. Their sugar content varies from 0 (proteins from S. officinalis) to 40% (protein from H. crepitans). The ribosome-inactivating proteins inhibit protein synthesis by rabbit reticulocyte lysate, the ID50 (concentration giving 50% inhibition) ranging from 1 ng/ml (a protein from S. officinalis) to 18 ng/ml (a protein from A. githago). Those which were tested (the proteins from S. officinalis and from A. githago) also inhibit polymerization of phenylalanine by isolated ribosomes, acting in an apparently catalytic manner. The protein from H. crepitans inhibited protein synthesis by HeLa cells, with an ID50 of 4 micrograms/ml, whereas the proteins from S. officinalis and from A. githago had an ID50 of more than 50-100 micrograms/ml. The ribosome-inactivating proteins from S. officinalis and from A. githago reduced the number of local lesions by tobacco-mosaic virus in the leaves of Nicotiana glutinosa. PMID:6667259

  15. Antibodies against ribosomal protein S29 (RPS29) fused with glutathione's transferase specially react with native RPS29 in mouse and human cells

    Institute of Scientific and Technical Information of China (English)

    Liu Jia; Zhang Junlei; Han Junfeng; Li Dongying; Jian Rui; Rao XianCai; Chen Wei; Wang Jiali; Xu Xiaofeng; Hu Zhen

    2011-01-01

    The ribosomal protein S29 also known as RPS29,is not only a component of the 40S subunit of ribosome,but also involved in embryonic development,oncogenesis and other pathologic conditions. However,rare commercial antibody against RPS29 restricts the discovery of precise physiological and pathological function of this protein. In this study,the whole RPS29 gene was inserted into plasmid pGEX-6p-1 to express glutathione's transferase (GST) fusion proteins in Escherichia coli (E. coli) strain BL21. High yields of soluble recombinant proteins were obtained. Mice were immunized with the recombinant RPS29 protein. The serum from the immunized mice could specially react with purified recombinant RPS29 proteins and native RPS29 proteins in CCE cells by western blotting,immunofluorescence staining and flow cytometric analysis. Further more the polyclonal antibodies also reacted specifically with human cell strain ECV304,which showed typical cytoplasmatic fluorescence. The polyclonal antibodies we prepared would be an available tool for studying the roles of RPS29 in embryonic development and human diseases.

  16. On peptide bond formation, translocation, nascent protein progression and the regulatory properties of ribosomes. Derived on 20 October 2002 at the 28th FEBS Meeting in Istanbul.

    Science.gov (United States)

    Agmon, Ilana; Auerbach, Tamar; Baram, David; Bartels, Heike; Bashan, Anat; Berisio, Rita; Fucini, Paola; Hansen, Harly A S; Harms, Joerg; Kessler, Maggie; Peretz, Moshe; Schluenzen, Frank; Yonath, Ada; Zarivach, Raz

    2003-06-01

    High-resolution crystal structures of large ribosomal subunits from Deinococcus radiodurans complexed with tRNA-mimics indicate that precise substrate positioning, mandatory for efficient protein biosynthesis with no further conformational rearrangements, is governed by remote interactions of the tRNA helical features. Based on the peptidyl transferase center (PTC) architecture, on the placement of tRNA mimics, and on the existence of a two-fold related region consisting of about 180 nucleotides of the 23S RNA, we proposed a unified mechanism integrating peptide bond formation, A-to-P site translocation, and the entrance of the nascent protein into its exit tunnel. This mechanism implies sovereign, albeit correlated, motions of the tRNA termini and includes a spiral rotation of the A-site tRNA-3' end around a local two-fold rotation axis, identified within the PTC. PTC features, ensuring the precise orientation required for the A-site nucleophilic attack on the P-site carbonyl-carbon, guide these motions. Solvent mediated hydrogen transfer appears to facilitate peptide bond formation in conjunction with the spiral rotation. The detection of similar two-fold symmetry-related regions in all known structures of the large ribosomal subunit, indicate the universality of this mechanism, and emphasizes the significance of the ribosomal template for the precise alignment of the substrates as well as for accurate and efficient translocation. The symmetry-related region may also be involved in regulatory tasks, such as signal transmission between the ribosomal features facilitating the entrance and the release of the tRNA molecules. The protein exit tunnel is an additional feature that has a role in cellular regulation. We showed by crystallographic methods that this tunnel is capable of undergoing conformational oscillations and correlated the tunnel mobility with sequence discrimination, gating and intracellular regulation. PMID:12787020

  17. Vaccination of dogs with six different candidate leishmaniasis vaccines composed of a chimerical recombinant protein containing ribosomal and histone protein epitopes in combination with different adjuvants.

    Science.gov (United States)

    Poot, J; Janssen, L H M; van Kasteren-Westerneng, T J; van der Heijden-Liefkens, K H A; Schijns, V E J C; Heckeroth, A

    2009-07-16

    Chimerical protein "Q", composed of antigenic ribosomal and histone sequences, in combination with live BCG is a promising canine leishmaniasis vaccine candidate; one of the few vaccine candidates that have been tested successfully in dogs. Unfortunately, live BCG is not an appropriate adjuvant for commercial application due to safety problems in dogs. In order to find a safe adjuvant with similar efficacy to live BCG, muramyl dipeptide, aluminium hydroxide, Matrix C and killed Propionibacterium acnes in combination with either E. coli- or baculovirus-produced recombinant JPCM5_Q protein were tested. Groups of five or seven dogs were vaccinated with six different adjuvant-antigen combinations and challenged with a high dose intravenous injection of Leishmania infantum JPC strain promastigotes. All candidate vaccines proved to be safe, and both humoral and cellular responses to the recombinant proteins were detected at the end of the prime-boost vaccination scheme. However, clinical and parasitological data obtained during the 10 month follow-up period indicated that protection was not induced by either of the six candidate vaccines. Although no direct evidence was obtained, our data suggest that live BCG may have a significant protective effect against challenge with L. infantum in dogs. PMID:19500553

  18. Surface Proteins of Streptococcus agalactiae and Related Proteins in Other Bacterial Pathogens.

    OpenAIRE

    Lindahl, Gunnar; Stålhammar-Carlemalm, Margaretha; Areschoug, Thomas

    2005-01-01

    Streptococcus agalactiae (group B Streptococcus) is the major cause of invasive bacterial disease, including meningitis, in the neonatal period. Although prophylactic measures have contributed to a substantial reduction in the number of infections, development of a vaccine remains an important goal. While much work in this field has focused on the S. agalactiae polysaccharide capsule, which is an important virulence factor that elicits protective immunity, surface proteins have received incre...

  19. Attachment sites of primary binding proteins L1, L2 and L23 on 23 S ribosomal RNA of Escherichia coli

    DEFF Research Database (Denmark)

    Egebjerg, Jan; Christiansen, Jan; Garrett, Roger Antony

    1991-01-01

    The attachment sites of the primary binding proteins L1, L2 and L23 on 23 S ribosomal RNA of Escherichia coli were examined by a chemical and ribonuclease footprinting method using several probes with different specificities. The results show that the sites are confined to localized RNA regions...... within the large ribonuclease-protected ribonucleoprotein fragments that were characterized earlier. They are as follows: 1. (1) L1 recognizes a tertiary structural motif in domain V centred on two interacting internal loops; the main protein interaction sites occur at the internal loop/helix junctions.2...

  20. A key role for the mRNA leader structure in translational control of ribosomal protein S1 synthesis in γ-proteobacteria

    OpenAIRE

    Tchufistova, Ludmila S.; Komarova, Anastassia V.; Boni, Irina V.

    2003-01-01

    The translation initiation region (TIR) of the Escherichia coli rpsA mRNA coding for ribosomal protein S1 is characterized by a remarkable efficiency in driving protein synthesis despite the absence of the canonical Shine–Dalgarno element, and by a strong and specific autogenous repression in the presence of free S1 in trans. The efficient and autoregulated E.coli rpsA TIR comprises not less than 90 nt upstream of the translation start and can be unambiguously folded into three irregular hair...

  1. The Interaction Pattern between a Homology Model of 40S Ribosomal S9 Protein of Rhizoctonia solani and 1-Hydroxyphenaize by Docking Study

    OpenAIRE

    Seema Dharni; Sanchita; Abdul Samad; Ashok Sharma; Dharani Dhar Patra

    2014-01-01

    1-Hydroxyphenazine (1-OH-PHZ), a natural product from Pseudomonas aeruginosa strain SD12, was earlier reported to have potent antifungal activity against Rhizoctonia solani. In the present work, the antifungal activity of 1-OH-PHZ on 40S ribosomal S9 protein was validated by molecular docking approach. 1-OH-PHZ showed interaction with two polar contacts with residues, Arg69 and Phe19, which inhibits the synthesis of fungal protein. Our study reveals that 1-OH-PHZ can be a potent inhibitor of ...

  2. Ribosome engineering to promote new crystal forms

    International Nuclear Information System (INIS)

    Truncation of ribosomal protein L9 in T. thermophilus allows the generation of new crystal forms and the crystallization of ribosome–GTPase complexes. Crystallographic studies of the ribosome have provided molecular details of protein synthesis. However, the crystallization of functional complexes of ribosomes with GTPase translation factors proved to be elusive for a decade after the first ribosome structures were determined. Analysis of the packing in different 70S ribosome crystal forms revealed that regardless of the species or space group, a contact between ribosomal protein L9 from the large subunit and 16S rRNA in the shoulder of a neighbouring small subunit in the crystal lattice competes with the binding of GTPase elongation factors to this region of 16S rRNA. To prevent the formation of this preferred crystal contact, a mutant strain of Thermus thermophilus, HB8-MRCMSAW1, in which the ribosomal protein L9 gene has been truncated was constructed by homologous recombination. Mutant 70S ribosomes were used to crystallize and solve the structure of the ribosome with EF-G, GDP and fusidic acid in a previously unobserved crystal form. Subsequent work has shown the usefulness of this strain for crystallization of the ribosome with other GTPase factors

  3. Effect of bacterial protein meal on protein and energy metabolism in growing chickens

    DEFF Research Database (Denmark)

    Hellwing, Anne Louise Frydendahl; Tauson, Anne-Helene; Skrede, Anders

    2006-01-01

    This experiment investigates the effect of increasing the dietary content of bacterial protein meal (BPM) on the protein and energy metabolism, and carcass chemical composition of growing chickens. Seventy-two Ross male chickens were allocated to four diets, each in three replicates with 0% (D0), 2......% (D2), 4% D4), and 6% BPM (D6), BPM providing up to 20% of total dietary N. Five balance experiments were conducted when the chickens were 3-7, 10-14, 17-21, 23-27, and 30-34 days old. During the same periods, 22-h respiration experiments (indirect calorimetry) were performed with troups of 6 chickens...... for protein and energy retention found in the balance and respiration experiments. It was concluded that the overall protein and energy metabolism as well as carcass composition were not influenced by a dietary content of up to 6% BPM corresponding to 20% of dietary N....

  4. A unique DNA repair and recombination gene (recN) sequence for identification and intraspecific molecular typing of bacterial wilt pathogen Ralstonia solanacearum and its comparative analysis with ribosomal DNA sequences

    Indian Academy of Sciences (India)

    Aundy Kumar; Thekkan Puthiyaveedu Prameela; Rajamma Suseelabhai

    2013-06-01

    Ribosomal gene sequences are a popular choice for identification of bacterial species and, often, for making phylogenetic interpretations. Although very popular, the sequences of 16S rDNA and 16-23S intergenic sequences often fail to differentiate closely related species of bacteria. The availability of complete genome sequences of bacteria, in the recent years, has accelerated the search for new genome targets for phylogenetic interpretations. The recently published full genome data of nine strains of R. solanacearum, which causes bacterial wilt of crop plants, has provided enormous genomic choices for phylogenetic analysis in this globally important plant pathogen. We have compared a gene candidate recN, which codes for DNA repair and recombination function, with 16S rDNA/16-23S intergenic ribosomal gene sequences for identification and intraspecific phylogenetic interpretations in R. solanacearum. recN gene sequence analysis of R. solanacearum revealed subgroups within phylotypes (or newly proposed species within plant pathogenic genus, Ralstonia), indicating its usefulness for intraspecific genotyping. The taxonomic discriminatory power of recN gene sequence was found to be superior to ribosomal DNA sequences. In all, the recN-sequence-based phylogenetic tree generated with the Bayesian model depicted 21 haplotypes against 15 and 13 haplotypes obtained with 16S rDNA and 16-23S rDNA intergenic sequences, respectively. Besides this, we have observed high percentage of polymorphic sites (S 23.04%), high rate of mutations (Eta 276) and high codon bias index (CBI 0.60), which makes the recN an ideal gene candidate for intraspecific molecular typing of this important plant pathogen.

  5. Mammalian mitochondrial ribosomal small subunit (MRPS) genes: A putative role in human disease.

    Science.gov (United States)

    Gopisetty, Gopal; Thangarajan, Rajkumar

    2016-09-01

    Mitochondria are prominently understood as power houses producing ATP the primary energy currency of the cell. However, mitochondria are also known to play an important role in apoptosis and autophagy, and mitochondrial dysregulation can lead to pathological outcomes. Mitochondria are known to contain 1500 proteins of which only 13 are coded by mitochondrial DNA and the rest are coded by nuclear genes. Protein synthesis in mitochondria involves mitochondrial ribosomes which are 55-60S particles and are composed of small 28S and large 39S subunits. A feature of mammalian mitoribosome which differentiate it from bacterial ribosomes is the increased protein content. The human mitochondrial ribosomal protein (MRP) gene family comprises of 30 genes which code for mitochondrial ribosomal small subunit and 50 genes for the large subunit. The present review focuses on the mitochondrial ribosomal small subunit genes (MRPS), presents an overview of the literature and data gleaned from publicly available gene and protein expression databases. The survey revealed aberrations in MRPS gene expression patterns in varied human diseases indicating a putative role in their etiology. PMID:27170550

  6. Erythromycin, roxithromycin, and clarithromycin: use of slow-binding kinetics to compare their in vitro interaction with a bacterial ribosomal complex active in peptide bond formation.

    Science.gov (United States)

    Dinos, George P; Connell, Sean R; Nierhaus, Knud H; Kalpaxis, Dimitrios L

    2003-03-01

    In a cell-free system derived from Escherichia coli, it is shown that clarithromycin and roxithromycin, like their parent compound erythromycin, do not inhibit the puromycin reaction (i.e., the peptide bond formation between puromycin and AcPhe-tRNA bound at the P-site of 70S ribosomes programmed with heteropolymeric mRNA). Nevertheless, all three antibiotics compete for binding on the ribosome with tylosin, a 16-membered ring macrolide that behaves as a slow-binding, slowly reversible inhibitor of peptidyltransferase. The mutually exclusive binding of these macrolides to ribosomes is also corroborated by the fact that they protect overlapping sites in domain V of 23S rRNA from chemical modification by dimethyl sulfate. From this competition effect, detailed kinetic analysis revealed that roxithromycin or clarithromycin (A), like erythromycin, reacts rapidly with AcPhe-tRNA.MF-mRNA x 70S ribosomal complex (C) to form the encounter complex CA which is then slowly isomerized to a more tight complex, termed C*A. The value of the overall dissociation constant, K, encompassing both steps of macrolide interaction with complex C, is 36 nM for erythromycin, 20 nM for roxithromycin, and 8 nM for clarithromycin. Because the off-rate constant of C*A complex does not significantly differ among the three macrolides, the superiority of clarithromycin as an inhibitor of translation in E. coli cells and many Gram-positive bacteria may be correlated with its greater rate of association with ribosomes. PMID:12606769

  7. Cloning, periplasmic expression, purification and structural characterization of human ribosomal protein L10; Clonagem, expressao, purificacao e caracterizacao estrutural da proteina ribossomal L10 humana recombinante

    Energy Technology Data Exchange (ETDEWEB)

    Pereira, Larissa Miranda

    2009-07-01

    The ribosomal protein L10 (RP L10) is a strong candidate to be included in the class of tumor suppressor proteins. This protein, also denominated as QM, is known to participate in the binding of ribosomal subunits 60S and 40S and the translation of mRNAs. It has a molecular weight that varies between 24 and 26 kDa and an isoelectric point of (pI) 10.5. The sequence of the protein QM is highly conserved in mammals, plants, invertebrates, insects and yeast which indicates its critical functions in a cell. As a tumor suppressor, RP L10 has been studied in strains of Wilm's tumor (WT-1) and tumor cells in the stomach, where was observed a decrease in the amount of its mRNA. More recently, the RP L10 was found in low amounts in the early stages of prostate adenoma and showed some mutation in ovarian cancer, what indicates its role as a suppressor protein in the development of these diseases. It has also been described that this protein interacts with c-Jun and c-Yes inhibiting growth factors and consequently, cell division. This work has an important role on the establishment of soluble expression of QM to give base information for further studies on expression that aim to evaluate the specific regions where it acts binding the 60S and 40S ribosomal subunits and translation, as well as its binding to proto-oncogenes. The cDNA for QM protein was amplified by PCR and cloned into periplasmic expression vector p3SN8. The QM protein was expressed in E. coli BL21 (DE3) in the region of cytoplasm and periplasm, the best condition was obtained from the expression of the recombinant plasmid QM p1813{sub Q}M at 25 degree C or 30 degree C, the soluble protein was obtained with small amounts of contaminants. The assays of secondary structure showed that the QM protein is predominantly alpha-helix, but when it loses the folding, this condition changes and the protein is replaced by {beta}- sheet feature. (author)

  8. Identification and Characterization of Inhibitors of Bacterial Enoyl-Acyl Carrier Protein Reductase

    OpenAIRE

    Ling, Losee L.; Xian, Jun; Ali, Syed; Geng, Bolin; Fan, Jun; Mills, Debra M.; Arvanites, Anthony C.; Orgueira, Hernan; Ashwell, Mark A.; Carmel, Gilles; Xiang, Yibin; Moir, Donald T.

    2004-01-01

    Bacterial enoyl-acyl carrier protein reductase (ENR) catalyzes an essential step in fatty acid biosynthesis. ENR is an attractive target for narrow-spectrum antibacterial drug discovery because of its essential role in metabolism and its sequence conservation across many bacterial species. In addition, the bacterial ENR sequence and structural organization are distinctly different from those of mammalian fatty acid biosynthesis enzymes. High-throughput screening to identify inhibitors of Esch...

  9. PROTEIN QUALITY CONTROL IN BACTERIAL CELLS: INTEGRATED NETWORKS OF CHAPERONES AND ATP-DEPENDENT PROTEASES.

    Energy Technology Data Exchange (ETDEWEB)

    FLANAGAN,J.M.BEWLEY,M.C.

    2002-10-01

    It is generally accepted that the information necessary to specify the native, functional, three-dimensional structure of a protein is encoded entirely within its amino acid sequence; however, efficient reversible folding and unfolding is observed only with a subset of small single-domain proteins. Refolding experiments often lead to the formation of kinetically-trapped, misfolded species that aggregate, even in dilute solution. In the cellular environment, the barriers to efficient protein folding and maintenance of native structure are even larger due to the nature of this process. First, nascent polypeptides must fold in an extremely crowded environment where the concentration of macromolecules approaches 300-400 mg/mL and on average, each ribosome is within its own diameter of another ribosome (1-3). These conditions of severe molecular crowding, coupled with high concentrations of nascent polypeptide chains, favor nonspecific aggregation over productive folding (3). Second, folding of newly-translated polypeptides occurs in the context of their vehtorial synthesis process. Amino acids are added to a growing nascent chain at the rate of {approx}5 residues per set, which means that for a 300 residue protein its N-terminus will be exposed to the cytosol {approx}1 min before its C-terminus and be free to begin the folding process. However, because protein folding is highly cooperative, the nascent polypeptide cannot reach its native state until a complete folding domain (50-250 residues) has emerged from the ribosome. Thus, for a single-domain protein, the final steps in ffolding are only completed post-translationally since {approx}40 residues of a nascent chain are sequestered within the exit channel of the ribosome and are not available for folding (4). A direct consequence of this limitation in cellular folding is that during translation incomplete domains will exist in partially-folded states that tend to expose hydrophobic residues that are prone to

  10. PROTEIN QUALITY CONTROL IN BACTERIAL CELLS: INTEGRATED NETWORKS OF CHAPERONES AND ATP-DEPENDENT PROTEASES.

    Energy Technology Data Exchange (ETDEWEB)

    FLANAGAN,J.M.; BEWLEY,M.C.

    2001-12-03

    It is generally accepted that the information necessary to specify the native, functional, three-dimensional structure of a protein is encoded entirely within its amino acid sequence; however, efficient reversible folding and unfolding is observed only with a subset of small single-domain proteins. Refolding experiments often lead to the formation of kinetically-trapped, misfolded species that aggregate, even in dilute solution. In the cellular environment, the barriers to efficient protein folding and maintenance of native structure are even larger due to the nature of this process. First, nascent polypeptides must fold in an extremely crowded environment where the concentration of macromolecules approaches 300-400 mg/mL and on average, each ribosome is within its own diameter of another ribosome (1-3). These conditions of severe molecular crowding, coupled with high concentrations of nascent polypeptide chains, favor nonspecific aggregation over productive folding (3). Second, folding of newly-translated polypeptides occurs in the context of their vehtorial synthesis process. Amino acids are added to a growing nascent chain at the rate of -5 residues per set, which means that for a 300 residue protein its N-terminus will be exposed to the cytosol {approx}1 min before its C-terminus and be free to begin the folding process. However, because protein folding is highly cooperative, the nascent polypeptide cannot reach its native state until a complete folding domain (50-250 residues) has emerged from the ribosome. Thus, for a single-domain protein, the final steps in folding are only completed post-translationally since {approx}40 residues of a nascent chain are sequestered within the exit channel of the ribosome and are not available for folding (4). A direct consequence of this limitation in cellular folding is that during translation incomplete domains will exist in partially-folded states that tend to expose hydrophobic residues that are prone to aggregation and

  11. PROTEIN QUALITY CONTROL IN BACTERIAL CELLS: INTEGRATED NETWORKS OF CHAPERONES AND ATP-DEPENDENT PROTEASES

    International Nuclear Information System (INIS)

    It is generally accepted that the information necessary to specify the native, functional, three-dimensional structure of a protein is encoded entirely within its amino acid sequence; however, efficient reversible folding and unfolding is observed only with a subset of small single-domain proteins. Refolding experiments often lead to the formation of kinetically-trapped, misfolded species that aggregate, even in dilute solution. In the cellular environment, the barriers to efficient protein folding and maintenance of native structure are even larger due to the nature of this process. First, nascent polypeptides must fold in an extremely crowded environment where the concentration of macromolecules approaches 300-400 mg/mL and on average, each ribosome is within its own diameter of another ribosome (1-3). These conditions of severe molecular crowding, coupled with high concentrations of nascent polypeptide chains, favor nonspecific aggregation over productive folding (3). Second, folding of newly-translated polypeptides occurs in the context of their vehtorial synthesis process. Amino acids are added to a growing nascent chain at the rate of -5 residues per set, which means that for a 300 residue protein its N-terminus will be exposed to the cytosol(approx)1 min before its C-terminus and be free to begin the folding process. However, because protein folding is highly cooperative, the nascent polypeptide cannot reach its native state until a complete folding domain (50-250 residues) has emerged from the ribosome. Thus, for a single-domain protein, the final steps in folding are only completed post-translationally since(approx)40 residues of a nascent chain are sequestered within the exit channel of the ribosome and are not available for folding (4). A direct consequence of this limitation in cellular folding is that during translation incomplete domains will exist in partially-folded states that tend to expose hydrophobic residues that are prone to aggregation and

  12. PAK1IP1, a ribosomal stress-induced nucleolar protein, regulates cell proliferation via the p53–MDM2 loop

    OpenAIRE

    Yu, Weishi; Qiu, Zhongwei; Gao, Na; Wang, Liren; Cui, Hengxiang; Qian, Yu; Jiang, Li; Luo, Jian; Yi, Zhengfang; Lu, Hua; Li, Dali; Liu, Mingyao

    2010-01-01

    Cell growth and proliferation are tightly controlled via the regulation of the p53–MDM2 feedback loop in response to various cellular stresses. In this study, we identified a nucleolar protein called PAK1IP1 as another regulator of this loop. PAK1IP1 was induced when cells were treated with chemicals that disturb ribosome biogenesis. Overexpression of PAK1IP1 inhibited cell proliferation by inducing p53-dependent G1 cell-cycle arrest. PAK1IP1 bound to MDM2 and inhibited its ability to ubiquit...

  13. Identification of a novel 70 kDa protein that binds to the core promoter element and is essential for ribosomal DNA transcription

    OpenAIRE

    Yamamoto, Kazuo; Koga, Atsuro; Yamamoto, Mika; Nishi, Yu-ichi; Tamura, Taka-aki; Nogi, Yasuhisa; Muramatsu, Masami

    2000-01-01

    Mammalian ribosomal RNA genes (rDNA) are transcribed by RNA polymerase I and at least two auxiliary factors, UBF and SL1/TFID/TIF-IB. It has also been reported that an additional factor(s) is required to reconstitute efficient initiation of rDNA transcription in vitro, depending upon the procedures of chromatographic separation. In an attempt to elucidate the molecular identity of such yet uncertain activities, we have developed agarose gel shift and UV cross-linking assays to detect proteins...

  14. Neisseria gonorrhoeae strain with high-level resistance to spectinomycin due to a novel resistance mechanism (mutated ribosomal protein S5) verified in Norway.

    Science.gov (United States)

    Unemo, Magnus; Golparian, Daniel; Skogen, Vegard; Olsen, Anne Olaug; Moi, Harald; Syversen, Gaute; Hjelmevoll, Stig Ove

    2013-02-01

    Gonorrhea may become untreatable, and new treatment options are essential. Verified resistance to spectinomycin is exceedingly rare. However, we describe a high-level spectinomycin-resistant (MIC, >1,024 μg/ml) Neisseria gonorrhoeae strain from Norway with a novel resistance mechanism. The resistance determinant was a deletion of codon 27 (valine) and a K28E alteration in the ribosomal protein 5S. The traditional spectinomycin resistance gene (16S rRNA) was wild type. Despite this exceedingly rare finding, spectinomycin available for treatment of ceftriaxone-resistant urogenital gonorrhea would be very valuable. PMID:23183436

  15. Normalization with genes encoding ribosomal proteins but not GAPDH provides an accurate quantification of gene expressions in neuronal differentiation of PC12 cells

    Directory of Open Access Journals (Sweden)

    Lim Qing-En

    2010-01-01

    Full Text Available Abstract Background Gene regulation at transcript level can provide a good indication of the complex signaling mechanisms underlying physiological and pathological processes. Transcriptomic methods such as microarray and quantitative real-time PCR require stable reference genes for accurate normalization of gene expression. Some but not all studies have shown that housekeeping genes (HGKs, β-actin (ACTB and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, which are routinely used for normalization, may vary significantly depending on the cell/tissue type and experimental conditions. It is currently unclear if these genes are stably expressed in cells undergoing drastic morphological changes during neuronal differentiation. Recent meta-analysis of microarray datasets showed that some but not all of the ribosomal protein genes are stably expressed. To test the hypothesis that some ribosomal protein genes can serve as reference genes for neuronal differentiation, a genome-wide analysis was performed and putative reference genes were identified based on stability of expressions. The stabilities of these potential reference genes were then analyzed by reverse transcription quantitative real-time PCR in six differentiation conditions. Results Twenty stably expressed genes, including thirteen ribosomal protein genes, were selected from microarray analysis of the gene expression profiles of GDNF and NGF induced differentiation of PC12 cells. The expression levels of these candidate genes as well as ACTB and GAPDH were further analyzed by reverse transcription quantitative real-time PCR in PC12 cells differentiated with a variety of stimuli including NGF, GDNF, Forskolin, KCl and ROCK inhibitor, Y27632. The performances of these candidate genes as stable reference genes were evaluated with two independent statistical approaches, geNorm and NormFinder. Conclusions The ribosomal protein genes, RPL19 and RPL29, were identified as suitable reference genes

  16. Co-expression of a modified maize ribosome-inactivating protein and a rice basic chitinase gene in transgenic rice plants confers enhanced resistance to sheath blight.

    Science.gov (United States)

    Kim, Ju-Kon; Jang, In-Cheol; Wu, Ray; Zuo, Wei-Neng; Boston, Rebecca S; Lee, Yong-Hwan; Ahn, Il-Pyung; Nahm, Baek Hie

    2003-08-01

    Chitinases, beta-1,3-glucanases, and ribosome-inactivating proteins are reported to have antifungal activity in plants. With the aim of producing fungus-resistant transgenic plants, we co-expressed a modified maize ribosome-inactivating protein gene, MOD1, and a rice basic chitinase gene, RCH10, in transgenic rice plants. A construct containing MOD1 and RCH10 under the control of the rice rbcS and Act1 promoters, respectively, was co-transformed with a plasmid containing the herbicide-resistance gene bar as a selection marker into rice by particle bombardment. Several transformants analyzed by genomic Southern-blot hybridization demonstrated integration of multiple copies of the foreign gene into rice chromosomes. Immunoblot experiments showed that MOD1 formed approximately 0.5% of the total soluble protein in transgenic leaves. RCH10 expression was examined using the native polyacrylamide-overlay gel method, and high RCH10 activity was observed in leaf tissues where endogenous RCH10 is not expressed. R1 plants were analyzed in a similar way, and the Southern-blot patterns and levels of transgene expression remained the same as in the parental line. Analysis of the response of R2 plants to three fungal pathogens of rice, Rhizoctonia solani, Bipolaris oryzae, and Magnaporthe grisea, indicated statistically significant symptom reduction only in the case of R. solani (sheath blight). The increased resistance co-segregated with herbicide tolerance, reflecting a correlation between the resistance phenotype and transgene expression. PMID:12885168

  17. PAK1IP1, a ribosomal stress-induced nucleolar protein, regulates cell proliferation via the p53–MDM2 loop

    Science.gov (United States)

    Yu, Weishi; Qiu, Zhongwei; Gao, Na; Wang, Liren; Cui, Hengxiang; Qian, Yu; Jiang, Li; Luo, Jian; Yi, Zhengfang; Lu, Hua; Li, Dali; Liu, Mingyao

    2011-01-01

    Cell growth and proliferation are tightly controlled via the regulation of the p53–MDM2 feedback loop in response to various cellular stresses. In this study, we identified a nucleolar protein called PAK1IP1 as another regulator of this loop. PAK1IP1 was induced when cells were treated with chemicals that disturb ribosome biogenesis. Overexpression of PAK1IP1 inhibited cell proliferation by inducing p53-dependent G1 cell-cycle arrest. PAK1IP1 bound to MDM2 and inhibited its ability to ubiquitinate and to degrade p53, consequently leading to the accumulation of p53 levels. Interestingly, knockdown of PAK1IP1 in cells also inhibited cell proliferation and induced p53-dependent G1 arrest. Deficiency of PAK1IP1 increased free ribosomal protein L5 and L11 which were required for PAK1IP1 depletion-induced p53 activation. Taken together, our results reveal that PAK1IP1 is a new nucleolar protein that is crucial for rRNA processing and plays a regulatory role in cell proliferation via the p53–MDM2 loop. PMID:21097889

  18. Surface topography of the Bacillus stearothermophilus ribosome

    International Nuclear Information System (INIS)

    The surface topography of the intact 70S ribosome and free 30S and 50S subunits from Bacillus stearothermophilus strain 2,184 was investigated by lactoperoxidase-catalyzed iodination. Two-dimensional polyacrylamide gel electrophoresis was employed to separate ribosomal proteins for analysis of their reactivity. Free 50S subunits incorporated about 18% more 125I than did 50S subunits derived from 70S ribosomes, whereas free 30S subunits and 30S subunits derived from 70S ribosomes incorporated similar amounts of 125I. Iodinated 70S ribosomes and subunits retained 62-78% of the protein synthesis activity of untreated particles and sedimentation profiles showed no gross conformational changes due to iodination. The proteins most reactive to enzymatic iodination were S4, S7, S10 and Sa of the small subunit and L2, L4, L5/9, L6 and L36 of the large subunit. Proteins S2, S3, S7, S13, Sa, L5/9, L10, L11 and L24/25 were labeled substantially more in the free subunits than in the 70S ribosome. Other proteins, including S5, S9, S12, S15/16, S18 and L36 were more extensively iodinated in the 70S ribosome than in the free subunits. The locations of tyrosine residues in some homologus ribosomal proteins from B. stearothermophilus and E. coli are compared. (orig.)

  19. Viral IRES RNA structures and ribosome interactions.

    Science.gov (United States)

    Kieft, Jeffrey S

    2008-06-01

    In eukaryotes, protein synthesis initiates primarily by a mechanism that requires a modified nucleotide 'cap' on the mRNA and also proteins that recruit and position the ribosome. Many pathogenic viruses use an alternative, cap-independent mechanism that substitutes RNA structure for the cap and many proteins. The RNAs driving this process are called internal ribosome-entry sites (IRESs) and some are able to bind the ribosome directly using a specific 3D RNA structure. Recent structures of IRES RNAs and IRES-ribosome complexes are revealing the structural basis of viral IRES' 'hijacking' of the protein-making machinery. It now seems that there are fundamental differences in the 3D structures used by different IRESs, although there are some common features in how they interact with ribosomes. PMID:18468443

  20. Anti-tumor activity and immunological modification of ribosome-inactivating protein (RIP) from Momordica charantia by covalent attachment of polyethylene glycol

    Institute of Scientific and Technical Information of China (English)

    Mengen Li; Yiwen Chen; Zhongyu Liu; Fubing Shen; Xiaoxiao Bian; Yanfa Meng

    2009-01-01

    Ribosome-inactivating proteins (RIPs) are a family of enzymes that depurinate rRNA and inhibit protein biosynthesis. Here we report the purification, apoptosis-inducing activity, and polyethylene glycol (PEG) modification of RIP from the bitter melon seeds. The protein has a homogenous N-terminal sequence of N-Asp-Val-Ser-Phe-Arg. Moreover, the RIP displayed strong apoptosis-inducing activity and suppressed cancer cell growth. This might be attributed to the acti-vation of caspases-3. To make it available for in vivo application, the immunogenicity of RIP was reduced by chemical modification with 20 kDa (mPEG)2-Lys-NHS. The inhibition activity of both PEGylated and non-PEGylated RIP against cancer cells was much stronger than against normal cells, and the antigenicity of PEGylated RIP was reduced significantly. Our results suggested that the PEGylated RIP might be potentially developed as anti-cancer drug.

  1. Transcriptional abundance is not the single force driving the evolution of bacterial proteins

    OpenAIRE

    Wei, Wen; Zhang, Tao; Lin, Dan; Yang, Zu-Jun; Guo, Feng-Biao

    2013-01-01

    Background Despite rapid progress in understanding the mechanisms that shape the evolution of proteins, the relative importance of various factors remain to be elucidated. In this study, we have assessed the effects of 16 different biological features on the evolutionary rates (ERs) of protein-coding sequences in bacterial genomes. Results Our analysis of 18 bacterial species revealed new correlations between ERs and constraining factors. Previous studies have suggested that transcriptional a...

  2. Ribosome biogenesis during skeletal muscle hypertrophy

    OpenAIRE

    von Walden, Ferdinand

    2014-01-01

    Muscle adaptation to chronic resistance exercise (RE) is the result of a cumulative effect on gene expression and protein content. Following a bout of RE, muscle protein synthesis increases and, if followed by consecutive bouts (training), protein accretion and muscle hypertrophy develops. The protein synthetic capacity of the muscle is dictated by ribosome content. Therefore, the general aim of this thesis is to investigate the regulation of ribosome biogenesis during skeletal muscle hypertr...

  3. Integration of bacterial expansin-like proteins into cellulosome promotes the cellulose degradation.

    Science.gov (United States)

    Chen, Chao; Cui, Zhenling; Song, Xiangfei; Liu, Ya-Jun; Cui, Qiu; Feng, Yingang

    2016-03-01

    Cellulosomes are multi-enzyme complexes assembled by cellulases and hemicellulases through dockerin-cohesin interactions, which are the most efficient system for the degradation of lignocellulosic resources in nature. Recent genomic analysis of a cellulosome-producing anaerobe Clostridium clariflavum DSM 19732 revealed that two expansin-like proteins, Clocl_1298 and Clocl_1862, contain a dockerin module, which suggests that they are components of the cellulosome. Bacterial expansin-like proteins do not have hydrolytic activities, but can facilitate the degradation of cellulosic biomass via synergistic effects with cellulases. In this study, the synergistic effect of the expansin-like proteins with both native and designer cellulosomes was investigated. The free expansin-like proteins, including expansin-like domains of Clocl_1298 and Clocl_1862, as well as a well-studied bacterial expansin-like protein BsEXLX1 from Bacillus subtilis, promoted the cellulose degradation by native cellulosomes, indicating the cellulosomal expansin-like proteins have the synergistic function. When they were integrated into a trivalent designer cellulosome, the synergistic effect was further amplified. The sequence and structure analyses indicated that these cellulosomal expansin-like proteins share the conserved functional mechanism with other bacterial expansin-like proteins. These results indicated that non-catalytic expansin-like proteins in the cellulosome can enhance the activity of the cellulosome in lignocellulose degradation. The involvement of functional expansin-like proteins in the cellulosome also implies new physiological functions of bacterial expansin-like proteins and cellulosomes. PMID:26521249

  4. EEVD motif of heat shock cognate protein 70 contributes to bacterial uptake by trophoblast giant cells

    Directory of Open Access Journals (Sweden)

    Kim Suk

    2009-12-01

    Full Text Available Abstract Background The uptake of abortion-inducing pathogens by trophoblast giant (TG cells is a key event in infectious abortion. However, little is known about phagocytic functions of TG cells against the pathogens. Here we show that heat shock cognate protein 70 (Hsc70 contributes to bacterial uptake by TG cells and the EEVD motif of Hsc70 plays an important role in this. Methods Brucella abortus and Listeria monocytogenes were used as the bacterial antigen in this study. Recombinant proteins containing tetratricopeptide repeat (TPR domains were constructed and confirmation of the binding capacity to Hsc70 was assessed by ELISA. The recombinant TPR proteins were used for investigation of the effect of TPR proteins on bacterial uptake by TG cells and on pregnancy in mice. Results The monoclonal antibody that inhibits bacterial uptake by TG cells reacted with the EEVD motif of Hsc70. Bacterial TPR proteins bound to the C-terminal of Hsc70 through its EEVD motif and this binding inhibited bacterial uptake by TG cells. Infectious abortion was also prevented by blocking the EEVD motif of Hsc70. Conclusions Our results demonstrate that surface located Hsc70 on TG cells mediates the uptake of pathogenic bacteria and proteins containing the TPR domain inhibit the function of Hsc70 by binding to its EEVD motif. These molecules may be useful in the development of methods for preventing infectious abortion.

  5. Molecular paleontology: a biochemical model of the ancestral ribosome

    OpenAIRE

    Hsiao, Chiaolong; Lenz, Timothy K.; Peters, Jessica K; Fang, Po-Yu; Schneider, Dana M.; Anderson, Eric J.; Preeprem, Thanawadee; Bowman, Jessica C.; O'Neill, Eric B.; Lie, Lively; Athavale, Shreyas S.; Gossett, J. Jared; Trippe, Catherine; Murray, Jason; Anton S. Petrov

    2013-01-01

    Ancient components of the ribosome, inferred from a consensus of previous work, were constructed in silico, in vitro and in vivo. The resulting model of the ancestral ribosome presented here incorporates ∼20% of the extant 23S rRNA and fragments of five ribosomal proteins. We test hypotheses that ancestral rRNA can: (i) assume canonical 23S rRNA-like secondary structure, (ii) assume canonical tertiary structure and (iii) form native complexes with ribosomal protein fragments. Footprinting exp...

  6. PKA-dependent phosphorylation of ribosomal protein S6 does not correlate with translation efficiency in striatonigral and striatopallidal medium-sized spiny neurons.

    Science.gov (United States)

    Biever, Anne; Puighermanal, Emma; Nishi, Akinori; David, Alexandre; Panciatici, Claire; Longueville, Sophie; Xirodimas, Dimitris; Gangarossa, Giuseppe; Meyuhas, Oded; Hervé, Denis; Girault, Jean-Antoine; Valjent, Emmanuel

    2015-03-11

    Ribosomal protein S6 (rpS6), a component of the 40S ribosomal subunit, is phosphorylated on several residues in response to numerous stimuli. Although commonly used as a marker for neuronal activity, its upstream mechanisms of regulation are poorly studied and its role in protein synthesis remains largely debated. Here, we demonstrate that the psychostimulant d-amphetamine (d-amph) markedly increases rpS6 phosphorylation at Ser235/236 sites in both crude and synaptoneurosomal preparations of the mouse striatum. This effect occurs selectively in D1R-expressing medium-sized spiny neurons (MSNs) and requires the cAMP/PKA/DARPP-32/PP-1 cascade, whereas it is independent of mTORC1/p70S6K, PKC, and ERK signaling. By developing a novel assay to label nascent peptidic chains, we show that the rpS6 phosphorylation induced in striatonigral MSNs by d-amph, as well as in striatopallidal MSNs by the antipsychotic haloperidol or in both subtypes by papaverine, is not correlated with the translation of global or 5' terminal oligopyrimidine tract mRNAs. Together, these results provide novel mechanistic insights into the in vivo regulation of the post-translational modification of rpS6 in the striatum and point out the lack of a relationship between PKA-dependent rpS6 phosphorylation and translation efficiency. PMID:25762659

  7. New ribosome-inactivating proteins with polynucleotide:adenosine glycosidase and antiviral activities from Basella rubra L. and bougainvillea spectabilis Willd.

    Science.gov (United States)

    Bolognesi, A; Polito, L; Olivieri, F; Valbonesi, P; Barbieri, L; Battelli, M G; Carusi, M V; Benvenuto, E; Del Vecchio Blanco, F; Di Maro, A; Parente, A; Di Loreto, M; Stirpe, F

    1997-12-01

    New single-chain (type 1) ribosome-inactivating proteins (RIPs) were isolated from the seeds of Basella rubra L. (two proteins) and from the leaves of Bougainvillea spectabilis Willd. (one protein). These RIPs inhibit protein synthesis both in a cell-free system, with an IC50 (concentration causing 50% inhibition) in the 10(-10) M range, and by various cell lines, with IC50S in the 10(-8)-10(-6) M range. All three RIPs released adenine not only from rat liver ribosomes but also from Escherichia coli rRNA, polyadenylic acid, herring sperm DNA, and artichoke mottled crinkle virus (AMCV) genomic RNA, thus being polynucleotide:adenosine glycosidases. The proteins from Basella rubra had toxicity to mice similar to that of most type 1 RIPs (Barbieri et al., 1993, Biochim Biophys Acta 1154: 237-282) with an LD50 (concentration that is 50% lethal) 32 mg.kg-1. The N-terminal sequence of the two RIPs from Basella rubra had 80-93% identity, whereas it differed from the sequence of the RIP from Bougainvillea spectabilis. When tested with antibodies against various RIPs, the RIPs from Basella gave some cross-reactivity with sera against dianthin 32, and weak cross-reactivity with momordin I and momorcochin-S, whilst the RIP from Bougainvillea did not cross-react with any antiserum tested. An RIP from Basella rubra and one from Bougainvillea spectabilis were tested for antiviral activity, and both inhibited infection of Nicotiana benthamiana by AMCV. PMID:9421927

  8. An RNA-binding complex involved in ribosome biogenesis contains a protein with homology to tRNA CCA-adding enzyme.

    Directory of Open Access Journals (Sweden)

    Jinzhong Lin

    2013-10-01

    Full Text Available A multitude of proteins and small nucleolar RNAs transiently associate with eukaryotic ribosomal RNAs to direct their modification and processing and the assembly of ribosomal proteins. Utp22 and Rrp7, two interacting proteins with no recognizable domain, are components of the 90S preribosome or the small subunit processome that conducts early processing of 18S rRNA. Here, we determine the cocrystal structure of Utp22 and Rrp7 complex at 1.97 Å resolution and the NMR structure of a C-terminal fragment of Rrp7, which is not visible in the crystal structure. The structure reveals that Utp22 surprisingly resembles a dimeric class I tRNA CCA-adding enzyme yet with degenerate active sites, raising an interesting evolutionary connection between tRNA and rRNA processing machineries. Rrp7 binds extensively to Utp22 using a deviant RNA recognition motif and an extended linker. Functional sites on the two proteins were identified by structure-based mutagenesis in yeast. We show that Rrp7 contains a flexible RNA-binding C-terminal tail that is essential for association with preribosomes. RNA-protein crosslinking shows that Rrp7 binds at the central domain of 18S rRNA and shares a neighborhood with two processing H/ACA snoRNAs snR30 and snR10. Depletion of snR30 prevents the stable assembly of Rrp7 into preribosomes. Our results provide insight into the evolutionary origin and functional context of Utp22 and Rrp7.

  9. Blocking of bacterial biofilm formation by a fish protein coating

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk; Klemm, Per

    2008-01-01

    proteinaceous coating is characterized with regards to its biofilm-reducing properties by using a range of urinary tract infectious isolates with various pathogenic and adhesive properties. The antiadhesive coating significantly reduced or delayed biofilm formation by all these isolates under every condition......Bacterial biofilm formation on inert surfaces is a significant health and economic problem in a wide range of environmental, industrial, and medical areas. Bacterial adhesion is generally a prerequisite for this colonization process and, thus, represents an attractive target for the development of...... biofilm-preventive measures. We have previously found that the preconditioning of several different inert materials with an aqueous fish muscle extract, composed primarily of fish muscle alpha-tropomyosin, significantly discourages bacterial attachment and adhesion to these surfaces. Here, this...

  10. Data presenting a modified bacterial expression vector for expressing and purifying Nus solubility-tagged proteins.

    Science.gov (United States)

    Gupta, Nidhi; Wu, Heng; Terman, Jonathan R

    2016-09-01

    Bacteria are the predominant source for producing recombinant proteins but while many exogenous proteins are expressed, only a fraction of those are soluble. We have found that a new actin regulatory enzyme Mical is poorly soluble when expressed in bacteria but the use of a Nus fusion protein tag greatly increases its solubility. However, available vectors containing a Nus tag have been engineered in a way that hinders the separation of target proteins from the Nus tag during protein purification. We have now used recombinant DNA approaches to overcome these issues and reengineer a Nus solubility tag-containing bacterial expression vector. The data herein present a modified bacterial expression vector useful for expressing proteins fused to the Nus solubility tag and separating such target proteins from the Nus tag during protein purification. PMID:27547802

  11. Organization of a large gene cluster encoding ribosomal proteins in the cyanobacterium Synechococcus sp. strain PCC 6301: comparison of gene clusters among cyanobacteria, eubacteria and chloroplast genomes.

    Science.gov (United States)

    Sugita, M; Sugishita, H; Fujishiro, T; Tsuboi, M; Sugita, C; Endo, T; Sugiura, M

    1997-08-11

    The structure of a large gene cluster containing 22 ribosomal protein (r-protein) genes of the cyanobacterium Synechococcus sp. strain PCC6301 is presented. Based on DNA and protein sequence analyses, genes encoding r-proteins L3, L4, L23, L2, S19, L22, S3, L16, L29, S17, L14, L24, L5, S8, L6, L18, S5, L15, L36, S13, S11, L17, SecY, adenylate kinase (AK) and the alpha subunit of RNA polymerase were identified. The gene order is similar to that of the E. coli S10, spc and alpha operons. Unlike the corresponding E. coli operons, the genes for r-proteins S4, S10, S14 and L30 are not present in this cluster. The organization of Synechococcus r-protein genes also resembles that of chloroplast (cp) r-protein genes of red and brown algal species. This strongly supports the endosymbiotic theory that the cp genome evolved from an ancient photosynthetic bacterium. PMID:9300823

  12. Imaging bacterial protein expression using genetically encoded sensors composed of RNA

    OpenAIRE

    Song, Wenjiao; Strack, Rita L.; Jaffrey, Samie R.

    2013-01-01

    We show that the difficulties in imaging the dynamics of protein expression in live bacterial cells can be overcome using fluorescent sensors based on Spinach, an RNA that activates the fluorescence of a small-molecule fluorophore. These RNAs selectively bind target proteins, and exhibit fluorescence increases that enable protein expression to be imaged in living cells. These sensors provide a general strategy to image protein expression in single bacteria in real-time.

  13. Transduced PEP-1-ribosomal protein S3 (rpS3) ameliorates 12-O-tetradecanoylphorbol-13-acetate-induced inflammation in mice

    International Nuclear Information System (INIS)

    This study investigated the preventive effect of ribosomal protein S3 (rpS3) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema in mice. A cell permeable expression vector PEP-1-rpS3 was constructed. Topical application of the vector markedly inhibited TPA-induced expression levels of cyclooxygenase-2 (COX-2) and pro-inflammatory cytokines. Application of PEP-1-rpS3 also resulted in a significant reduction in the activation of nuclear factor-kappa B (NF-kB) and mitogen-activated protein kinase (MAPK) in TPA-treated ears. These results indicate that PEP-1-rpS3 inhibits inflammatory response cytokines and enzymes by blocking NF-kB and MAPK, prompting the suggestion that PEP-1-rpS3 can be used as a therapeutic agent against skin inflammation.

  14. The trp RNA-binding attenuation protein of Bacillus subtilis regulates translation of the tryptophan transport gene trpP (yhaG) by blocking ribosome binding.

    Science.gov (United States)

    Yakhnin, Helen; Zhang, Hong; Yakhnin, Alexander V; Babitzke, Paul

    2004-01-01

    Expression of the Bacillus subtilis tryptophan biosynthetic genes (trpEDCFBA and pabA [trpG]) is regulated in response to tryptophan by TRAP, the trp RNA-binding attenuation protein. TRAP-mediated regulation of the tryptophan biosynthetic genes includes a transcription attenuation and two distinct translation control mechanisms. TRAP also regulates translation of trpP (yhaG), a single-gene operon that encodes a putative tryptophan transporter. Its translation initiation region contains triplet repeats typical of TRAP-regulated mRNAs. We found that regulation of trpP and pabA is unaltered in a rho mutant strain. Results from filter binding and gel mobility shift assays demonstrated that TRAP binds specifically to a segment of the trpP transcript that includes the untranslated leader and translation initiation region. While the affinities of TRAP for the trpP and pabA transcripts are similar, TRAP-mediated translation control of trpP is much more extensive than for pabA. RNA footprinting revealed that the trpP TRAP binding site consists of nine triplet repeats (five GAG, three UAG, and one AAG) that surround and overlap the trpP Shine-Dalgarno (S-D) sequence and translation start codon. Results from toeprint and RNA-directed cell-free translation experiments indicated that tryptophan-activated TRAP inhibits TrpP synthesis by preventing binding of a 30S ribosomal subunit. Taken together, our results establish that TRAP regulates translation of trpP by blocking ribosome binding. Thus, TRAP coordinately regulates tryptophan synthesis and transport by three distinct mechanisms: attenuation transcription of the trpEDCFBA operon, promoting formation of the trpE S-D blocking hairpin, and blocking ribosome binding to the pabA and trpP transcripts. PMID:14702295

  15. The diagnostic value of c-reactive protein estimation in differentiating bacterial from viral meningitis

    International Nuclear Information System (INIS)

    Objective: To evaluate the efficacy of serum and CSF C-reactive protein (C-rp) in differentiating bacterial from viral meningitis. Design: An observational, respective hospital-based study. Place and duration of study: It was conducted at the Department of Medicine and Department of Pediatrics, Shaikh Zayed Postgraduate Medical Institute Lahore, Over a Period of one year between march, 1999 and March, 2000. Subject and Methods: A randomized group of thirty patients, who presented with clinical features, suggestive of meningitis, were included in the study. C-reactive protein determinations were performed by latex agglutination method on the serum and cerebrospinal fluid (CSF) of these patients. Results: In the present study, c-reactive protein was found to be a more sensitive test for differentiating bacterial from non-bacterial meningitis on initial examination than the usual conventional methods used to diagnose bacterial meningitis. CSF C-reactive protein had a greater sensitivity (92% as compared to serum C-reactive protein (71%). Conclusion: C-reactive protein determination in CSF was found to be a useful indicator of bacterial meningitis that can be used to distinguish it from viral meningitis. (author)

  16. Extinction, applied after retrieval of auditory fear memory, selectively increases zinc-finger protein 268 and phosphorylated ribosomal protein S6 expression in prefrontal cortex and lateral amygdala.

    Science.gov (United States)

    Tedesco, Vincenzo; Roquet, Rheall F; DeMis, John; Chiamulera, Cristiano; Monfils, Marie-H

    2014-11-01

    Retrieval of consolidated memories induces a labile phase during which memory can be disrupted or updated through a reconsolidation process. A central component of behavioral updating during reconsolidation using a retrieval-extinction manipulation (Ret+Ext) is the synaptic removal of a calcium-permeable-α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (CP-AMPARs) in the lateral amygdala-a metabotropic GluR1 receptor (mGluR1) dependent mechanism. In the present study, we investigate the effect of Ret+Ext on the expression of molecular markers that could play a role in the reconsolidation process. Specifically, we tested the effects of Ret+Ext on the global expression of zinc-finger 268 protein (Zif268), a marker previously found to be implicated in memory reconsolidation, to confirm its occurrence after retrieval (Ret) and Ret+Ext. We also evaluated the global expression of phosphorylated ribosomal protein S6 (rpS6P), here proposed as a marker of the mGluR1-mediated memory process induced by Ret+Ext. The expression of both markers (zif268, rpS6P) was assessed by immunolocalization in prelimbic cortex (PRL), infralimbic cortex (IL), ventral subdivision of the lateral amygdala (LA) and hippocampus CA1 (CA1) in fear-conditioned rats. Our results showed that retrieval and Ret+Ext, but not extinction alone, increased Zif268 expression in prefrontal cortex and lateral amygdala. Ret+Ext, but not retrieval, retrieval followed by context exposure or extinction alone, increased the expression of rpS6P in prefrontal cortex and LA. In summary, (i) Zif268 increased after retrieval confirming that reconsolidation is engaged in our conditions, (ii) Zif268 increased after Ret+Ext confirming that it does not simply reflect an extinction or reconsolidation disruption (Zif268 level of expression should be lower in both cases) and (iii) rpS6P increased after Ret+Ext, but not after extinction, suggesting, as expected, a potential mGluR1 mediated molecular mechanism specific

  17. Viral IRES RNA structures and ribosome interactions

    OpenAIRE

    Kieft, Jeffrey S.

    2008-01-01

    In eukaryotes, protein synthesis initiates primarily by a mechanism that requires a modified nucleotide ‘cap’ on the mRNA and also proteins that recruit and position the ribosome. Many pathogenic viruses use an alternative, cap-independent mechanism that substitutes RNA structure for the cap and many proteins. The RNAs driving this process are called internal ribosome-entry sites (IRESs) and some are able to bind the ribosome directly using a specific 3D RNA structure. Recent structures of IR...

  18. Interactions That Drive Sec-Dependent Bacterial Protein Transport†

    OpenAIRE

    Rusch, Sharyn L.; Kendall, Debra A.

    2007-01-01

    Understanding the transport of hydrophilic proteins across biological membranes continues to be an important undertaking. The general secretory (Sec) pathway in Escherichia coli transports the majority of E. coli proteins from their point of synthesis in the cytoplasm to their sites of final localization, associating sequentially with a number of protein components of the transport machinery. The targeting signals for these substrates must be discriminated from those of proteins transported v...

  19. Mutations in the leader region of ribosomal RNA operons cause structurally defective 30 S ribosomes as revealed by in vivo structural probing.

    Science.gov (United States)

    Balzer, M; Wagner, R

    1998-02-27

    The biogenesis of functional ribosomes is regulated in a very complex manner, involving different proteins and RNA molecules. RNAs are not only essential components of both ribosomal subunits but also transiently interacting factors during particle formation. In eukaryotes snoRNAs act as molecular chaperones to assist maturation, modification and assembly. In a very similar way highly conserved leader sequences of bacterial rRNA operons are involved in the correct formation of 30 S ribosomal subunits. Certain mutations in the rRNA leader region cause severe growth defects due to malfunction of ribosomes which are assembled from such transcription units. To understand how the leader sequences act to facilitate the formation of the correct 30 S subunits we performed in vivo chemical probing to assess structural differences between ribosomes assembled either from rRNA transcribed from wild-type operons or from operons which contain mutations in the rRNA leader region. Cells transformed with plasmids containing the respective rRNA operons were reacted with dimethylsulphate (DMS). Ribosomes were isolated by sucrose gradient centrifugation and modified nucleotides within the 16 S rRNA were identified by primer extension reaction. Structural differences between ribosomes from wild-type and mutant rRNA operons occur in several clusters within the 16 S rRNA secondary structure. The most prominent differences are located in the central domain including the universally conserved pseudoknot structure which connects the 5', the central and the 3' domain of 16 S rRNA. Two other clusters with structural differences fall in the 5' domain where the leader had been shown to interact with mature 16 S rRNA and within the ribosomal protein S4 binding site. The other differences in structure are located in sites which are also known as sites for the action of several antibiotics. The data explain the functional defects of ribosomes from rRNA operons with leader mutations and help to

  20. Ribosome dynamics and the evolutionary history of ribosomes

    Science.gov (United States)

    Fox, George E.; Paci, Maxim; Tran, Quyen; Petrov, Anton S.; Williams, Loren D.

    2015-09-01

    The ribosome is a dynamic nanomachine responsible for coded protein synthesis. Its major subsystems were essentially in place at the time of the last universal common ancestor (LUCA). Ribosome evolutionary history thus potentially provides a window into the pre- LUCA world. This history begins with the origins of the peptidyl transferase center where the actual peptide is synthesized and then continues over an extended timeframe as additional functional centers including the GTPase center are added. The large ribosomal RNAs (rRNAs) have grown over time by an accretion process and a model exists that proposes a relative age of each accreted element. We have compared atomic resolution ribosome structures before and after EF-G bound GTP hydrolysis and thereby identified the location of 23 pivot points in the large rRNAs that facilitate ribosome dynamics. Pivots in small subunit helices h28 and h44 appear to be especially central to the process and according to the accretion model significantly older than the other helices containing pivots. Overall, the results suggest that ribosomal dynamics occurred in two phases. In the first phase, an inherently mobile h28/h44 combination provided the flexibility needed to create a dynamic ribosome that was essentially a Brownian machine. This addition likely made coded peptide synthesis possible by facilitating movement of a primitive mRNA. During the second phase, addition of pivoting elements and the creation of a factor binding site allowed the regulation of the inherent motion created by h28/h44. All of these events likely occurred before LUCA.

  1. Protein folding: understanding the role of water and the low Reynolds number environment as the peptide chain emerges from the ribosome and folds.

    Science.gov (United States)

    Sen, Siddhartha; Voorheis, H Paul

    2014-12-21

    The mechanism of protein folding during early stages of the process has three determinants. First, moving water molecules obey the rules of low Reynolds number physics without an inertial component. Molecular movement is instantaneous and size insensitive. Proteins emerging from the ribosome move and rotate without an external force if they change shape, forming and propagating helical structures that increases translocational efficiency. Forward motion ceases when the shape change or propelling force ceases. Second, application of quantum field theory to water structure predicts the spontaneous formation of low density coherent units of fixed size that expel dissolved atmospheric gases. Structured water layers with both coherent and non-coherent domains, form a sheath around the new protein. The surface of exposed hydrophobic amino acids is protected from water contact by small nanobubbles of dissolved atmospheric gases, 5 or 6 molecules on average, that vibrate, attracting even widely separated resonating nanobubbles. This force results from quantum effects, appearing only when the system is within and interacts with an oscillating electromagnetic field. The newly recognized quantum force sharply bends the peptide and is part of a dynamic field determining the pathway of protein folding. Third, the force initiating the tertiary folding of proteins arises from twists at the position of each hydrophobic amino acid, that minimizes surface exposure of the hydrophobic amino acids and propagates along the protein. When the total bend reaches 360°, the leading segment of water sheath intersects the trailing segment. This steric self-intersection expels water from overlapping segments of the sheath and by Newton׳s second law moves the polypeptide chain in an opposite direction. Consequently, with very few exceptions that we enumerate and discuss, tertiary structures are absent from proteins without hydrophobic amino acids, which control the early stages of protein

  2. Meat, dairy and plant proteins alter bacterial composition of rat gut bacteria

    OpenAIRE

    Yingying Zhu; Xisha Lin; Fan Zhao; Xuebin Shi; He Li; Yingqiu Li; Weiyun Zhu; Xinglian Xu; Chunbao Lu; Guanghong Zhou

    2015-01-01

    Long-term consumption of red meat has been considered a potential risk to gut health, but this is based on clinic investigations, excessive intake of fat, heme and some injurious compounds formed during cooking or additions to processed meat products. Whether intake of red meat protein affects gut bacteria and the health of the host remains unclear. In this work, we compared the composition of gut bacteria in the caecum, by sequencing the V4-V5 region of 16S ribosomal RNA gene, obtained from ...

  3. The DNA virus white spot syndrome virus uses an internal ribosome entry site for translation of the highly expressed nonstructural protein ICP35.

    Science.gov (United States)

    Kang, Shih-Ting; Wang, Han-Ching; Yang, Yi-Ting; Kou, Guang-Hsiung; Lo, Chu-Fang

    2013-12-01

    Although shrimp white spot syndrome virus (WSSV) is a large double-stranded DNA virus (∼300 kbp), it expresses many polycistronic mRNAs that are likely to use internal ribosome entry site (IRES) elements for translation. A polycistronic mRNA encodes the gene of the highly expressed nonstructural protein ICP35, and here we use a dual-luciferase assay to demonstrate that this protein is translated cap independently by an IRES element located in the 5' untranslated region of icp35. A deletion analysis of this region showed that IRES activity was due to stem-loops VII and VIII. A promoterless assay, a reverse transcription-PCR together with quantitative real-time PCR analysis, and a stable stem-loop insertion upstream of the Renilla luciferase open reading frame were used, respectively, to rule out the possibility that cryptic promoter activity, abnormal splicing, or read-through was contributing to the IRES activity. In addition, a Northern blot analysis was used to confirm that only a single bicistronic mRNA was expressed. The importance of ICP35 to viral replication was demonstrated in a double-stranded RNA (dsRNA) interference knockdown experiment in which the mortality of the icp35 dsRNA group was significantly reduced. Tunicamycin was used to show that the α subunit of eukaryotic initiation factor 2 is required for icp35 IRES activity. We also found that the intercalating drug quinacrine significantly inhibited icp35 IRES activity in vitro and reduced the mortality rate and viral copy number in WSSV-challenged shrimp. Lastly, in Sf9 insect cells, we found that knockdown of the gene for the Spodoptera frugiperda 40S ribosomal protein RPS10 decreased icp35 IRES-regulated firefly luciferase activity but had no effect on cap-dependent translation. PMID:24089551

  4. High efficacy of a 20 amino acid peptide of the acidic ribosomal protein P0 against the cattle tick, Rhipicephalus microplus.

    Science.gov (United States)

    Rodríguez-Mallon, Alina; Encinosa, Pedro E; Méndez-Pérez, Lídice; Bello, Yamil; Rodríguez Fernández, Rafmary; Garay, Hilda; Cabrales, Ania; Méndez, Luis; Borroto, Carlos; Estrada, Mario Pablo

    2015-06-01

    Current strategies to control cattle ticks use integrated control programs (ICP) that include vaccination. Reduction in the use of chemicals and in the cost of tick control, the delay or elimination of acaricide resistance and the decreasing of environmental pollution are the advantages of using these programs. This integrated program is potentially applicable to all genotypes of chemical resistant ticks. However, the problem here is to improve the efficacy of anti-tick vaccines. The P0 protein is a structural component of the ribosome of all organisms. We have identified an immunogenic region of ribosomal protein P0 from Rhipicephalus spp. ticks that is not very conserved compared to the orthologous protein in their hosts. A synthetic 20 amino acid peptide from this sequence was effective as a vaccine against Rhipicephalus sanguineus infestations in an immunization and challenge experiment using rabbits. In this paper, the same peptide used as vaccine against the cattle tick Rhipicephalus Boophilus microplus shows a significant diminution in the number of engorged females recovered, in the weight of females and the weight of egg masses. The number of eggs hatched was also significantly reduced for the vaccinated group, with an overall effectivity for the antigen pP0 of 96%. These results, together with the conserved sequence of the P0 peptide among ticks, suggest that this antigen could be a good broad spectrum vaccine candidate. It would be expected to be active against many species of ticks and thus has promise in an ICP for effective control of ticks and thereby to improve the efficiency and productivity of the livestock industry. PMID:25958782

  5. Visualizing translocation and localization of bacterial type III effector proteins using a genetically encoded reporter system

    OpenAIRE

    Gawthorne, Jayde A.; Audry, Laurent; McQuitty, Claire; Dean, Paul; Christie, John M.; Enninga, Jost; Andrew J. Roe

    2016-01-01

    Bacterial Type Three Secretion System (T3SS) effector proteins are critical determinants of infection for many animal and plant pathogens. However, monitoring of the translocation and delivery of these important virulence determinants has proved to be technically challenging. Here, we used a genetically engineered LOV (light-oxygen-voltage) sensing domain derivative to monitor the expression, translocation and localization of bacterial T3SS effectors. We found the Escherichia coli O157:H7 bac...

  6. Identification of the oligonucleotide and oligopeptide involved in an RNA-protein crosslink induced by ultraviolet irradiation of Escherichia coli 30 S ribosomal subunits

    International Nuclear Information System (INIS)

    When 30 S ribosomal subunits are irradiated with ultraviolet light, it has been found that an RNA-protein crosslinking reaction occurs whose primary target is protein S7. This paper describes the identification of the oligopeptide and oligonucleotide at the crosslinking point, by direct analysis (a) of the peptide remaining attached to an oligonucleotide (after total digestion of the RNA in the crosslinked complex with ribonucleases A and Tperpendicular, followed by digestion with trypsin), and (b) of the nucleotides remaining attached to the crosslinked protein (after digestion of the RNA in the complex with ribonuclease Tperpendicular alone). The crosslinking site was found to lie within a single short peptide, Ser-Met-Ala-Leu-Arg (positions 113 to 117 in the S7 sequence), with methionine as the probable amino acid concerned. The principal RNA site was found to lie within an oligonucleotide three to six bases long, the bracketed portion of the partially ordered sequence C-U-A-C-[A-A-U-G.G.C]-G in section P of the 16 S RNA. The methodology involved has been designed with a view to being generally applicable in future RNA-protein crosslinking studies, where several proteins are simultaneously attached to the RNA. (author)

  7. Ribosomal studies on the 70S ribosome of E.coli by means of neutron scattering

    International Nuclear Information System (INIS)

    Ribosomes are ribonucleo-protein complexes, which catalyse proteinbiosynthesis in all living organisms. Currently, most of the structural models of the prokaryotic 70S ribosome rely on electron microscopy and describe mainly the outer shape of the particle. Neutron scattering can provide information on the internal structure of the ribosome. Parts of the structure can be contrasted for neutrons by means of an isotopic exchange of the naturally occurring hydrogen (1H) for deuterium (2H), allowing direct measurements in situ. Specifically deuterium-labeled ribosomes (E. coli) were prepared and analysed with neutron scattering. The biochemical methods were established and combined to a generally applicable preparation system. This allows labeling of all ribosomal components in any combination. A systematic analysis of the protein and RNA phases resulted in the development of a new model for the 70S ribosome. This model describes not only the outer shape of the particle, but displays also an experimentally determined internal protein-RNA distribution and the border of subunits for the first time (four-phase model; resolution: 50A). Models of the 70S ribosome from other studies were evaluated and ranked according to consistency with the measured scattering data. Applying a new neutron scattering technique of particular sensitivity, the proton-spin contrast-variation, single proteins could be measured and localized. The positions of the proteins S6 and S10 were determined, providing the first coordinates of protein mass centers within the 70S ribosome. (orig.)

  8. Identification of nucleosome assembly protein 1 (NAP1) as an interacting partner of plant ribosomal protein S6 (RPS6) and a positive regulator of rDNA transcription

    Energy Technology Data Exchange (ETDEWEB)

    Son, Ora [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Kim, Sunghan [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921 (Korea, Republic of); Shin, Yun-jeong [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Kim, Woo-Young [College of Pharmacy, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Koh, Hee-Jong, E-mail: heejkoh@snu.ac.kr [Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921 (Korea, Republic of); Cheon, Choong-Ill, E-mail: ccheon@sookmyung.ac.kr [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of)

    2015-09-18

    The ribosomal protein S6 (RPS6) is a downstream component of the signaling mediated by the target of rapamycin (TOR) kinase that acts as a central regulator of the key metabolic processes, such as protein translation and ribosome biogenesis, in response to various environmental cues. In our previous study, we identified a novel role of plant RPS6, which negatively regulates rDNA transcription, forming a complex with a plant-specific histone deacetylase, AtHD2B. Here we report that the Arabidopsis RPS6 interacts additionally with a histone chaperone, nucleosome assembly protein 1(AtNAP1;1). The interaction does not appear to preclude the association of RPS6 with AtHD2B, as the AtNAP1 was also able to interact with AtHD2B as well as with an RPS6-AtHD2B fusion protein in the BiFC assay and pulldown experiment. Similar to a positive effect of the ribosomal S6 kinase 1 (AtS6K1) on rDNA transcription observed in this study, overexpression or down regulation of the AtNAP1;1 resulted in concomitant increase and decrease, respectively, in rDNA transcription suggesting a positive regulatory role played by AtNAP1 in plant rDNA transcription, possibly through derepression of the negative effect of the RPS6-AtHD2B complex. - Highlights: • Nucleosome assembly protein 1 (AtNAP1) interacts with RPS6 as well as with AtHD2B. • rDNA transcription is regulated S6K1. • Overexpression or down regulation of AtNAP1 results in concomitant increase or decrease in rDNA transcription.

  9. Identification of nucleosome assembly protein 1 (NAP1) as an interacting partner of plant ribosomal protein S6 (RPS6) and a positive regulator of rDNA transcription

    International Nuclear Information System (INIS)

    The ribosomal protein S6 (RPS6) is a downstream component of the signaling mediated by the target of rapamycin (TOR) kinase that acts as a central regulator of the key metabolic processes, such as protein translation and ribosome biogenesis, in response to various environmental cues. In our previous study, we identified a novel role of plant RPS6, which negatively regulates rDNA transcription, forming a complex with a plant-specific histone deacetylase, AtHD2B. Here we report that the Arabidopsis RPS6 interacts additionally with a histone chaperone, nucleosome assembly protein 1(AtNAP1;1). The interaction does not appear to preclude the association of RPS6 with AtHD2B, as the AtNAP1 was also able to interact with AtHD2B as well as with an RPS6-AtHD2B fusion protein in the BiFC assay and pulldown experiment. Similar to a positive effect of the ribosomal S6 kinase 1 (AtS6K1) on rDNA transcription observed in this study, overexpression or down regulation of the AtNAP1;1 resulted in concomitant increase and decrease, respectively, in rDNA transcription suggesting a positive regulatory role played by AtNAP1 in plant rDNA transcription, possibly through derepression of the negative effect of the RPS6-AtHD2B complex. - Highlights: • Nucleosome assembly protein 1 (AtNAP1) interacts with RPS6 as well as with AtHD2B. • rDNA transcription is regulated S6K1. • Overexpression or down regulation of AtNAP1 results in concomitant increase or decrease in rDNA transcription

  10. Proteolytic activation of human pancreatitis associated protein is required for peptidoglycan binding and bacterial aggregation

    OpenAIRE

    Medveczky, Péter; Szmola, Richárd; Sahin-Tóth, Miklós

    2009-01-01

    Pancreatitis associated protein (PAP) is a 16 kDa lectin-like protein, which becomes robustly upregulated in the pancreatic juice during acute pancreatitis. Trypsin cleaves the N terminus of PAP, which in turn forms insoluble fibrils. PAP and its paralog the pancreatic stone protein induce bacterial aggregation and, more recently, PAP was shown to bind to the peptidoglycan of Gram positive bacteria and exert a direct bactericidal effect. However, the role of N-terminal processing in the antib...

  11. An overview of pre-ribosomal RNA processing in eukaryotes

    OpenAIRE

    Henras, Anthony K.; Plisson-Chastang, Célia; O'Donohue, Marie-Françoise; Chakraborty, Anirban; Gleizes, Pierre-Emmanuel

    2014-01-01

    Ribosomal RNAs are the most abundant and universal noncoding RNAs in living organisms. In eukaryotes, three of the four ribosomal RNAs forming the 40S and 60S subunits are borne by a long polycistronic pre-ribosomal RNA. A complex sequence of processing steps is required to gradually release the mature RNAs from this precursor, concomitant with the assembly of the 79 ribosomal proteins. A large set of trans-acting factors chaperone this process, including small nucleolar ribonucleoparticles. ...

  12. Essential bacterial helicases that counteract the toxicity of recombination proteins

    OpenAIRE

    Petit, Marie-Agnès; Ehrlich, Dusko

    2002-01-01

    PcrA, Rep and UvrD are three closely related bacterial helicases with a DExx signature. PcrA is encoded by Gram-positive bacteria and is essential for cell growth. Rep and UvrD are encoded by Gram-negative bacteria, and mutants lacking both helicases are also not viable. To understand the non-viability of the helicase mutants, we characterized spontaneous extragenic suppressors of a Bacillus subtilis pcrA null mutation. Here we report that one of these suppressors maps in recF and that previo...

  13. Bacterial S-layer protein coupling to lipids

    DEFF Research Database (Denmark)

    Weygand, M.; Wetzer, B.; Pum, D.;

    1999-01-01

    -filled cavities near its center. The protein volume fraction reaches maxima of >60% in two horizontal sections of the S-layer, close to the lipid monolayer and close to the free subphase. In between it drops to similar to 20%. Four S-layer protein monomers are located within the unit cell of a square lattice with...

  14. BACTERIAL SOLUTE TRANSPORT PROTEINS IN THEIR LIPID ENVIRONMENT

    NARCIS (Netherlands)

    TVELD, GI; DRIESSEN, AJM; KONINGS, WN; Veld, Gerda in 't

    1993-01-01

    The cytoplasmic membrane of bacteria is a selective barrier that restricts entry and exit of solutes. Transport of solutes across this membrane is catalyzed by specific membrane proteins. Integral membrane proteins usually require specific lipids for optimal activity and are inhibited by other lipid

  15. High-Throughput Screening of Bacterial Protein Localization

    OpenAIRE

    Werner, John N.; Gitai, Zemer

    2010-01-01

    The ever-increasing number of sequenced genomes and subsequent sequence-based analysis has provided tremendous insight into cellular processes; however, the ability to experimentally manipulate this genomic information in the laboratory requires the development of new high-throughput methods. To translate this genomic information into information on protein function, molecular and cell biological techniques are required. One strategy to gain insight into protein function is to observe where e...

  16. Potential Natural Products for Alzheimer’s Disease: Targeted Search Using the Internal Ribosome Entry Site of Tau and Amyloid-β Precursor Protein

    Directory of Open Access Journals (Sweden)

    Yun-Chieh Tasi

    2015-04-01

    Full Text Available Overexpression of the amyloid precursor protein (APP and the hyperphosphorylation of the tau protein are vital in the understanding of the cause of Alzheimer’s disease (AD. As a consequence, regulation of the expression of both APP and tau proteins is one important approach in combating AD. The APP and tau proteins can be targeted at the levels of transcription, translation and protein structural integrity. This paper reports the utilization of a bi-cistronic vector containing either APP or tau internal ribosome entry site (IRES elements flanked by β-galactosidase gene (cap-dependent and secreted alkaline phosphatase (SEAP (cap-independent to discern the mechanism of action of memantine, an N-methyl-d-aspartate (NMDA receptor antagonist. Results indicate that memantine could reduce the activity of both the APP and tau IRES at a concentration of ~10 μM (monitored by SEAP activity without interfering with the cap-dependent translation as monitored by the β-galactosidase assay. Western blot analysis of the tau protein in neuroblastoma (N2A and rat hippocampal cells confirmed the halting of the expression of the tau proteins. We also employed this approach to identify a preparation named NB34, extracts of Boussingaultia baselloides (madeira-vine fermented with Lactobacillus spp., which can function similarly to memantine in both IRES of APP and Tau. The water maze test demonstrated that NB34 could improve the spatial memory of a high fat diet induced neurodegeneration in apolipoprotein E-knockout (ApoE−/− mice. These results revealed that the bi-cistronic vector provided a simple, and effective platform in screening and establishing the mechanistic action of potential compounds for the treatment and management of AD.

  17. A bacterial two-hybrid system that utilizes Gateway cloning for rapid screening of protein-protein interactions.

    Science.gov (United States)

    Karna, S L Rajasekhar; Zogaj, Xhavit; Barker, Jeffrey R; Seshu, Janakiram; Dove, Simon L; Klose, Karl E

    2010-11-01

    Comprehensive clone sets representing the entire genome now exist for a large number of organisms. The Gateway entry clone sets are a particularly useful means to study gene function, given the ease of introduction into any Gateway-suitable destination vector. We have adapted a bacterial two-hybrid system for use with Gateway entry clone sets, such that potential interactions between proteins encoded within these clone sets can be determined by new destination vectors. We show that utilizing the Gateway clone sets for Francisella tularensis and Vibrio cholerae, known interactions between F. tularensis IglA and IglB and V. cholerae VipA and VipB could be confirmed with these destination vectors. Moreover, the introduction of unique tags into each vector allowed for visualization of the expressed hybrid proteins via Western immunoblot. This Gateway-suitable bacterial two-hybrid system provides a new tool for rapid screening of protein-protein interactions. PMID:21091448

  18. A simple yeast-based strategy to identify host cellular processes targeted by bacterial effector proteins.

    Directory of Open Access Journals (Sweden)

    Eran Bosis

    Full Text Available Bacterial effector proteins, which are delivered into the host cell via the type III secretion system, play a key role in the pathogenicity of gram-negative bacteria by modulating various host cellular processes to the benefit of the pathogen. To identify cellular processes targeted by bacterial effectors, we developed a simple strategy that uses an array of yeast deletion strains fitted into a single 96-well plate. The array is unique in that it was optimized computationally such that despite the small number of deletion strains, it covers the majority of genes in the yeast synthetic lethal interaction network. The deletion strains in the array are screened for hypersensitivity to the expression of a bacterial effector of interest. The hypersensitive deletion strains are then analyzed for their synthetic lethal interactions to identify potential targets of the bacterial effector. We describe the identification, using this approach, of a cellular process targeted by the Xanthomonas campestris type III effector XopE2. Interestingly, we discover that XopE2 affects the yeast cell wall and the endoplasmic reticulum stress response. More generally, the use of a single 96-well plate makes the screening process accessible to any laboratory and facilitates the analysis of a large number of bacterial effectors in a short period of time. It therefore provides a promising platform for studying the functions and cellular targets of bacterial effectors and other virulence proteins.

  19. Identification of Dominant Immunogenic Bacteria and Bacterial Proteins in Periodontitis

    DEFF Research Database (Denmark)

    Agerbæk, Mette Rylev; Haubek, Dorte; Birkelund, Svend;

    Marginal periodontitis is considered an infectious disease that triggers host inflammatory responses resulting in destruction of the periodontium. A complex biofilm of bacteria is associated with periodontitis. Some species have been identified as putative pathogens such as Porphyromonas gingivalis...... (P.g) and Actinobacillus actinomycetemcomitans (A.a), but the identity of dominate immunogens of these bacteria is poorly elucidated. The aim of the study was to identify dominant immunogenic proteins of P.g and A.a in patients suffering from chronic and aggressive periodontitis by proteomic analysis...... will be able to identify immunodominant proteins and potentially important virulence factors of putative periodontal pathogens....

  20. Low expression of the X-linked ribosomal protein S4 in human serous epithelial ovarian cancer is associated with a poor prognosis

    International Nuclear Information System (INIS)

    The X-linked ribosomal protein S4 (RPS4X), which is involved in cellular translation and proliferation, has previously been identified as a partner of the overexpressed multifunctional protein YB-1 in several breast cancer cells. Depletion of RPS4X results in consistent resistance to cisplatin in such cell lines. As platinum-based chemotherapy is a standard first line therapy used to treat patients with ovarian cancer, we evaluated the prognostic value of RPS4X and YB-1 at the protein level in specimen from 192 high-grade serous epithelial ovarian cancer patients. Immunohistochemistry studies indicated that high expression of RPS4X was associated with a lower risk of death and later disease progression (HR = 0.713, P = 0.001 and HR = 0.761, P = 0.001, respectively) as compared to low expression of RPS4X. In contrast, YB-1 was not significantly associated with either recurrence or survival time in this cohort. Finally, the depletion of RPS4X with different siRNAs in two different ovarian cancer cell lines reduced their proliferative growth rate but more importantly increased their resistance to cisplatin. Altogether, these results suggest that the levels of RPS4X could be a good indicator for resistance to platinum-based therapy and a prognostic marker for ovarian cancer. Our study also showed that RPS4X is an independent prognostic factor in patients with serous epithelial ovarian cancer

  1. Expression of Ribosomal RNA and Protein Genes in Human Embryonic Stem Cells Is Associated With the Activating H3K4me3 Histone Mark.

    Science.gov (United States)

    Zaidi, Sayyed K; Boyd, Joseph R; Grandy, Rodrigo A; Medina, Ricardo; Lian, Jane B; Stein, Gary S; Stein, Janet L

    2016-09-01

    Embryonic stem cells (ESCs) exhibit unrestricted and indefinite, but stringently controlled, proliferation, and can differentiate into any lineage in the body. In the current study, we test the hypothesis that expression of ribosomal RNA (rRNA) and ribosomal protein genes (RPGs) contribute to the ability of hESCs to proliferate indefinitely. Consistent with the accelerated growth rate of hESCs, we find that hESC lines H1 and H9 both exhibit significantly higher levels of rRNA when compared to a panel of normal and cancer human cell lines. Although many RPGs are expressed at levels that comparable to other human cell lines, a few RPGs also exhibit higher expression levels. In situ nuclear run-on assays reveal that both nucleoli in hESCs actively transcribe nascent rRNA. Employing genome-wide chromatin immunoprecipitation-deep sequencing and bioinformatics approaches, we discovered that, RPGs are dominantly marked by the activating H3K4me3 histone mark in the G1, M, and G2 phases of the cell cycle. Interestingly, the rDNA repeats are marked by the activating H3K4me3 only in the M phase, and repressive H3K27me3 histone mark in all three cell cycle phases. Bioinformatics analyses also reveal that Myc, a known regulator of cell growth and proliferation, occupies both the rRNA genes and RPGs. Functionally, down-regulation of Myc expression by siRNA results in a concomitant decrease in rRNA levels. Together, our results show that expression of rRNA, which is regulated by the Myc pluripotency transcription factor, and of RPGs in hESCs is associated with the activating H3K4me3 modification. J. Cell. Physiol. 231: 2007-2013, 2016. © 2016 Wiley Periodicals, Inc. PMID:26755341

  2. Specific dose-dependent damage of Lieberkuehn crypts promoted by large doses of type 2 ribosome-inactivating protein nigrin b intravenous injection to mice

    International Nuclear Information System (INIS)

    Nigrin b is a non-toxic type 2 ribosome-inactivating protein as active as ricin at ribosomal level but 105 and 5 x 103 times less toxic for animal cell cultures and mice, respectively, than ricin. The purpose of the present study was to analyze the effects of intravenous injection of large amounts of nigrin b to the mouse. Injection through the tail vein of 16 mg/kg body weight killed all mice studied before 2 days. Analysis of several major tissues by light microscopy did not reveal gross nigrin b-promoted changes, except in the intestines which appeared highly damaged. As a consequence of the injury, the villi and crypt structures of the small intestine disappeared, leading to profuse bleeding and death. In contrast, intravenous injection of 5 mg/kg body weight was not lethal to mice but did trigger reversible toxic effects. In both cases, lethal and sub-lethal doses, the target of nigrin b appeared to be the highly proliferating stem cells of the intestinal crypts, which had undergone apoptotic changes. In contrast to nigrin b, the injection of 3 μg/kg of ricin kills all mice in 5 days but does not trigger apoptosis in the crypts. Therefore, the effect seen with sub-lethal nigrin b concentrations seems to be specific. Nigrin b killed COLO 320 human colon adenocarcinoma cells with an IC50 of 3.1 x 10-8 M and the effect was parallel to the extent of DNA fragmentation of these cells. Accordingly, despite the low general toxicity exerted by nigrin b as compared with ricin, intravenous injection of large amounts of nigrin b is able to kill mouse intestinal stem cells without threatening the lives of the animals, thereby opening a door for its use for the targeting of intestinal stem cells

  3. Prediction of Bacterial Virulent Proteins with Composition Moment Vector Feature Encoding Method

    Directory of Open Access Journals (Sweden)

    Gök Murat

    2016-01-01

    Full Text Available Prediction of bacterial virulent proteins is critical for vaccine development and understanding of virulence mechanisms in pathogens. For this purpose, a number of feature encoding methods based on sequences and evolutionary information of a given protein have been proposed and applied with some classifier algorithms so far. In this paper, we performed composition moment vector (CMV, which includes information about both composition and position of amino acid in the protein sequence to predict bacterial virulent proteins. The tests were validated in three different independent datasets. Experimental results show that CMV feature encoding method leads to better classification performance in terms of accuracy, sensitivity, f-measure and the Matthews correlation coefficient (MCC scores on diverse classifiers.

  4. Biochemical characterization of three mycobacterial ribosomal fractions.

    Science.gov (United States)

    Portelance, V; Beaudet, R

    1983-02-01

    The induction of antituberculous immunity by crude ribosomal fractions isolated from Mycobacterium tuberculosis strain H37Ra, M. bovis strain BCG, and M. smegmatis was studied in CF-1 mice. Levels of antituberculous immunity similar to that induced by live BCG were induced by the BCG and H37Ra ribosomal fractions whereas that isolated from M. smegmatis was found to be inactive. Electrophoresis of the three ribosomal fractions in sodium dodecyl sulfate - polyacylamide gels followed by differential staining showed the two active ribosomal fractions to be similar in their proteins, carbohydrate-containing substances, and lipid profiles. The inactive smegmatis ribosomal fraction differed mainly from the active ones on the basis of its carbohydrate-containing substances profile and by the absence of lipids. The polysaccharides and the ribosomes present in the H37Ra ribosomal fractions were purified by affinity chromatography on concanavalin A - Sepharose 4B. Each purified preparation showed no or only low antituberculous activity when injected separately, but when mixed together a high protection was observed. The formation of complexes between the ribosomes and the polysaccharide fraction was suggested and appears to be necessary for the induction of antituberculous immunity. PMID:6189570

  5. Polyacrylamide Slab Gel Electrophoresis of Soluble Proteins for Studies of Bacterial Floras

    OpenAIRE

    Moore, W. E. C.; Hash, D E; Holdeman, Lillian V.; Cato, Elizabeth P.

    1980-01-01

    A polyacrylamide slab gel electrophoresis procedure was used to compare cellular proteins from bacterial isolates of gingival crevice floras. Isolates with identical protein patterns consistently were shown to be members of the same species. When used to screen isolates, the procedure reduced total analytical time and expense without sacrificing accuracy, and it provided additional verification of the identity of strains characterized by conventional phenotypic tests.

  6. Rapid and widely disseminated acute phase protein response after experimental bacterial infection of pigs

    OpenAIRE

    Skovgaard, Kerstin; Mortensen, Shila; Boye, Mette; Poulsen, Karin T.; Campbell, Fiona M; Eckersall, P. David; Heegaard, Peter M.H.

    2009-01-01

    International audience The acute phase protein response is a well-described generalized early host response to tissue injury, inflammation and infection, observed as pronounced changes in the concentrations of a number of circulating serum proteins. The biological function of this response and its interplay with other parts of innate host defence reactions remain somewhat elusive. In order to gain new insight into this early host defence response in the context of bacterial infection we st...

  7. Procalcitonin and C-reactive protein as markers of bacterial infection in patients with solid tumours

    DEFF Research Database (Denmark)

    Diness, Laura V; Maraldo, Maja V; Mortensen, Christiane E;

    2014-01-01

    infection. In this prospective study, we wanted to investigate the value of procalcitonin (PCT) compared with C-reactive protein (CRP) as an indicator of bacterial infection in adult patients with solid tumours. METHODS: A total of 41 patients with solid tumours admitted to hospital due to fever or clinical...

  8. Niobium Uptake and Release by Bacterial Ferric Ion Binding Protein

    Directory of Open Access Journals (Sweden)

    Yanbo Shi

    2010-01-01

    Full Text Available Ferric ion binding proteins (Fbps transport FeIII across the periplasm and are vital for the virulence of many Gram negative bacteria. Iron(III is tightly bound in a hinged binding cleft with octahedral coordination geometry involving binding to protein side chains (including tyrosinate residues together with a synergistic anion such as phosphate. Niobium compounds are of interest for their potential biological activity, which has been little explored. We have studied the binding of cyclopentadienyl and nitrilotriacetato NbV complexes to the Fbp from Neisseria gonorrhoeae by UV-vis spectroscopy, chromatography, ICP-OES, mass spectrometry, and Nb K-edge X-ray absorption spectroscopy. These data suggest that NbV binds strongly to Fbp and that a dinuclear NbV centre can be readily accommodated in the interdomain binding cleft. The possibility of designing niobium-based antibiotics which block iron uptake by pathogenic bacteria is discussed.

  9. Proteinaceous determinants of surface colonization in bacteria: Bacterial adhesion and biofilm formation from a protein secretion perspective

    Directory of Open Access Journals (Sweden)

    MickaelDesvaux

    2013-10-01

    Full Text Available Bacterial colonization of biotic or abiotic surfaces results from two quite distinct physiological processes, namely bacterial adhesion and biofilm formation. Broadly speaking, a biofilm is defined as the sessile development of microbial cells. Biofilm formation arises following bacterial adhesion but not all single bacterial cells adhering reversibly or irreversibly engage inexorably into a sessile mode of growth. Among molecular determinants promoting bacterial colonization, surface proteins are the most functionally diverse active components. To be present on the bacterial cell surface, though, a protein must be secreted in the first place. Considering the close association of secreted proteins with their cognate secretion systems, the secretome (which refers both to the secretion systems and their protein substrates is a key concept to apprehend the protein secretion and related physiological functions. The protein secretion systems are here considered in light of the differences in the cell-envelope architecture between diderm-LPS (archetypal Gram-negative, monoderm (archetypal Gram-positive and diderm-mycolate (archetypal acid-fast bacteria. Besides, their cognate secreted proteins engaged in the bacterial colonization process are regarded from single protein to supramolecular protein structure as well as the non-classical protein secretion. This state-of-the-art on the complement of the secretome (the secretion systems and their cognate effectors involved in the surface colonization process in diderm-LPS and monoderm bacteria paves the way for future research directions in the field.

  10. 16S ribosomal RNA-based methods to monitor changes in the hindgut bacterial community of piglets after oral administration of Lactobacillus sobrius S1.

    Science.gov (United States)

    Su, Yong; Yao, Wen; Perez-Gutierrez, Odette N; Smidt, Hauke; Zhu, Wei-Yun

    2008-04-01

    16S ribosomal RNA (rRNA) gene based PCR/denaturing gradient gel electrophoresis (DGGE) and real-time PCR were used to monitor the changes in the composition of microbiota in the hindgut of piglets after oral administration of Lactobacillus sobrius S1. Six litters of neonatal piglets were divided randomly into control group and treatment group. At 7, 9, and 11 days of age, piglets in the treatment group orally received a preparation of L. sobrius S1. At 7, 14, 21(weaning), 24, and 35 days of age, one piglet from each litter was sacrificed and digesta samples of hindgut were collected. DGGE analysis of 16S rRNA gene V6-V8 region for all bacteria showed that several populations present in the hindgut of piglets, represented by far-migrating bands, disappeared after weaning. Most of these bands corresponded to Lactobacillus spp. as revealed by sequence analysis. Quantitative real-time PCR specific for lactobacilli further demonstrated that the number of lactobacilli population tended to decrease after the piglets were weaned. Drastic changes of L. amylovorus and L. sobrius in total Lactobacillus populations were also observed in the colon of piglets around weaning, as monitored by 16S rRNA gene V2-V3 region based Lactobacillus-specific PCR-DGGE. Species-specific real-time PCR also revealed that the population of L. sobrius declined apparently in the colon of piglets after weaning. No remarkable changes in the overall microbial community in the hindgut were found between control and treatment groups. However, comparison of DGGE profiles between the two groups revealed a specific band related to Clostridium disporicum that was found in treatment group on day 14. On day 35, a specific band appeared only in the control group, representing a population most closely related to Streptococcus suis (99%). Real-time PCR showed that L. sobrius 16S rRNA gene copies in treatment group were relatively higher than in the control group (10(8.45) vs. 10(6.83)) on day 35, but no

  11. Crystallization and preliminary X-ray diffraction data analysis of stenodactylin, a highly toxic type 2 ribosome-inactivating protein from Adenia stenodactyla

    International Nuclear Information System (INIS)

    Stenodactylin is a type 2 RIP from the caudex of Adenia stenodactyla. Stenodactylin crystallization and preliminary X-ray diffraction data analysis are reported. Ribosome-inactivating proteins (RIPs) inhibit protein synthesis and induce cell death by removing a single adenine from a specific rRNA loop. They can be divided into two main groups: type 1 and type 2 RIPs. Type 1 RIPs are single-chain enzymes with N-glycosidase activity. Type 2 RIPs contain two chains (A and B) linked by a disulfide bond. The A chain has RIP enzymatic activity, whereas the B chain shows lectin activity and is able to bind to glycosylated receptors on the cell surface. Stenodactylin is a type 2 RIP from the caudex of Adenia stenodactyla from the Passifloraceae family that has been recently purified and characterized. It shows a strong enzymatic activity towards several substrates and is more cytotoxic than other toxins of the same type. Here, the crystallization and preliminary X-ray diffraction data analysis of stenodactylin are reported. This RIP forms crystals that diffract to high resolution (up to 2.15 Å). The best data set was obtained by merging data collected from two crystals. Stenodactylin crystals belonged to the centred monoclinic space group C2 and contained two molecules in the asymmetric unit

  12. A novel method for simultaneous production of two ribosome-inactivating proteins, α-MMC and MAP30, from Momordica charantia L.

    Directory of Open Access Journals (Sweden)

    Yao Meng

    Full Text Available Alpha-momorcharin (α-MMC and momordica anti-HIV protein (MAP30 from Momordica charantia L. have been confirmed to possess anti-tumor and anti-virus activities. Traditional purification methods of these two ribosome-inactivating proteins (RIPs were separate which was time consuming and cost effective as well as low efficient. In order to obtain sufficient samples for researches, a strategy combining ion-exchange and gel filtration chromatography was developed and optimized in this study. Using this novel purification method, averagely 1162 mg of α-MMC and 535 mg of MAP30 were obtained from 400 g of Momordica charantia L seeds. The homogeneities of them were assessed by electrophoresis analysis. Determination of molecular weights of α-MMC and MAP30 were 28.585 kDa and 29.094 kDa by MALDI-TOF/TOF and pI were 9.02 and 9.12, respectively. The single glycoproteins were identified by Periodate-Schiff's base (PAS and the saccharide content was tested to be 1.25% and 1.1% by anthrone-sulfuric acid method. Biological activities were evidenced by their ability to inhibit proliferation of lung adenocarcinoma A549 cell and to convert supercoiled plasmid pUC18 into relaxed forms. Finally, we also found that both two RIPs exhibited no superoxide dismutase (SOD activity.

  13. Comparative Immunohistochemical Analysis of Ochratoxin A Tumourigenesis in Rats and Urinary Tract Carcinoma in Humans; Mechanistic Significance of p-S6 Ribosomal Protein Expression

    Directory of Open Access Journals (Sweden)

    Sarah Pinder

    2012-09-01

    Full Text Available Ochratoxin A (OTA is considered to be a possible human urinary tract carcinogen, based largely on a rat model, but no molecular genetic changes in the rat carcinomas have yet been defined. The phosphorylated-S6 ribosomal protein is a marker indicating activity of the mammalian target of rapamycin, which is a serine/threonine kinase with a key role in protein biosynthesis, cell proliferation, transcription, cellular metabolism and apoptosis, while being functionally deregulated in cancer. To assess p-S6 expression we performed immunohistochemistry on formalin-fixed and paraffin-embedded tumours and normal tissues. Marked intensity of p-S6 expression was observed in highly proliferative regions of rat renal carcinomas and a rare angiosarcoma, all of which were attributed to prolonged exposure to dietary OTA. Only very small OTA-generated renal adenomas were negative for p-S6. Examples of rat subcutaneous fibrosarcoma and testicular seminoma, as well as of normal renal tissue, showed no or very weak positive staining. In contrast to the animal model, human renal cell carcinoma, upper urinary tract transitional cell carcinoma from cases of Balkan endemic nephropathy, and a human angiosarcoma were negative for p-S6. The combined findings are reminiscent of constitutive changes in the rat tuberous sclerosis gene complex in the Eker strain correlated with renal neoplasms, Therefore rat renal carcinogenesis caused by OTA does not obviously mimic human urinary tract tumourigenesis.

  14. Cotranscription of two genes necessary for ribosomal protein L11 methylation (prmA) and pantothenate transport (panF) in Escherichia coli K-12.

    Science.gov (United States)

    Vanet, A; Plumbridge, J A; Alix, J H

    1993-11-01

    Genetic complementation and enzyme assays have shown that the DNA region between panF, which encodes pantothenate permease, and orf1, the first gene of the fis operon, encodes prmA, the genetic determinant for the ribosomal protein L11 methyltransferase. Sequencing of this region identified one long open reading frame that encodes a protein of 31,830 Da and corresponds to the prmA gene. We found, both in vivo and in vitro, that prmA is expressed from promoters located upstream of panF and thus that the panF and prmA genes constitute a bifunctional operon. We located the major 3' end of prmA transcripts 90 nucleotides downstream of the stop codon of prmA in the DNA region upstream of the fis operon, a region implicated in the control of the expression of the fis operon. Although no promoter activity was detected immediately upstream of prmA, S1 mapping detected 5' ends of mRNA in this region, implying that some mRNA processing occurs within the bicistronic panF-prmA mRNA. PMID:8226664

  15. An internal ribosome entry site element directs the synthesis of the 80 kDa isoforms of protein 4.1R

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    Correas Isabel

    2008-12-01

    Full Text Available Abstract Background In red blood cells, protein 4.1 (4.1R is an 80 kDa protein that stabilizes the spectrin-actin network and anchors it to the plasma membrane through its FERM domain. While the expression pattern of 4.1R in mature red cells is relatively simple, a rather complex array of 4.1R protein isoforms varying in N-terminal extensions, internal sequences and subcellular locations has been identified in nucleated cells. Among these, 135 kDa and 80 kDa isoforms have different N-terminal extensions and are expressed either from AUG1- or AUG2-containing mRNAs, respectively. These two types of mRNAs, varying solely by presence/absence of 17 nucleotides (nt which contain the AUG1 codon, are produced by alternative splicing of the 4.1R pre-mRNA. It is unknown whether the 699 nt region comprised between AUG1 and AUG2, kept as a 5' untranslated region in AUG2-containing mRNAs, plays a role on 4.1R mRNA translation. Results By analyzing the in vitro expression of a panel of naturally occurring 4.1R cDNAs, we observed that all AUG1/AUG2-containing cDNAs gave rise to both long, 135 kDa, and short, 80 kDa, 4.1R isoforms. More importantly, similar results were also observed in cells transfected with this set of 4.1R cDNAs. Mutational studies indicated that the short isoforms were not proteolytic products of the long isoforms but products synthesized from AUG2. The presence of a cryptic promoter in the 4.1R cDNA sequence was also discounted. When a 583 nt sequence comprised between AUG1 and AUG2 was introduced into bicistronic vectors it directed protein expression from the second cistron. This was also the case when ribosome scanning was abolished by introduction of a stable hairpin at the 5' region of the first cistron. Deletion analysis of the 583 nt sequence indicated that nucleotides 170 to 368 are essential for expression of the second cistron. The polypyrimidine tract-binding protein bound to the 583 nt active sequence but not to an inactive 3

  16. Solenopsis invicta virus 3: mapping of structural proteins, ribosomal frameshifting, and similarities to Acyrthosiphon pisum virus and Kelp fly virus.

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    Steven M Valles

    Full Text Available Solenopsis invicta virus 3 (SINV-3 is a positive-sense single-stranded RNA virus that infects the red imported fire ant, Solenopsis invicta. We show that the second open reading frame (ORF of the dicistronic genome is expressed via a frameshifting mechanism and that the sequences encoding the structural proteins map to both ORF2 and the 3' end of ORF1, downstream of the sequence that encodes the RNA-dependent RNA polymerase. The genome organization and structural protein expression strategy resemble those of Acyrthosiphon pisum virus (APV, an aphid virus. The capsid protein that is encoded by the 3' end of ORF1 in SINV-3 and APV is predicted to have a jelly-roll fold similar to the capsid proteins of picornaviruses and caliciviruses. The capsid-extension protein that is produced by frameshifting, includes the jelly-roll fold domain encoded by ORF1 as its N-terminus, while the C-terminus encoded by the 5' half of ORF2 has no clear homology with other viral structural proteins. A third protein, encoded by the 3' half of ORF2, is associated with purified virions at sub-stoichiometric ratios. Although the structural proteins can be translated from the genomic RNA, we show that SINV-3 also produces a subgenomic RNA encoding the structural proteins. Circumstantial evidence suggests that APV may also produce such a subgenomic RNA. Both SINV-3 and APV are unclassified picorna-like viruses distantly related to members of the order Picornavirales and the family Caliciviridae. Within this grouping, features of the genome organization and capsid domain structure of SINV-3 and APV appear more similar to caliciviruses, perhaps suggesting the basis for a "Calicivirales" order.

  17. The economics of ribosome biosynthesis in yeast.

    Science.gov (United States)

    Warner, J R

    1999-11-01

    In a rapidly growing yeast cell, 60% of total transcription is devoted to ribosomal RNA, and 50% of RNA polymerase II transcription and 90% of mRNA splicing are devoted to ribosomal proteins (RPs). Coordinate regulation of the approximately 150 rRNA genes and 137 RP genes that make such prodigious use of resources is essential for the economy of the cell. This is entrusted to a number of signal transduction pathways that can abruptly induce or silence the ribosomal genes, leading to major implications for the expression of other genes as well. PMID:10542411

  18. Effect of Dietary Protein Levels on Composition of Odorous Compounds and Bacterial Ecology in Pig Manure

    OpenAIRE

    Cho, Sungback; Hwang, Okhwa; Park, Sungkwon

    2015-01-01

    This study was performed to investigate the effect of different levels of dietary crude protein (CP) on composition of odorous compounds and bacterial communities in pig manure. A total of 48 male pigs (average initial body weight 45 kg) fed diets containing three levels of dietary CP (20%, 17.5%, and 15%) and their slurry samples were collected from the pits under the floor every week for one month. Changes in composition of odorous compounds and bacterial communities were analyzed by gas ch...

  19. Excretion of purine base derivatives after intake of bacterial protein meal in pigs

    DEFF Research Database (Denmark)

    Hellwing, Anne Louise Frydendahl; Tauson, Anne-Helene; Skrede, A.

    2007-01-01

    Bacterial protein meal has a high content ofprotein but also of RNA and DNA. Sixteen barrows were allocated to four diets containing increasing levels of bacterial protein meal (BPM), from weaning to 80 kg live weight, to evaluate whether the RNA and DNA contents of BPM influenced the retention...... of nitrogen. It was hypothesised that an increased intake of RNA and DNA would lead to an increased urinary excretion of purine base derivatives and increased plasma concentrations. Retention of nitrogen was unaffected by dietary content of BPM (P=0.08) and the urinary excretion of purine base derivatives...... increased with increasing dietary content of BPM. No differences in fasting plasma concentration of uric acid, xanthine and hypoxanthine were observed. It can therefore be concluded that increasing levels of dietary BPM maintained protein accretion and led to changes in excretion of purine detrivatices...

  20. Effect of sulfur analogue of lysine on bacterial protein biosynthesis

    International Nuclear Information System (INIS)

    S-(beta-Aminoethyl)-L-cysteine, a sulfur analogue of lysine inhibited strongly growth of Escherichia coli A-19, and weakly that of Corynebacterium sp. isolated from soil, but did not inhibit growth of Aerobacter aerogenes. In Corynebacterium sp. the inhibitory effect was markedly enhanced in the presence of L-threonine. The inhibition of growth by S-(beta-aminoethyl)-L-cysteine was rapidly reversed by the addition of L-lysine. S-(beta-Aminoethyl)-L-cysteine inhibited protein synthesis and the activity of lysyl-tRNA synthetase from E. coli and A. aerogenes. All the other lysine analogues tested inhibited the activity of enzyme, but S-(beta-aminoethyl)-L-cysteine derivatives, S-(beta-N-acetyl-aminoethyl)-L-cysteine and S-(beta-aminoethyl)-alpha-N-acetyl-L-cysteine were not effective. (auth.)

  1. Flexibility in targeting and insertion during bacterial membrane protein biogenesis

    International Nuclear Information System (INIS)

    The biogenesis of Escherichia coli inner membrane proteins (IMPs) is assisted by targeting and insertion factors such as the signal recognition particle (SRP), the Sec-translocon and YidC with translocation of (large) periplasmic domains energized by SecA and the proton motive force (pmf). The use of these factors and forces is probably primarily determined by specific structural features of an IMP. To analyze these features we have engineered a set of model IMPs based on endogenous E. coli IMPs known to follow distinct targeting and insertion pathways. The modified model IMPs were analyzed for altered routing using an in vivo protease mapping approach. The data suggest a facultative use of different combinations of factors

  2. Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily.

    Science.gov (United States)

    Matsunaga, James; Barocchi, Michele A; Croda, Julio; Young, Tracy A; Sanchez, Yolanda; Siqueira, Isadora; Bolin, Carole A; Reis, Mitermayer G; Riley, Lee W; Haake, David A; Ko, Albert I

    2003-08-01

    Proteins with bacterial immunoglobulin-like (Big) domains, such as the Yersinia pseudotuberculosis invasin and Escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. Here, we report the identification and characterization of a new family of Big domain proteins, referred to as Lig (leptospiral Ig-like) proteins, in pathogenic Leptospira. Screening of L. interrogans and L. kirschneri expression libraries with sera from leptospirosis patients identified 13 lambda phage clones that encode tandem repeats of the 90 amino acid Big domain. Two lig genes, designated ligA and ligB, and one pseudogene, ligC, were identified. The ligA and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or 11 Big domain repeats and, in the case of ligB, a unique carboxy-terminal non-repeat domain. The organization of ligC is similar to that of ligB but contains mutations that disrupt the reading frame. The lig sequences are present in pathogenic but not saprophytic Leptospira species. LigA and LigB are expressed by a variety of virulent leptospiral strains. Loss of Lig protein and RNA transcript expression is correlated with the observed loss of virulence during culture attenuation of pathogenic strains. High-pressure freeze substitution followed by immunocytochemical electron microscopy confirmed that the Lig proteins were localized to the bacterial surface. Immunoblot studies with patient sera found that the Lig proteins are a major antigen recognized during the acute host infection. These observations demonstrate that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis. PMID:12890019

  3. Human Mitochondrial Ribosomal Protein MRPL12 Interacts Directly with Mitochondrial RNA Polymerase to Modulate Mitochondrial Gene Expression*

    OpenAIRE

    Wang, Zhibo; Cotney, Justin; Shadel, Gerald S.

    2007-01-01

    The core human mitochondrial transcription machinery comprises a single subunit bacteriophage-related RNA polymerase, POLRMT, the high mobility group box DNA-binding protein h-mtTFA/TFAM, and two transcriptional co-activator proteins, h-mtTFB1 and h-mtTFB2 that also have rRNA methyltransferase activity. Recapitulation of specific initiation of transcription in vitro can be achieved by a complex of POLRMT, h-mtTFA, and either h-mtTFB1 or h-mtTFB2. However, the nature of mitochondrial transcrip...

  4. Nuclear-localized Calcineurin Homologous Protein CHP1 Interacts with Upstream Binding Factor and Inhibits Ribosomal RNA Synthesis*

    OpenAIRE

    Jiménez-Vidal, Maite; Srivastava, Jyoti; Putney, Luanna K; Barber, Diane L.

    2010-01-01

    Calcineurin homologous protein 1 (CHP1) is a widely expressed, 22-kDa myristoylated EF-hand Ca2+-binding protein that shares a high degree of similarity with the regulatory B subunit of calcineurin (65%) and with calmodulin (59%). CHP1 localizes to the plasma membrane, the Golgi apparatus, and the nucleus and functions to regulate trafficking of early secretory vesicles, activation of T cells, and expression and transport of the Na-H exchanger NHE1. Although CHP1 contains nuclear export signa...

  5. Ribosomal protein L10 is encoded in the mitochondrial genome of many land plants and green algae

    OpenAIRE

    Bonen Linda; Mower Jeffrey P

    2009-01-01

    Abstract Background The mitochondrial genomes of plants generally encode 30-40 identified protein-coding genes and a large number of lineage-specific ORFs. The lack of wide conservation for most ORFs suggests they are unlikely to be functional. However, an ORF, termed orf-bryo1, was recently found to be conserved among bryophytes suggesting that it might indeed encode a functional mitochondrial protein. Results From a broad survey of land plants, we have found that the orf-bryo1 gene is also ...

  6. Short hairpin RNA library-based functional screening identified ribosomal protein L31 that modulates prostate cancer cell growth via p53 pathway.

    Directory of Open Access Journals (Sweden)

    Yojiro Maruyama

    Full Text Available Androgen receptor is a primary transcription factor involved in the proliferation of prostate cancer cells. Thus, hormone therapy using antiandrogens, such as bicalutamide, is a first-line treatment for the disease. Although hormone therapy initially reduces the tumor burden, many patients eventually relapse, developing tumors with acquired endocrine resistance. Elucidation of the molecular mechanisms underlying endocrine resistance is therefore a fundamental issue for the understanding and development of alternative therapeutics for advanced prostate cancer. In the present study, we performed short hairpin RNA (shRNA-mediated functional screening to identify genes involved in bicalutamide-mediated effects on LNCaP prostate cancer cells. Among such candidate genes selected by screening using volcano plot analysis, ribosomal protein L31 (RPL31 was found to be essential for cell proliferation and cell-cycle progression in bicalutamide-resistant LNCaP (BicR cells, based on small interfering RNA (siRNA-mediated knockdown experiments. Of note, RPL31 mRNA is more abundantly expressed in BicR cells than in parental LNCaP cells, and clinical data from ONCOMINE and The Cancer Genome Altas showed that RPL31 is overexpressed in prostate carcinomas compared with benign prostate tissues. Intriguingly, protein levels of the tumor suppressor p53 and its targets, p21 and MDM2, were increased in LNCaP and BicR cells treated with RPL31 siRNA. We observed decreased degradation of p53 protein after RPL31 knockdown. Moreover, the suppression of growth and cell cycle upon RPL31 knockdown was partially recovered with p53 siRNA treatment. These results suggest that RPL31 is involved in bicalutamide-resistant growth of prostate cancer cells. The shRNA-mediated functional screen in this study provides new insight into the molecular mechanisms and therapeutic targets of advanced prostate cancer.

  7. Identification of beta-subunit of bacterial RNA-polymerase--a non-species-specific bacterial protein--as target of antibodies in primary biliary cirrhosis.

    Science.gov (United States)

    Roesler, Kai-Wolfgang; Schmider, Wolfgang; Kist, Manfred; Batsford, Stephen; Schiltz, Emile; Oelke, Mathias; Tuczek, Anja; Dettenborn, Therese; Behringer, Dirk; Kreisel, Wolfgang

    2003-03-01

    Several observations suggest that bacteria induce autoimmunity in primary biliary cirrhosis (PBC). Since no PBC-specific bacterial species could be identified, it can be speculated that the triggers are non-species-specific bacterial proteins. This hypothesis would imply that several or even all bacterial species can trigger PBC. Therefore, we investigated whether PBC exhibits immune reactions to non-species-specific bacterial antigens. Yersinia enterocolitica O3 was screened for the presence of proteins that were labeled by immunoblotting using PBC sera. We focused our investigations on a 160-kDa protein, which was further enriched and characterized by partial N-terminal amino acid sequencing. The prevalence of antibodies to this protein was determined by immunoblotting in a variety of diseases. The 160-kDa protein was identified as the beta-subunit of bacterial RNA-polymerase, a highly conserved bacterial protein with a very high degree of sequence identity among all bacterial species. Antibodies to the beta-subunit of bacterial RNA polymerase were specific for this protein. Until now no mammalian protein could be found that cross-reacts with these antibodies. The prevalence of antibodies to the beta-subunit of bacterial RNA polymerase (ARPA) using the protein from Yersinia enterocolitica O3 (serum dilution 1:1000) was: healthy controls (HC, N = 101) 7.9%, primary biliary cirrhosis (PBC, N = 61) 32.8%, autoimmune hepatitis type 1 (AIH, N = 46) 26.1%, alcoholic liver cirrhosis (ALC, N = 44) 9.1%, Crohn's disease (CD, N = 38) 7.9%, ulcerative colitis (UC, N = 24) 8.3%, primary sclerosing cholangitis + UC (PSC/UC, N = 11) 0%, acute yersiniosis (Yers, N = 36) 19.4%, acute infection with Campylobacter jejuni (Camp, N = 10) 0%, acute Q-fever (QF, N = 16) 6.25%, chronic hepatitis C (HCV, N = 39) 7.7%, c-ANCA-positive vasculitis (Vasc, N = 40) 15%, systemic lupus erythematosus (SLE, N = 28) 10.7%, and malaria tropica (MT, N = 24) 16.7%. There was no significant

  8. Immunogenicity Analysis of a Novel Subunit Vaccine Candidate Molecule-Recombinant L7/L12 Ribosomal Protein of Brucella suis.

    Science.gov (United States)

    Du, Zhi-Qiang; Li, Xin; Wang, Jian-Ying

    2016-08-01

    Brucella was an intracellular parasite, which could infect special livestock and humans. After infected by Brucella, livestock's reproductive system could be affected and destroyed resulting in huge economic losses. More seriously, it could be contagious from livestock to humans. So far, there is no available vaccine which is safe enough for humans. On this point, subunit vaccine has become the new breakthrough of conquering brucellosis. In this study, Brucella rL7/L12-BLS fusion protein was used as an antigen to immunize rabbits to detect the immunogenicity. The results of antibody level testing assay of rabbit antiserum indicated rL7/L12-BLS fusion protein could elicit rabbits to produce high-level IgG. And gamma interferon (IFN-γ) concentrations in rabbit antiserum were obviously up-regulated in both the rL7/L12 group and rL7/L12-BLS group. Besides, the results of quantitative real-time PCR (qRT-PCR) showed the IFN-γ gene's expression levels of both the rL7/L12 group and rL7/L12-BLS group were obviously up-regulated. All these results suggested Brucella L7/L12 protein was an ideal subunit vaccine candidate and possessed good immunogenicity. And Brucella lumazine synthase (BLS) molecule was a favorable transport vector for antigenic protein. PMID:27075455

  9. The use of fed batch approaches to maximise yields in bacterial fermentation and protein expression

    International Nuclear Information System (INIS)

    A fermentation facility for the scale up of bacterial and yeast fermentations has been set up at the University of Queensland under the auspices of the ARC Special Research Centre for Functional and Applied Genomics. A major application is the production of recombinant proteins for determination of tertiary structures by X-ray crystallography or nuclear magnetic resonance. For this purpose, large amounts of protein arc needed and the yield from a single fermentation run is crucial to success within constrained laboratory budgets. To achieve maximal yields we are optimising fed batch approaches in bacterial fermentation. Fed batch offers many advantages over batch cultures. Coupled with the ability to monitor online the internal conditions of the fermentation including pH and dissolved oxygen and stirrer cascading functions it is possible to ensure that the nutritional environment of the microorganism is optimised for its growth and or for optimal protein expression. The poster will describe some of our experience in setting up fed batch fermentations and successful applications of fed batches to increasing protein yield. It will also outline services that are available to academic groups outside the University of Queensland For structure determination and functional studies, the production of radiolabelled proteins can also be an advantage. We will describe initial experiments aimed at coupling the principles of fed batch fermentation to the introduction of carbon or nitrogen isotopes into the recombinant protein

  10. Phase variation of Opa proteins of Neisseria meningitidis and the effects of bacterial transformation

    Indian Academy of Sciences (India)

    Manish Sadarangani; J Claire Hoe; Katherine Makepeace; Peter Van Der Ley; Andrew J Pollard

    2016-03-01

    Opa proteins are major proteins involved in meningococcal colonization of the nasopharynx and immune interactions. Opa proteins undergo phase variation (PV) due to the presence of the 5′-CTCTT-3′ coding repeat (CR) sequence. The dynamics of PV of meningococcal Opa proteins is unknown. Opa PV, including the effect of transformation on PV, was assessed using a panel of Opa-deficient strains of Neisseria meningitidis. Analysis of Opa expression from UK disease-causing isolates was undertaken. Different opagenes demonstrated variable rates of PV, between 6.4 ×10–4 and 6.9 ×10–3 per cell per generation. opa genes with a longer CR tract had a higher rate of PV (r2=0.77, p=0.1212). Bacterial transformation resulted in a 180-fold increase in PV rate. The majority of opagenes in UK disease isolates (315/463, 68.0%) were in the ‘on’ phase, suggesting the importance of Opa proteins during invasive disease. These data provide valuable information for the first time regarding meningococcal Opa PV. The presence of Opa PV in meningococcal populations and high expression of Opa among invasive strains likely indicates the importance of this protein in bacterial colonization in the human nasopharynx. These findings have potential implications for development of vaccines derived from meningococcal outer membranes.

  11. The Elav-like protein HuR exerts translational control of viral internal ribosome entry sites

    International Nuclear Information System (INIS)

    The human embryonic-lethal abnormal vision (ELAV)-like protein, HuR, has been recently found to be involved in the regulation of protein synthesis. In this study we show that HuR participates in the translational control of the HIV-1 and HCV IRES elements. HuR functions as a repressor of HIV-1 IRES activity and acts as an activator of the HCV IRES. The effect of HuR was evaluated in three independent experimental systems, rabbit reticulocyte lysate, HeLa cells, and Xenopus laevis oocytes, using both overexpression and knockdown approaches. Furthermore, results suggest that HuR mediated regulation of HIV-1 and HCV IRESes does not require direct binding of the protein to the RNA nor does it need the nuclear translocation of the IRES-containing RNAs. Finally, we show that HuR has a negative impact on post-integration steps of the HIV-1 replication cycle. Thus, our observations yield novel insights into the role of HuR in the post-transcriptional regulation of HCV and HIV-1 gene expression.

  12. Crystal structure of the Campylobacter jejuni Cj0090 protein reveals a novel variant of the immunoglobulin fold among bacterial lipoproteins.

    Science.gov (United States)

    Paek, Seonghee; Kawai, Fumihiro; Choi, Kyoung-Jae; Yeo, Hye-Jeong

    2012-12-01

    Bacterial lipoproteins play an important role in bacterial pathogenesis and physiology. The genome of Campylobacter jejuni, a major foodborn pathogen, is predicted to contain over 20 lipoproteins. However, the functions of the majority of C. jejuni lipoproteins remain unknown. The Cj0090 protein is encoded by a lipoprotein operon composed of cj0089, cj0090, and cj0091. Here, we report the crystal structure of Cj0090 at 1.9 Å resolution, revealing a novel variant of the immunoglobulin fold with β-sandwich architecture. The structure suggests that Cj0090 may be involved in protein-protein interactions, consistent with a possible role for bacterial lipoproteins. PMID:22987763

  13. Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance

    OpenAIRE

    Cashman Kathleen A; Ferro Philip J; Sampey Darryl B; Goba Augustine; Fair Joseph N; Matschiner Alex; Branco Luis M; Schoepp Randal J; Tesh Robert B; Bausch Daniel G; Garry Robert F; Guttieri Mary C

    2008-01-01

    Abstract Background There is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, recombinant Lassa virus (LASV) proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for expression and purification of recombinant LASV nucleoprotein (NP), glycoprotein 1 (GP1), and glycoprotein 2 (GP2). Results Full-length NP and the ...

  14. In vitro estimation of rumen protein degradability using 35S to label the bacterial mass

    International Nuclear Information System (INIS)

    An experiment was carried out in order to simplify a previously developed 15N-method for in vitro estimation of rumen protein degradability. Casein (Cas), whole soybeans (Sb) heated at 120oC for 20 min (SbTherm) and sunflower (Sfl) were incubated at 39oC for 4 hours in a water bathshaker with the following media: McDougall's buffer, strained and enriched with particle associated bacteria rumen fluid (2:1), rapidly (maltose, sucrose, glucose) and more slowly (pectin, soluble starch) degradable carbohydrates with final concentration of 815 mg/100 ml and 21.7 μCi/100 ml of35S (from Na235SO4). After the incubation had been ceased, a bacterial fraction was isolated through differential centrifugation and specific activity of bacterial (Bac) and high speed total solids (TS) nitrogen was measured. The ratio was used to calculate bacterial mass in TS and through the Kjeldahl nitrogen concentration in TS - the net bacterial growth (against control vessels without protein). The level of ammonia-N in the supernate after blank correction was used to find the ammonia-N released from protein degradation. The data showed that the rate (and extend) of degradation for the Cas (as a standard protein) was lower compared to those obtained through the 15N-method but it was higher than the rate derived through another in vitro method. The Cas equivalent of the Sb was higher than the figure we found in a previous experiment with solvent extracted soybean meal suggesting that the 35S-method underestimated the degradability of the Cas. After being tested on a wider range of foodstuffs, the proposed 35S-method might be considered as an alternative procedure which is less laborous than the 15N-method. (author)

  15. Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA

    OpenAIRE

    Belcourt, Michael F.; Penketh, Philip G.; Hodnick, William F.; Johnson, David A.; David H Sherman; Rockwell, Sara; Sartorelli, Alan C.

    1999-01-01

    The mitomycin C-resistance gene, mcrA, of Streptomyces lavendulae produces MCRA, a protein that protects this microorganism from its own antibiotic, the antitumor drug mitomycin C. Expression of the bacterial mcrA gene in mammalian Chinese hamster ovary cells causes profound resistance to mitomycin C and to its structurally related analog porfiromycin under aerobic conditions but produces little change in drug sensitivity under hypoxia. The mitomycins are prodrugs that are enzymatically reduc...

  16. Dependence of Bacterial Protein Adhesins on Toll-Like Receptors for Proinflammatory Cytokine Induction

    OpenAIRE

    Hajishengallis, George; Martin, Michael; Sojar, Hakimuddin T.; Sharma, Ashu; Schifferle, Robert E.; DeNardin, Ernesto; Russell, Michael W.; Genco, Robert J.

    2002-01-01

    Toll-like receptors (TLRs) are important signal transducers that mediate inflammatory reactions induced by microbes through pattern recognition of virulence molecules such as lipopolysaccharide (LPS) and lipoproteins. We investigated whether proinflammatory cytokine responses induced by certain bacterial protein adhesins may also depend on TLRs. In differentiated THP-1 mononuclear cells stimulated by LPS-free recombinant fimbrillin (rFimA) from Porphyromonas gingivalis, cytokine release was a...

  17. Slow Onset Inhibition of Bacterial β-Ketoacyl-acyl Carrier Protein Synthases by Thiolactomycin*

    OpenAIRE

    Machutta, Carl A.; Bommineni, Gopal R.; Luckner, Sylvia R.; Kapilashrami, Kanishk; Ruzsicska, Bela; Simmerling, Carlos; Kisker, Caroline; Tonge, Peter J.

    2009-01-01

    Thiolactomycin (TLM), a natural product thiolactone antibiotic produced by species of Nocardia and Streptomyces, is an inhibitor of the β-ketoacyl-acyl carrier protein synthase (KAS) enzymes in the bacterial fatty acid synthase pathway. Using enzyme kinetics and direct binding studies, TLM has been shown to bind preferentially to the acyl-enzyme intermediates of the KASI and KASII enzymes from Mycobacterium tuberculosis and Escherichia coli. These studies, which utilized acyl-enzyme mimics in...

  18. Structural basis of a rationally rewired protein-protein interface critical to bacterial signaling.

    Science.gov (United States)

    Podgornaia, Anna I; Casino, Patricia; Marina, Alberto; Laub, Michael T

    2013-09-01

    Two-component signal transduction systems typically involve a sensor histidine kinase that specifically phosphorylates a single, cognate response regulator. This protein-protein interaction relies on molecular recognition via a small set of residues in each protein. To better understand how these residues determine the specificity of kinase-substrate interactions, we rationally rewired the interaction interface of a Thermotoga maritima two-component system, HK853-RR468, to match that found in a different two-component system, Escherichia coli PhoR-PhoB. The rewired proteins interacted robustly with each other, but no longer interacted with the parent proteins. Analysis of the crystal structures of the wild-type and mutant protein complexes and a systematic mutagenesis study reveal how individual mutations contribute to the rewiring of interaction specificity. Our approach and conclusions have implications for studies of other protein-protein interactions and protein evolution and for the design of novel protein interfaces. PMID:23954504

  19. Bioinformatic evidence for a widely distributed, ribosomally produced electron carrier precursor, its maturation proteins, and its nicotinoprotein redox partners

    Directory of Open Access Journals (Sweden)

    Haft Daniel H

    2011-01-01

    Full Text Available Abstract Background Enzymes in the radical SAM (rSAM domain family serve in a wide variety of biological processes, including RNA modification, enzyme activation, bacteriocin core peptide maturation, and cofactor biosynthesis. Evolutionary pressures and relationships to other cellular constituents impose recognizable grammars on each class of rSAM-containing system, shaping patterns in results obtained through various comparative genomics analyses. Results An uncharacterized gene cluster found in many Actinobacteria and sporadically in Firmicutes, Chloroflexi, Deltaproteobacteria, and one Archaeal plasmid contains a PqqE-like rSAM protein family that includes Rv0693 from Mycobacterium tuberculosis. Members occur clustered with a strikingly well-conserved small polypeptide we designate "mycofactocin," similar in size to bacteriocins and PqqA, precursor of pyrroloquinoline quinone (PQQ. Partial Phylogenetic Profiling (PPP based on the distribution of these markers identifies the mycofactocin cluster, but also a second tier of high-scoring proteins. This tier, strikingly, is filled with up to thirty-one members per genome from three variant subfamilies that occur, one each, in three unrelated classes of nicotinoproteins. The pattern suggests these variant enzymes require not only NAD(P, but also the novel gene cluster. Further study was conducted using SIMBAL, a PPP-like tool, to search these nicotinoproteins for subsequences best correlated across multiple genomes to the presence of mycofactocin. For both the short chain dehydrogenase/reductase (SDR and iron-containing dehydrogenase families, aligning SIMBAL's top-scoring sequences to homologous solved crystal structures shows signals centered over NAD(P-binding sites rather than over substrate-binding or active site residues. Previous studies on some of these proteins have revealed a non-exchangeable NAD cofactor, such that enzymatic activity in vitro requires an artificial electron acceptor such

  20. Recombinant expression and purification of "virus-like" bacterial encapsulin protein cages.

    Science.gov (United States)

    Rurup, W Frederik; Cornelissen, Jeroen J L M; Koay, Melissa S T

    2015-01-01

    Ultracentrifugation, particularly the use of sucrose or cesium chloride density gradients, is a highly reliable and efficient technique for the purification of virus-like particles and protein cages. Since virus-like particles and protein cages have a unique size compared to cellular macromolecules and organelles, the rate of migration can be used as a tool for purification. Here we describe a detailed protocol for the purification of recently discovered virus-like assemblies called bacterial encapsulins from Thermotoga maritima and Brevibacterium linens. PMID:25358773

  1. The circadian clock coordinates ribosome biogenesis.

    Directory of Open Access Journals (Sweden)

    Céline Jouffe

    Full Text Available Biological rhythms play a fundamental role in the physiology and behavior of most living organisms. Rhythmic circadian expression of clock-controlled genes is orchestrated by a molecular clock that relies on interconnected negative feedback loops of transcription regulators. Here we show that the circadian clock exerts its function also through the regulation of mRNA translation. Namely, the circadian clock influences the temporal translation of a subset of mRNAs involved in ribosome biogenesis by controlling the transcription of translation initiation factors as well as the clock-dependent rhythmic activation of signaling pathways involved in their regulation. Moreover, the circadian oscillator directly regulates the transcription of ribosomal protein mRNAs and ribosomal RNAs. Thus the circadian clock exerts a major role in coordinating transcription and translation steps underlying ribosome biogenesis.

  2. The immunological properties of Brucella ribosomal preparations.

    Science.gov (United States)

    Corbel, M J

    1976-01-01

    Ribosomes were isolated from Brucella abortus strains 19 and 45/20 by disruption of the cells followed by differential ultracentrifugation. The ribosome preparations contained 2-3 components reacting in immunodiffusion tests but were free of detectable lipopolysaccharide-protein agglutinogen. They crossreacted with antisera to Br. abortus, Br. melitensis, Br. suis and Br. ovis and elicited intradermal delayed hypersensitivity reactions in animals infected with Br. abortus, Br. melitensis or Br. suis. The ribosomes were antigenic in rabbits, guinea pigs and mice. Those from Br. abortus S19 induced agglutinins reaction with smooth brucella strains whereas those from Br. abortus 45/20 induced agglutinins reacting with rough brucella strains. Cattle vaccinated with S19 or 45/20 vaccines or infected with Br. abortus developed pricipitins to ribosomal components at an early stage in the immune response. PMID:816681

  3. [Comparison of histone-like proteins from blue-green algae with ribosomal basic proteins of alga and wheat germ histones].

    Science.gov (United States)

    Gofshteĭn, L V; Iurina, N P; Romashkin, V I; Oparin, A I

    1975-01-01

    Histone-like proteins was found in blue-green alga Anacystis nidulans, which has no nucleus. F2b2, F2a2, F2a1 fractions were found in histone-like algae proteins and no fraction F1. Content of basic amino acids (arginine being prevailing in algae protein) is quite identical in histone-like algae proteins and in wheat germs histones, while the content of acid amino acids is considerably higher in algae. The presence in procaryotic cells of basic proteins similar in a number of properties to histones of higher organisms suggests that these proteins are evolutionary precursors of eucaryotic histones. PMID:813782

  4. Secreted and immunogenic proteins produced by the honeybee bacterial pathogen, Paenibacillus larvae.

    Science.gov (United States)

    Antúnez, Karina; Anido, Matilde; Evans, Jay D; Zunino, Pablo

    2010-03-24

    American Foulbrood is a severe disease affecting larvae of honeybee Apis mellifera, causing significant decrease in the honeybee population, beekeeping industries and agricultural production. In spite of its importance, little is known about the virulence factors secreted by Paenibacillus larvae during larval infection. The aim of the present work was to perform a first approach to the identification and characterization of P. larvae secretome. P. larvae secreted proteins were analyzed by SDS-PAGE and identified by MALDI-TOF. Protein toxicity was evaluated using an experimental model based on feeding of A. mellifera larvae and immunogenicity was evaluated by Western blot, using an antiserum raised against cells and spores of P. larvae. Ten different proteins were identified among P. larvae secreted proteins, including proteins involved in transcription, metabolism, translation, cell envelope, transport, protein folding, degradation of polysaccharides and motility. Although most of these proteins are cytosolic, many of them have been previously detected in the extracellular medium of different Bacillus spp. cultures and have been related to virulence. The secreted proteins resulted highly toxic and immunogenic when larvae were exposed using an experimental model. This is the first description of proteins secreted by the honeybee pathogen P. larvae. This information may be relevant for the elucidation of bacterial pathogenesis mechanisms. PMID:19781868

  5. Ribosome-dependent activation of stringent control.

    Science.gov (United States)

    Brown, Alan; Fernández, Israel S; Gordiyenko, Yuliya; Ramakrishnan, V

    2016-06-01

    In order to survive, bacteria continually sense, and respond to, environmental fluctuations. Stringent control represents a key bacterial stress response to nutrient starvation that leads to rapid and comprehensive reprogramming of metabolic and transcriptional patterns. In general, transcription of genes for growth and proliferation is downregulated, while those important for survival and virulence are upregulated. Amino acid starvation is sensed by depletion of the aminoacylated tRNA pools, and this results in accumulation of ribosomes stalled with non-aminoacylated (uncharged) tRNA in the ribosomal A site. RelA is recruited to stalled ribosomes and activated to synthesize a hyperphosphorylated guanosine analogue, (p)ppGpp, which acts as a pleiotropic secondary messenger. However, structural information about how RelA recognizes stalled ribosomes and discriminates against aminoacylated tRNAs is missing. Here we present the cryo-electron microscopy structure of RelA bound to the bacterial ribosome stalled with uncharged tRNA. The structure reveals that RelA utilizes a distinct binding site compared to the translational factors, with a multi-domain architecture that wraps around a highly distorted A-site tRNA. The TGS (ThrRS, GTPase and SpoT) domain of RelA binds the CCA tail to orient the free 3' hydroxyl group of the terminal adenosine towards a β-strand, such that an aminoacylated tRNA at this position would be sterically precluded. The structure supports a model in which association of RelA with the ribosome suppresses auto-inhibition to activate synthesis of (p)ppGpp and initiate the stringent response. Since stringent control is responsible for the survival of pathogenic bacteria under stress conditions, and contributes to chronic infections and antibiotic tolerance, RelA represents a good target for the development of novel antibacterial therapeutics. PMID:27279228

  6. The role of bacterial antizyme: From an inhibitory protein to AtoC transcriptional regulator

    Directory of Open Access Journals (Sweden)

    Kyriakidis Dimitrios A

    2004-06-01

    Full Text Available Abstract This review considers the role of bacterial antizyme in the regulation of polyamine biosynthesis and gives new perspectives on the involvement of antizyme in other significant cellular mechanisms. Antizyme is a protein molecule induced by the end product of the enzymic reaction that it inhibits, in a non-competitive manner. The bacterial ornithine decarboxylase is regulated by nucleotides, phosphorylation and antizyme. The inhibition of ornithine decarboxylase by antizyme can be relieved to different degrees by DNA or by a variety of synthetic nucleic acid polymers, attributed to a specific interaction between nucleic acid and antizyme. Recently, this interplay between bacterial antizyme and nucleic acid was determined by discerning an additional function to antizyme that proved to be the atoC gene product, encoding the response regulator of the bacterial two-component system AtoS-AtoC. The gene located just upstream of atoC encodes the sensor kinase, named AtoS, that modulates AtoC activity. AtoC regulates expression of atoDAEB operon which is involved in short-chain fatty acid metabolism. Antizyme is thus referred to as AtoC, functioning both as a post-translational and transcriptional regulator. Also, the AtoS-AtoC signal transduction system in E. coli has a positive regulatory role on poly-(R-3-hydroxybutyrate biosynthesis. The properties and gene structural similarities of antizymes from different organisms were compared. It was revealed that conserved domains are present mostly in the C-domain of all antizymes. BLAST analysis of the E. coli antizyme protein (AtoC showed similarities around 69–58% among proteobacteria, g-proteobacteria, enterobacteria and the thermophilic bacterium Thermus thermophilus. A working hypothesis is proposed for the metabolic role of antizyme (AtoC describing the significant biological implications of this protein molecule. Whether antizymes exist to other enzymes in different tissues, meeting the

  7. Molecular Profiling of Activated Neurons by Phosphorylated Ribosome Capture

    OpenAIRE

    Knight, Zachary A.; Tan, Keith; Birsoy, Kivanc; Schmidt, Sarah; Garrison, Jennifer L.; Wysocki, Robert W.; Emiliano, Ana; Ekstrand, Mats I.; Friedman, Jeffrey M.

    2012-01-01

    The mammalian brain is composed of thousands of interacting neural cell types. Systematic approaches to establish the molecular identity of functional populations of neurons would advance our understanding of neural mechanisms controlling behavior. Here, we show that ribosomal protein S6, a structural component of the ribosome, becomes phosphorylated in neurons activated by a wide range of stimuli. We show that these phosphorylated ribosomes can be captured from mouse brain homogenates, there...

  8. Bacterial Protein Characterization of Streptococcus agalactiae by SDS-page Method for Subclinical Mastitis Irradiated Vaccine Materials in Dairy Cattle

    OpenAIRE

    B.J. Tuasikal; I.W.T. Wibawan2; F.H. Pasaribu2; S. Estuningsih2

    2012-01-01

    A study have been conducted to isolate and characterize bacterial protein S. agalactiae, which is antigenic and can be used to test immunogenicity of vaccine in order to manufacture irradiated mastitis (inflammation of the udder) vaccine in ruminant. The study aims to determine the Molecular Weight (MW) bacterial protein S. agalactiae irradiation, which can be used to test the nature of its antigenic caharacteristic. The character of S. agalactiae antigenic stimulates antibody induction of th...

  9. Bioactivities of Jc-SCRIP, a type 1 ribosome-inactivating protein from Jatropha curcas seed coat.

    Science.gov (United States)

    Nuchsuk, Chanthakan; Wetprasit, Nuanchawee; Roytrakul, Sittiruk; Choowongkomon, Kiattawee; T-Thienprasert, Nattanan; Yokthongwattana, Chotika; Arpornsuwan, Theerakul; Ratanapo, Sunanta

    2013-10-01

    In this study, a type 1 RIP, designated as Jc-SCRIP, was first isolated from the seed coat of Jatropha curcas Linn. It was purified by ammonium sulfate precipitation and chromatography on DEAE-Sephacel™ and CM-cellulose columns. Purification fold of Jc-SCRIP increased 113.8 times, and the yield was 1.13% of the total protein in the final step. It was shown to be a monomeric glycoprotein with a molecular mass of 38 938 Da, as determined by MALDI-TOF/MS. It exhibited hemagglutination activity and possessed strong N-glycosidase activity. The antimicrobial activity of Jc-SCRIP was tested against nine human pathogenic bacteria and one fungus; the most potent inhibitory activity was against Staphylococcus epidermidis ATCC 12228, with minimum inhibitory concentration value of 0.20 μm. Jc-SCRIP demonstrated in vitro cytotoxicity against human breast adenocarcinoma cell line (MCF-7), a colon adenocarcinoma (SW620), and a liver carcinoma cell line (HepG2), with IC50 values of 0.15, 0.25, and 0.40 mm, respectively. The results suggested that Jc-SCRIP may be a potential natural antimicrobial and anticancer agent in medical applications. PMID:23773434

  10. Biochemical characterization and bacterial expression of an odorant-binding protein from Locusta migratoria.

    Science.gov (United States)

    Ban, L; Scaloni, A; D'Ambrosio, C; Zhang, L; Yahn, Y; Pelosi, P

    2003-02-01

    Analysis of soluble proteins from different body parts of Locusta migratoria revealed a fast-migrating component in native electrophoresis, unique to antennae of both sexes. N-terminal sequence analysis and cloning identified this protein as a member of the insect odorant-binding proteins, carrying a well-conserved six-cysteine motif. Mass spectrometry analysis confirmed the occurrence of two distinct polypeptide species determined by nucleotide sequencing and demonstrated that the cysteine residues are paired in an interlocked fashion. The protein was expressed in a bacterial system with yields of about 10 mg/l of culture, mostly present as inclusion bodies. However, this recombinant product was solubilized after disulfide reduction. Air oxidation yielded a species with all disulfides spontaneously formed as in the native counterpart. Both native and recombinant proteins migrated as a dimer in gel filtration chromatography. Ligand binding was measured, using N-phenyl-1-naphthylamine as the fluorescent probe; the affinity of other ligands was measured in competitive binding assays. The protein exhibited great resistance to thermal denaturation even following prolonged treatment at 100 degrees C. A structural model for this dimeric species was generated on the basis of its sequence homology with Bombyx mori pheromone-binding protein, whose three-dimensional structure has been resolved as an unbound species and in complex with its physiological ligand. This is the first report of an odorant-binding protein identified and characterized from Orthoptera. PMID:12678502

  11. Immunomodulation by microbial ribosomes

    Directory of Open Access Journals (Sweden)

    W. Domzig

    1987-01-01

    Full Text Available Over the past twenty years, many authors have reported evidence of the immunoprotective capacity of ribosomes isolated from bacteria, fungi and parasites. Since 1971 we have explored the protective capacity of ribosomes isolated from a large variety of microorganisms responsible for human and animal diseases. More recently, using monoclonal antibodies raised against ribosomes and then selected for their ability to confer passive immunity to mice, we have studied the mechanism of the protection induced by ribosomes. These studies, in parallel with the development of a technology for the large scale production of ribosomes, have allowed us to achieve a new regard for ribosomal vaccines for use in human. The general concept of ribosomal vaccines in presented and examples of two such vaccines are described with data on the specific protection that they induce in mice against experimental infections with Klebsiella peneumoniae, Streptococcus pneumoniae, S. pyogenes and Haemophilus influenzae for the first one, and against Candida albicans type A and type B for the second one. Because of their high immunogenicity and their innocuity these vaccines represent a decisive improvement over classical microbial vaccines.

  12. Object-adapted trapping and shape-tracking to probe a bacterial protein chain motor

    Science.gov (United States)

    Roth, Julian; Koch, Matthias; Rohrbach, Alexander

    2015-03-01

    The helical bacterium Spiroplasma is a motile plant and anthropod pathogen which swims by propagating pairs of kinks along its cell body. As a well suited model system for bacterial locomotion, understanding the cell's molecular motor is of vital interest also regarding the combat of bacterial diseases. The extensive deformations related to these kinks are caused by a contractile cytoskeletal protein ribbon representing a linear motor in contrast to common rotary motors as, e.g., flagella. We present new insights into the working of this motor through experiments with object-adapted optical traps and shape-tracking techniques. We use the given laser irradiation from the optical trap to hinder bacterial energy (ATP) production through the production of O2 radicals. The results are compared with experiments performed under the influence of an O2-Scavenger and ATP inhibitors, respectively. Our results show clear dependences of the kinking properties on the ATP concentration inside the bacterium. The experiments are supported by a theoretical model which we developed to describe the switching of the ribbon's protein subunits.

  13. Effect of Dietary Protein Levels on Composition of Odorous Compounds and Bacterial Ecology in Pig Manure.

    Science.gov (United States)

    Cho, Sungback; Hwang, Okhwa; Park, Sungkwon

    2015-09-01

    This study was performed to investigate the effect of different levels of dietary crude protein (CP) on composition of odorous compounds and bacterial communities in pig manure. A total of 48 male pigs (average initial body weight 45 kg) fed diets containing three levels of dietary CP (20%, 17.5%, and 15%) and their slurry samples were collected from the pits under the floor every week for one month. Changes in composition of odorous compounds and bacterial communities were analyzed by gas chromatography and 454 FLX titanium pyrosequencing systems, respectively. Levels of phenols, indoles, short chain fatty acid and branched chain fatty acid were lowest (pp-cresol and skatole with Bacteroides, acetic acid and butyric acid with AM982595_g of Porphyromonadaceae family, and propionic acid with Tissierella. Taken together, administration of 15% CP showed less production of odorous compounds than 20% CP group and this result might be associated with the changes in bacterial communities especially whose roles in protein metabolism. PMID:26194219

  14. Synchronization of cytoplasmic and transferred mitochondrial ribosomal protein gene expression in land plants is linked to Telo-box motif enrichment

    Directory of Open Access Journals (Sweden)

    Zhu Xin-Guang

    2011-06-01

    Full Text Available Abstract Background Chloroplasts and mitochondria evolved from the endosymbionts of once free-living eubacteria, and they transferred most of their genes to the host nuclear genome during evolution. The mechanisms used by plants to coordinate the expression of such transferred genes, as well as other genes in the host nuclear genome, are still poorly understood. Results In this paper, we use nuclear-encoded chloroplast (cpRPGs, as well as mitochondrial (mtRPGs and cytoplasmic (euRPGs ribosomal protein genes to study the coordination of gene expression between organelles and the host. Results show that the mtRPGs, but not the cpRPGs, exhibit strongly synchronized expression with euRPGs in all investigated land plants and that this phenomenon is linked to the presence of a telo-box DNA motif in the promoter regions of mtRPGs and euRPGs. This motif is also enriched in the promoter regions of genes involved in DNA replication. Sequence analysis further indicates that mtRPGs, in contrast to cpRPGs, acquired telo-box from the host nuclear genome. Conclusions Based on our results, we propose a model of plant nuclear genome evolution where coordination of activities in mitochondria and chloroplast and other cellular functions, including cell cycle, might have served as a strong selection pressure for the differential acquisition of telo-box between mtRPGs and cpRPGs. This research also highlights the significance of physiological needs in shaping transcriptional regulatory evolution.

  15. Differential inhibitory potencies and mechanisms of the type I ribosome inactivating protein marmorin on estrogen receptor (ER)-positive and ER-negative breast cancer cells.

    Science.gov (United States)

    Pan, Wen Liang; Wong, Jack Ho; Fang, Evandro Fei; Chan, Yau Sang; Ye, Xiu Juan; Ng, Tzi Bun

    2013-05-01

    Breast cancer is the second most common cancer with a high incidence rate worldwide. One of the promising therapeutic approaches on breast cancer is to use the drugs that target the estrogen receptor (ER). In the present investigation, marmorin, a type I ribosome inactivating protein from the mushroom Hypsizigus marmoreus, inhibited the survival of breast cancer in vitro and in vivo. It evinced more potent cytotoxicity toward estrogen receptor (ER)-positive MCF7 breast cancer cells than ER-negative MDA-MB-231 cells. Further study disclosed that marmorin undermined the expression level of estrogen receptor α (ERα) and significantly inhibited the proliferation of MCF7 cells induced by 17β-estradiol. Knockdown of ERα in MCF7 cells significantly attenuated the inhibitory effect of marmorin on proliferation, suggesting that the ERα-mediated pathway was implicated in the suppressive action of marmorin on ER-positive breast cancer cells. Moreover, marmorin induced time-dependent apoptosis in both MCF7 and MDA-MB-231 cells. It brought about G2/M-phase arrest, mitochondrial membrane potential depolarization and caspase-9 activation in MCF7 cells, and to a lesser extent in MDA-MB-231 cells. Marmorin triggered the death receptor apoptotic pathway (e.g. caspase-8 activation) and endoplasmic reticulum stress (ERS, as evidenced by phosphorylation of PERK and IRE1α, cleavage of caspase-12, and up-regulation of CHOP expression) in both MCF7 and MDA-MB-231 cells. In summary, marmorin exhibited inhibitory effect on breast cancer partially via diminution of ERα and apoptotic pathways mediated by mitochondrial, death receptor and ERS. The results advocate that marmorin is a potential candidate for breast cancer therapy. PMID:23274857

  16. Mechanisms of Host-Pathogen Protein Complex Formation and Bacterial Immune Evasion of Streptococcus suis Protein Fhb.

    Science.gov (United States)

    Li, Xueqin; Liu, Peng; Gan, Shuzhen; Zhang, Chunmao; Zheng, Yuling; Jiang, Yongqiang; Yuan, Yuan

    2016-08-12

    Streptococcus suis serotype 2 (S. suis 2)-induced sepsis and meningitis are often accompanied by bacteremia. The evasion of polymorphonuclear leukocyte-mediated phagocytic clearance is central to the establishment of bacteremia caused by S. suis 2 and is facilitated by the ability of factor H (FH)-binding protein (Fhb) to bind FH on the bacterial surface, thereby impeding alternative pathway complement activation and phagocytic clearance. Here, C3b/C3d was found to bind to Fhb, along with FH, forming a large immune complex. The formation of this immune complex was mediated by domain II of Fhb via electrostatic and hydrophobic interactions, which, to our knowledge, is a new type of interaction. Interestingly, Fhb was found to be associated with the cell envelope and also present in the culture supernatant, where secreted Fhb inhibited complement activation via interactions with domain II, thereby enhancing antiphagocytic clearance by polymorphonuclear leukocytes. Thus, Fhb is a multifunctional bacterial protein, which binds host complement component C3 as well as FH and interferes with innate immune recognition in a secret protein manner. S. suis 2 therefore appears to have developed a new strategy to combat host innate immunity and enhance survival in host blood. PMID:27342778

  17. Chirality Switching by Martensitic Transformation in Protein Cylindrical Crystals: Application to Bacterial Flagella

    Science.gov (United States)

    Komai, Ricardo Kiyohiro

    Martensitic transformations provide unique engineering properties that, when designed properly, become important parts of new technology. Martensitic transformations have been studied for many years in traditional alloys (iron, steel, titanium, etc.), however there is still much to be learned in regards to these transformations in biological materials. Olson and Hartman showed in 1982 that these transformations are also observed in bacterial flagella and T4 bacteriophage viral sheaths, allowing for propulsion of bacteria in a fluid environment and, for the virus, is responsible for the infection mechanism. This work demonstrates, using the bacterial flagella as an example, that these transformations can be modelled using thermodynamic methods that are also used to model the transformations in alloys. This thesis work attempts to explain the transformations that occur in bacterial flagella, which are capable of small strain, highly reversible martensitic transformations. The first stress/temperature phase diagrams of these flagella were created by adding the mechanical energy of the transformation of the flagella to limited chemical thermodynamics information of the transformation. Mechanical energy is critical to the transformation process because the bacterial body applies a torque to the radius of the flagella. Finally, work has begun and will be completed in regards to understanding the kinetics of the transformation of the flagella. The motion of the transformation interface can be predicted by using a Landau-Ginzburg model. The crystallography of the transformation in bacterial flagella is also being computed to determine the invariant lines of transformation that occur within this cylindrical crystal. This work has shown that it is possible to treat proteins in a similar manner that alloys are treated when using thermodynamic modelling. Much can be learned from translating what is known regarding phase transformations in hard material systems to soft, organic

  18. Cyclic enterobacterial common antigen: Potential contaminant of bacterially expressed protein preparations

    International Nuclear Information System (INIS)

    We have previously reported the identification of the cyclic enterobacterial common antigen (ECACYC) polysaccharide in E. coli strains commonly used for heterologous protein expression (PJA Erbel et al., J. Bacteriol.185 (2003): 1995). Following this initial report, interactions among several NMR groups established that characteristic N-acetyl signals of ECACYC have been observed in 15N-1H HSQC spectra of samples of various bacterially-expressed proteins suggesting that this water-soluble carbohydrate is a common contaminant. We provide NMR spectroscopic tools to recognize ECACYC in protein samples, as well as several methods to remove this contaminant. Early recognition of ECA-based NMR signals will prevent time-consuming analyses of this copurifying carbohydrate

  19. Bacterial Ortholog of Mammalian Translocator Protein (TSPO) with Virulence Regulating Activity

    Science.gov (United States)

    Chapalain, Annelise; Chevalier, Sylvie; Orange, Nicole; Murillo, Laurence; Papadopoulos, Vassilios; Feuilloley, Marc G. J.

    2009-01-01

    The translocator protein (TSPO), previously designated as peripheral-type benzodiazepine receptor, is a protein mainly located in the outer mitochondrial membrane of eukaryotic cells. TSPO is implicated in major physiological functions and functionally associated with other proteins such as the voltage-dependent anionic channel, also designated as mitochondrial porin. Surprisingly, a TSPO-related protein was identified in the photosynthetic bacterium Rhodobacter sphaeroides but it was initially considered as a relict of evolution. In the present study we cloned a tspO gene in Pseudomonas fluorescens MF37, a non-photosynthetic eubacterium and we used bioinformatics tools to identify TSPO in the genome of 97 other bacteria. P. fluorescens TSPO was recognized by antibodies against mouse protein and by PK 11195, an artificial ligand of mitochondrial TSPO. As in eukaryotes, bacterial TSPO appears functionally organized as a dimer and the apparent Kd for PK 11195 is in the same range than for its eukaryotic counterpart. When P. fluorescens MF37 was treated with PK 11195 (10−5 M) adhesion to living or artificial surfaces and biofilm formation activity were increased. Conversely, the apoptotic potential of bacteria on eukaryotic cells was significantly reduced. This effect of PK11195 was abolished in a mutant of P. fluorescens MF37 deficient for its major outer membrane porin, OprF. The present results demonstrate the existence of a bacterial TSPO that shares common structural and functional characteristics with its mammalian counterpart. This protein, apparently involved in adhesion and virulence, reveals the existence of a possible new inter kingdom signalling system and suggests that the human microbiome should be involuntarily exposed to the evolutionary pressure of benzodiazepines and related molecules. This discovery also represents a promising opportunity for the development of alternative antibacterial strategies. PMID:19564920

  20. Bacterial ortholog of mammalian translocator protein (TSPO with virulence regulating activity.

    Directory of Open Access Journals (Sweden)

    Annelise Chapalain

    Full Text Available The translocator protein (TSPO, previously designated as peripheral-type benzodiazepine receptor, is a protein mainly located in the outer mitochondrial membrane of eukaryotic cells. TSPO is implicated in major physiological functions and functionally associated with other proteins such as the voltage-dependent anionic channel, also designated as mitochondrial porin. Surprisingly, a TSPO-related protein was identified in the photosynthetic bacterium Rhodobacter sphaeroides but it was initially considered as a relict of evolution. In the present study we cloned a tspO gene in Pseudomonas fluorescens MF37, a non-photosynthetic eubacterium and we used bioinformatics tools to identify TSPO in the genome of 97 other bacteria. P. fluorescens TSPO was recognized by antibodies against mouse protein and by PK 11195, an artificial ligand of mitochondrial TSPO. As in eukaryotes, bacterial TSPO appears functionally organized as a dimer and the apparent Kd for PK 11195 is in the same range than for its eukaryotic counterpart. When P. fluorescens MF37 was treated with PK 11195 (10(-5 M adhesion to living or artificial surfaces and biofilm formation activity were increased. Conversely, the apoptotic potential of bacteria on eukaryotic cells was significantly reduced. This effect of PK11195 was abolished in a mutant of P. fluorescens MF37 deficient for its major outer membrane porin, OprF. The present results demonstrate the existence of a bacterial TSPO that shares common structural and functional characteristics with its mammalian counterpart. This protein, apparently involved in adhesion and virulence, reveals the existence of a possible new inter kingdom signalling system and suggests that the human microbiome should be involuntarily exposed to the evolutionary pressure of benzodiazepines and related molecules. This discovery also represents a promising opportunity for the development of alternative antibacterial strategies.

  1. Bacterial ortholog of mammalian translocator protein (TSPO) with virulence regulating activity.

    Science.gov (United States)

    Chapalain, Annelise; Chevalier, Sylvie; Orange, Nicole; Murillo, Laurence; Papadopoulos, Vassilios; Feuilloley, Marc G J

    2009-01-01

    The translocator protein (TSPO), previously designated as peripheral-type benzodiazepine receptor, is a protein mainly located in the outer mitochondrial membrane of eukaryotic cells. TSPO is implicated in major physiological functions and functionally associated with other proteins such as the voltage-dependent anionic channel, also designated as mitochondrial porin. Surprisingly, a TSPO-related protein was identified in the photosynthetic bacterium Rhodobacter sphaeroides but it was initially considered as a relict of evolution. In the present study we cloned a tspO gene in Pseudomonas fluorescens MF37, a non-photosynthetic eubacterium and we used bioinformatics tools to identify TSPO in the genome of 97 other bacteria. P. fluorescens TSPO was recognized by antibodies against mouse protein and by PK 11195, an artificial ligand of mitochondrial TSPO. As in eukaryotes, bacterial TSPO appears functionally organized as a dimer and the apparent Kd for PK 11195 is in the same range than for its eukaryotic counterpart. When P. fluorescens MF37 was treated with PK 11195 (10(-5) M) adhesion to living or artificial surfaces and biofilm formation activity were increased. Conversely, the apoptotic potential of bacteria on eukaryotic cells was significantly reduced. This effect of PK11195 was abolished in a mutant of P. fluorescens MF37 deficient for its major outer membrane porin, OprF. The present results demonstrate the existence of a bacterial TSPO that shares common structural and functional characteristics with its mammalian counterpart. This protein, apparently involved in adhesion and virulence, reveals the existence of a possible new inter kingdom signalling system and suggests that the human microbiome should be involuntarily exposed to the evolutionary pressure of benzodiazepines and related molecules. This discovery also represents a promising opportunity for the development of alternative antibacterial strategies. PMID:19564920

  2. iTRAQ Protein Profile Differential Analysis of Dormant and Germinated Grassbur Twin Seeds Reveals that Ribosomal Synthesis and Carbohydrate Metabolism Promote Germination Possibly Through the PI3K Pathway.

    Science.gov (United States)

    Zhang, Guo-Liang; Zhu, Yue; Fu, Wei-Dong; Wang, Peng; Zhang, Rui-Hai; Zhang, Yan-Lei; Song, Zhen; Xia, Gui-Xian; Wu, Jia-He

    2016-06-01

    Grassbur is a destructive and invasive weed in pastures, and its burs can cause gastric damage to animals. The strong adaptability and reproductive potential of grassbur are partly due to a unique germination mechanism whereby twin seeds develop in a single bur: one seed germinates, but the other remains dormant. To investigate the molecular mechanism of seed germination in twin seeds, we used isobaric tags for relative and absolute quantitation (iTRAQ) to perform a dynamic proteomic analysis of germination and dormancy. A total of 1,984 proteins were identified, 161 of which were considered to be differentially accumulated. The differentially accumulated proteins comprised 102 up-regulated and 59 down-regulated proteins. These proteins were grouped into seven functional categories, ribosomal proteins being the predominant group. The authenticity and accuracy of the results were confirmed by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time reverse transcription-PCR (qPCR). A dynamic proteomic analysis revealed that ribosome synthesis and carbohydrate metabolism affect seed germination possibly through the phosphoinositide 3-kinase (PI3K) pathway. As the PI3K pathway is generally activated by insulin, analyses of seeds treated with exogenous insulin by qPCR, ELISA and iTRAQ confirmed that the PI3K pathway can be activated, which suppresses dormancy and promotes germination in twin grassbur seeds. Together, these results show that the PI3K pathway may play roles in stimulating seed germination in grassbur by modulating ribosomal synthesis and carbohydrate metabolism. PMID:27296714

  3. Structure of a mitochondrial ribosome with minimal RNA.

    Science.gov (United States)

    Sharma, Manjuli R; Booth, Timothy M; Simpson, Larry; Maslov, Dmitri A; Agrawal, Rajendra K

    2009-06-16

    The Leishmania tarentolae mitochondrial ribosome (Lmr) is a minimal ribosomal RNA (rRNA)-containing ribosome. We have obtained a cryo-EM map of the Lmr. The map reveals several features that have not been seen in previously-determined structures of eubacterial or eukaryotic (cytoplasmic or organellar) ribosomes to our knowledge. Comparisons of the Lmr map with X-ray crystallographic and cryo-EM maps of the eubacterial ribosomes and a cryo-EM map of the mammalian mitochondrial ribosome show that (i) the overall structure of the Lmr is considerably more porous, (ii) the topology of the intersubunit space is significantly different, with fewer intersubunit bridges, but more tunnels, and (iii) several of the functionally-important rRNA regions, including the alpha-sarcin-ricin loop, have different relative positions within the structure. Furthermore, the major portions of the mRNA channel, the tRNA passage, and the nascent polypeptide exit tunnel contain Lmr-specific proteins, suggesting that the mechanisms for mRNA recruitment, tRNA interaction, and exiting of the nascent polypeptide in Lmr must differ markedly from the mechanisms deduced for ribosomes in other organisms. Our study identifies certain structural features that are characteristic solely of mitochondrial ribosomes and other features that are characteristic of both mitochondrial and chloroplast ribosomes (i.e., organellar ribosomes). PMID:19497863

  4. Regulation of ribosome biogenesis in maize embryonic axes during germination.

    Science.gov (United States)

    Villa-Hernández, J M; Dinkova, T D; Aguilar-Caballero, R; Rivera-Cabrera, F; Sánchez de Jiménez, E; Pérez-Flores, L J

    2013-10-01

    Ribosome biogenesis is a pre-requisite for cell growth and proliferation; it is however, a highly regulated process that consumes a great quantity of energy. It requires the coordinated production of rRNA, ribosomal proteins and non-ribosomal factors which participate in the processing and mobilization of the new ribosomes. Ribosome biogenesis has been studied in yeast and animals; however, there is little information about this process in plants. The objective of the present work was to study ribosome biogenesis in maize seeds during germination, a stage characterized for its fast growth, and the effect of insulin in this process. Insulin has been reported to accelerate germination and to induce seedling growth. It was observed that among the first events reactivated just after 3 h of imbibition are the rDNA transcription and the pre-rRNA processing and that insulin stimulates both of them (40-230%). The transcript of nucleolin, a protein which regulates rDNA transcription and pre-rRNA processing, is among the messages stored in quiescent dry seeds and it is mobilized into the polysomal fraction during the first hours of imbibition (6 h). In contrast, de novo ribosomal protein synthesis was low during the first hours of imbibition (3 and 6 h) increasing by 60 times in later stages (24 h). Insulin increased this synthesis (75%) at 24 h of imbibition; however, not all ribosomal proteins were similarly regulated. In this regard, an increase in RPS6 and RPL7 protein levels was observed, whereas RPL3 protein levels did not change even though its transcription was induced. Results show that ribosome biogenesis in the first stages of imbibition is carried out with newly synthesized rRNA and ribosomal proteins translated from stored mRNA. PMID:23806421

  5. Viability of adhered bacterial cells: tracking MinD protein oscillations

    Science.gov (United States)

    Barrett, Matt; Colville, Keegan; Schultz-Nielsen, Chris; Jericho, Manfred; Dutcher, John

    2010-03-01

    To study bacterial cells using atomic force microscopy, it is necessary to immobilize the cells on a substrate. Because bacterial cells and common substrates such as glass and mica have a net negative charge, positively charged polymers such as poly-L-lysine (PLL) and polyethyleneimine (PEI) are commonly used as adhesion layers. However, the use of adhesion polymers could stress the cell and even render it inviable. Viable E. coli cells use oscillations of Min proteins along the axis of the rod-shaped cells to ensure accurate cell division. By tagging MinD proteins with GFP, oscillations can be observed using fluorescence microscopy. For a healthy cell in an ideal environment, the oscillation period is measured to be ˜40 s. Prior experiments have shown that PLL increases the oscillation period significantly (up to 80%). In the present study, we have used epifluorescence and total internal reflection fluorescence (TIRF) to track MinD protein oscillations in E. coli bacteria adhered to a variety of positively charged polymers on mica as a function of polymer surface coverage.

  6. Role of acute-phase proteins in interleukin-1-induced nonspecific resistance to bacterial infections in mice.

    OpenAIRE

    Vogels, M.T.E.; L. Cantoni; Carelli, M.; Sironi, M; Ghezzi, P; van der Meer, J. W M

    1993-01-01

    Treatment with a single low dose (80 to 800 ng) of interleukin-1 (IL-1) 24 h before a lethal bacterial challenge of granulocytopenic and normal mice enhances nonspecific resistance. Since IL-1 induces secretion of acute-phase proteins, liver proteins which possess several detoxifying effects, we investigated the role of these proteins in the IL-1-induced protection. Inhibition of liver protein synthesis with D-galactosamine (GALN) completely inhibited the IL-1-induced synthesis of acute-phase...

  7. Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins

    OpenAIRE

    Lin Jin; Jong Hyun Ham; Rosemary Hage; Wanying Zhao; Jaricelis Soto-Hernández; Sang Yeol Lee; Seung-Mann Paek; Min Gab Kim; Charles Boone; Coplin, David L.; David Mackey

    2016-01-01

    Bacterial AvrE-family Type-III effector proteins (T3Es) contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii, w...

  8. Structural and functional characterization of the bacterial ferrous homeostasis protein FeoA

    OpenAIRE

    Vieira, Vanessa Cristina de Carvalho

    2012-01-01

    O objectivo deste trabalho intitulado ““Structural and functional characterization of the bacterial ferrous homeostasis protein FeoA” consistiu na determinação da estrutura e função da proteína FeoA da bacteria E.coli. A principal via bacteriana de entrada do ferro ferroso é através do sistema Feo que deriva das palavras inglesas ferrous iron transport. O ferro é um elemento essencial para a maioria dos organismos participando em vias metabólicas essenciais. Os sistemas de importação ...

  9. Branched signal wiring of an essential bacterial cell-cycle phosphotransfer protein

    OpenAIRE

    Blair, Jimmy A.; Xu, Qingping; Childers, W. Seth; Mathews, Irimpan I.; Kern, Justin W.; Eckart, Michael; Deacon, Ashley M.; Shapiro, Lucy

    2013-01-01

    Vital to bacterial survival is the faithful propagation of cellular signals, and in Caulobacter crescentus ChpT is an essential mediator within the cell cycle circuit. ChpT functions as a histidine-containing phosphotransfer protein (HPt) that shuttles a phosphoryl group from the receiver domain of CckA, the upstream hybrid histidine kinase (HK), to one of two downstream response regulators (RRs)—CtrA or CpdR—that controls cell cycle progression. To understand how ChpT interacts with multiple...

  10. Heterologously expressed bacterial and human multidrug resistance proteins confer cadmium resistance to Escherichia coli

    OpenAIRE

    Achard-Joris, M; van Saparoea, HBV; Driessen, AJM; Bourdineaud, JP; Bourdineaud, Jean-Paul

    2005-01-01

    The human MDR1 gene is induced by cadmium exposure although no resistance to this metal is observed in human cells overexpressing hMDR1. To access the role of MDR proteins in cadmium resistance, human MDR1, Lactococcus lactis lmrA, and Oenococcus oeni omrA were expressed in an Escherichia coli tolC mutant strain which proved to be hypersensitive to cadmium. Both the human and bacterial MDR genes conferred cadmium resistance to E. coli up to 0.4 mM concentration. Protection was abolished by 10...

  11. Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family.

    Science.gov (United States)

    Broussard, Tyler C; Miller, Darcie J; Jackson, Pamela; Nourse, Amanda; White, Stephen W; Rock, Charles O

    2016-03-18

    Fatty acid kinase (Fak) is a ubiquitous Gram-positive bacterial enzyme consisting of an ATP-binding protein (FakA) that phosphorylates the fatty acid bound to FakB. In Staphylococcus aureus, Fak is a global regulator of virulence factor transcription and is essential for the activation of exogenous fatty acids for incorporation into phospholipids. The 1.2-Å x-ray structure of S. aureus FakB2, activity assays, solution studies, site-directed mutagenesis, and in vivo complementation were used to define the functions of the five conserved residues that define the FakB protein family (Pfam02645). The fatty acid tail is buried within the protein, and the exposed carboxyl group is bound by a Ser-93-fatty acid carboxyl-Thr-61-His-266 hydrogen bond network. The guanidinium of the invariant Arg-170 is positioned to potentially interact with a bound acylphosphate. The reduced thermal denaturation temperatures of the T61A, S93A, and H266A FakB2 mutants illustrate the importance of the hydrogen bond network in protein stability. The FakB2 T61A, S93A, and H266A mutants are 1000-fold less active in the Fak assay, and the R170A mutant is completely inactive. All FakB2 mutants form FakA(FakB2)2 complexes except FakB2(R202A), which is deficient in FakA binding. Allelic replacement shows that strains expressing FakB2 mutants are defective in fatty acid incorporation into phospholipids and virulence gene transcription. These conserved residues are likely to perform the same critical functions in all bacterial fatty acid-binding proteins. PMID:26774272

  12. The pneumococcal serine-rich repeat protein is an intra-species bacterial adhesin that promotes bacterial aggregation in vivo and in biofilms.

    Directory of Open Access Journals (Sweden)

    Carlos J Sanchez

    Full Text Available The Pneumococcal serine-rich repeat protein (PsrP is a pathogenicity island encoded adhesin that has been positively correlated with the ability of Streptococcus pneumoniae to cause invasive disease. Previous studies have shown that PsrP mediates bacterial attachment to Keratin 10 (K10 on the surface of lung cells through amino acids 273-341 located in the Basic Region (BR domain. In this study we determined that the BR domain of PsrP also mediates an intra-species interaction that promotes the formation of large bacterial aggregates in the nasopharynx and lungs of infected mice as well as in continuous flow-through models of mature biofilms. Using numerous methods, including complementation of mutants with BR domain deficient constructs, fluorescent microscopy with Cy3-labeled recombinant (rBR, Far Western blotting of bacterial lysates, co-immunoprecipitation with rBR, and growth of biofilms in the presence of antibodies and competitive peptides, we determined that the BR domain, in particular amino acids 122-166 of PsrP, promoted bacterial aggregation and that antibodies against the BR domain were neutralizing. Using similar methodologies, we also determined that SraP and GspB, the Serine-rich repeat proteins (SRRPs of Staphylococcus aureus and Streptococcus gordonii, respectively, also promoted bacterial aggregation and that their Non-repeat domains bound to their respective SRRPs. This is the first report to show the presence of biofilm-like structures in the lungs of animals infected with S. pneumoniae and show that SRRPs have dual roles as host and bacterial adhesins. These studies suggest that recombinant Non-repeat domains of SRRPs (i.e. BR for S. pneumoniae may be useful as vaccine antigens to protect against Gram-positive bacteria that cause infection.

  13. Blood parameters in growing pigs fed increasing levels of bacterial protein meal

    DEFF Research Database (Denmark)

    Hellwing, Anne Louise Frydendahl; Tauson, Anne-Helene; Skrede, Anders

    2007-01-01

    The experiment investigated the effects of increasing dietary levels of bacterial protein meal (BPM) on various blood parameters reflecting protein and fat metabolism, liver function, and purine base metabolism in growing pigs. Sixteen barrows were allocated to four different experimental diets....... The control diet was based on soybean meal. In the other three diets soybean meal was replaced with increasing levels of BPM, approximately 17%, 35%, and 50% of the nitrogen being derived from BPM. Blood samples from the jugular vein were taken when the body weights of the pigs were approximately 10 kg, 21 kg......, 45 kg, and 77 kg. The blood parameters reflecting fat metabolism and liver funtion were not affected by diet. Both the plasma albumin and uric acid concentrations tended to decrease (P = 0.07 and 0.01, respectively) with increasing dietary BPM content, whereas the plasma glucose concentration tended...

  14. Decreased Bacterial Attachment and Protein Adsorption to Coatings Produced by Low Enegy Plasma Polymerization

    DEFF Research Database (Denmark)

    Andersen, T.E.; Kingshott, Peter; Benter, M.;

    Introduction Silicone rubber is among the most biocompatible materials available, exhibiting low levels of extractables, absence of plasticizers and additives and fairly low activation of blood thrombogenesis components. However untreated silicone rubber does not efficiently resist protein...... with a surface less prone to the adsorption of biological matter. In the current study two different hydrophilic nanoscale coatings were produced by low energy plasma polymerization [3] and investigated· f()rl()w ... pr()tein adsorption and bacterial attachment properties. Methods were setup to enable...... the measurement of both initial adhesion of clinically isolated bacteria on silicone and subsequent biofilm formation during prolonged growth under liquid flow. The extend of adsorption of relevant proteins to the surfaces was also investigated using quartz crystal microbalance with dissipation (QCM...

  15. Reconstitution of nanomachine driving the assembly of proteins into bacterial outer membranes

    International Nuclear Information System (INIS)

    Over 9.5 million people die each year due to infectious diseases caused by pathogens. Many species of pathogenic bacteria require nanomachines acting like a molecular pump that shuttle key disease-causing molecules (proteins) from inside bacteria cells to the outside surface, priming the bacteria for infections. How such proteins are assembled remains an important question in biology. If we can inhibit the nanomachines function in transporting specific violence factors, it would disable the disease process. Therefore it is crucial to understand how the proteins are transported through the nanomachines from the periplasm to the extracellular space. Measuring the activity of the component parts of membrane-embedded nanomachines in solution is a major technological challenge. The translocation assembly module (the TAM) is a nanomachine required for virulence of bacterial pathogens. We have reconstituted a membrane containing the TAM onto a gold surface for characterization by Quartz Crystal Microbalance with Dissipation (QCM-D) and Magnetic Contrast Neutron Reflectrometry (MCNR). We show that dynamic movements within the TamA component of the TAM are initiated in the presence of a substrate protein, Ag43, and that these movements recapitulate an initial stage in membrane protein assembly. The reconstituted system provides a powerful new means to study molecular movements in biological membranes, and the technology is widely applicable to studying the dynamics of diverse cellular nanomachines.

  16. Mitochondrial-encoded membrane protein transcripts are pyrimidine-rich while soluble protein transcripts and ribosomal RNA are purine-rich

    Directory of Open Access Journals (Sweden)

    Samuels David C

    2005-09-01

    Full Text Available Abstract Background Eukaryotic organisms contain mitochondria, organelles capable of producing large amounts of ATP by oxidative phosphorylation. Each cell contains many mitochondria with many copies of mitochondrial DNA in each organelle. The mitochondrial DNA encodes a small but functionally critical portion of the oxidative phosphorylation machinery, a few other species-specific proteins, and the rRNA and tRNA used for the translation of these transcripts. Because the microenvironment of the mitochondrion is unique, mitochondrial genes may be subject to different selectional pressures than those affecting nuclear genes. Results From an analysis of the mitochondrial genomes of a wide range of eukaryotic species we show that there are three simple rules for the pyrimidine and purine abundances in mitochondrial DNA transcripts. Mitochondrial membrane protein transcripts are pyrimidine rich, rRNA transcripts are purine-rich and the soluble protein transcripts are purine-rich. The transitions between pyrimidine and purine-rich regions of the genomes are rapid and are easily visible on a pyrimidine-purine walk graph. These rules are followed, with few exceptions, independent of which strand encodes the gene. Despite the robustness of these rules across a diverse set of species, the magnitude of the differences between the pyrimidine and purine content is fairly small. Typically, the mitochondrial membrane protein transcripts have a pyrimidine richness of 56%, the rRNA transcripts are 55% purine, and the soluble protein transcripts are only 53% purine. Conclusion The pyrimidine richness of mitochondrial-encoded membrane protein transcripts is partly driven by U nucleotides in the second codon position in all species, which yields hydrophobic amino acids. The purine-richness of soluble protein transcripts is mainly driven by A nucleotides in the first codon position. The purine-richness of rRNA is also due to an abundance of A nucleotides. Possible

  17. Study on the Immunotoxicity of Ribosome Inhibiting Protein of Momordica Charantia%苦瓜蛋白RIP的免疫毒性研究

    Institute of Scientific and Technical Information of China (English)

    万莉; 孟延发; 沈富兵; 郑崛村; 邓念华; 张立实

    2009-01-01

    目的 分离纯化苦瓜籽核糖体失活蛋白(RIP),对RIP在动物体内急性毒性和免疫毒性进行研究.方法 采用离子交换层析、凝胶过滤层析等方法提取苦瓜籽RIP,对其进行小鼠急性毒性实验,确定小鼠半数致死量(LD_(50)),并通过豚鼠全身主动过敏试验和大鼠被动皮肤过敏试验,探讨苦瓜籽RIP的免疫毒性. 结果苦瓜籽RIP对小鼠的LD_(50)为25.2 mg/kg,全身主动过敏试验中,RIP高、低剂量组豚鼠出现明显过敏反应,多数迅速死亡,被动皮肤过敏试验中,RIP组大鼠皮肤出现明显蓝斑. 结论 苦瓜籽核糖体失活蛋白具有较强的免疫毒性.%Objective To separate and purify ribosome inhibiting protein (RIP) from Momordica charantia (bitter melon) seeds and to evaluate its acute toxicity and immunotoxicity in animal. Methods Ion exchange chromatography and gel filtration chromatography were applied in the separating and purifying of RIP from Momordica charantia seeds. Then the acute toxicity testing of RIP in mice was conducted to obtain its half lethal dose (LD_(50)). Active systemic anaphylaxis (ASA) test in guinea pig and passive cutaneous anaphylaxis test (PCA) in rat were performed to evaluate its immunotoxicity. Results The LD_(50) (iv) in mice of RIP was 25. 2 mg/kg in ASA. guinea pigs of the higher and lower RIP group all appeared stong allergic responses and most of them died quickly. In PCA. obvious blue dye in skin were observed in SD rats of the RIP group. Conclusion RIP getting from Momordica charantia seeds had a relatively strong immunotoxicity in animals.

  18. The β1 adrenergic effects of antibodies against the C-terminal end of the ribosomal P2β protein of Trypanosoma cruzi associate with a specific pattern of epitope recognition

    Science.gov (United States)

    Bergami, P Lopez; Gómez, KA; Levy, GV; Grippo, V; Baldi, A; Levin, MJ

    2005-01-01

    BALB/c mice immunized with recombinant Trypanosoma cruzi ribosomal P2β protein (TcP2β) develop a strong and specific antibody response against its 13 residue-long C-terminal epitope (peptide R13: EEEDDDMGFGLFD) that has a concomitant β1-adrenergic stimulating activity. However, other animals that undergo similar immunizations seem tolerant to this epitope. To evaluate further the antibody response against the ribosomal P proteins, 25 BALB/c and 25 Swiss mice were immunized with TcP2β. From the 50 animals, 31 developed a positive anti-R13 response, whereas 19 were non-responsive. From the 31 anti-R13 positive mice, 25 had anti-R13 antibodies that recognized the discontinuous motif ExDDxGF, and their presence correlated with the recording of supraventricular tachycardia. The other six had anti-R13 antibodies but with a normal electrocardiographic recording. These anti-R13 antibodies recognized the motif DDxGF shared by mammals and T. cruzi and proved to be a true anti-P autoantibody because they were similar to those elicited in Swiss, but not in BALB/c mice, by immunization with the C-terminal portion of the mouse ribosomal P protein. Our results show that the recognition of the glutamic acid in position 3 of peptide R13 defines the ability of anti-R13 antibodies to react with the motif AESDE of the second extracellular loop of the β1-adrenergic receptor, setting the molecular basis for their pathogenic β1 adrenoceptor stimulating activity. PMID:16178868

  19. Identification of the interactome between fish plasma proteins and Edwardsiella tarda reveals tissue-specific strategies against bacterial infection.

    Science.gov (United States)

    Li, Hui; Huang, Xiaoyan; Zeng, Zaohai; Peng, Xuan-Xian; Peng, Bo

    2016-09-01

    Elucidating the complex pathogen-host interaction is essential for a comprehensive understanding of how these remarkable agents invade their hosts and how the hosts defend against these invaders. During the infection, pathogens interact intensively with host to enable their survival, which can be revealed through their interactome. Edwardsiella tarda is a Gram-negative bacterial pathogen causing huge economic loss in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans. E. tarda is an ideal model for host-pathogen investigation as it infects fish in three distinct steps: entering the host, circulating through the blood and establishing infection. We adopted a previous established proteomic approach that inactivated E. tarda cells and covalent crosslink fish plasma proteins were used to capture plasma proteins and bacterial outer membrane proteins, respectively. By the combinatorial use of proteomic and biochemical approaches, six plasma proteins and seven outer membrane proteins (OMPs) were identified. Interactions among these proteins were validated with protein-array, far-Western blotting and co-immunoprecipitation. At last, seventeen plasma protein-bacteria protein-protein interaction were confirmed to be involved in the interaction network, forming a complex interactome. Compared to our previous results, different host proteins were detected, whereas some of the bacterial proteins were similar, which indicates that hosts adopt tissue-specific strategies to cope with the same pathogen during infection. Thus, our results provide a robust demonstration of both bacterial initiators and host receptors or interacting proteins to further explore infection and anti-infective mechanisms between hosts and microbes. PMID:27458055

  20. Ribosome evolution: Emergence of peptide synthesis machinery

    Indian Academy of Sciences (India)

    Koji Tamura

    2011-12-01

    Proteins, the main players in current biological systems, are produced on ribosomes by sequential amide bond (peptide bond) formations between amino-acid-bearing tRNAs. The ribosome is an exquisite super-complex of RNA-proteins, containing more than 50 proteins and at least 3 kinds of RNAs. The combination of a variety of side chains of amino acids (typically 20 kinds with some exceptions) confers proteins with extraordinary structure and functions. The origin of peptide bond formation and the ribosome is crucial to the understanding of life itself. In this article, a possible evolutionary pathway to peptide bond formation machinery (proto-ribosome) will be discussed, with a special focus on the RNA minihelix (primordial form of modern tRNA) as a starting molecule. Combining the present data with recent experimental data, we can infer that the peptidyl transferase center (PTC) evolved from a primitive system in the RNA world comprising tRNA-like molecules formed by duplication of minihelix-like small RNA.

  1. Crystal structure of the Campylobacter jejuni Cj0090 protein reveals a novel variant of the immunoglobulin fold among bacterial lipoproteins

    OpenAIRE

    Paek, Seonghee; Kawai, Fumihiro; Choi, Kyoung-Jae; Yeo, Hye-Jeong

    2012-01-01

    Bacterial lipoproteins play an important role in bacterial pathogenesis and physiology. The genome of Campylobacter jejuni, a major foodborn pathogen, is predicted to contain over 20 lipoproteins. However, the functions of the majority of C. jejuni lipoproteins remain unknown. The Cj0090 protein is encoded by a lipoprotein operon composed of cj0089, cj0090, and cj0091. Here, we report the crystal structure of Cj0090 at 1.9 Å resolution, revealing a novel variant of the immunoglobulin fold wit...

  2. The use of C-reactive protein in predicting bacterial co-Infection in children with bronchiolitis

    OpenAIRE

    Mohamad Fares; Sawsan Mourad; Mariam Rajab; Nahida Rifai

    2011-01-01

    Background: Bronchiolitis is a potentially life-threatening respiratory illness commonly affecting children who are less than two years of age. Patients with viral lower respiratory tract infection are at risk for co-bacterial infection. Aim: The aim of our study was to evaluate the use of C-reactive protein (CRP) in predicting bacterial co-infection in patients hospitalized for bronchiolitis and to correlate the results with the use of antibiotics. Patients and Methods: This is a prospective...

  3. Urokinase-targeted recombinant bacterial protein toxins-a rationally designed and engineered anticancer agent for cancer therapy

    Institute of Scientific and Technical Information of China (English)

    Yizhen LIU; Shi-Yan LI

    2009-01-01

    Urokinase-targeted recombinant bacterial protein toxins are a sort of rationally designed and engineered anticancer recombinant fusion proteins representing a novel class of agents for cancer therapy.Bacterial protein toxins have long been known as the primary virulence factor(s) for a variety of pathogenic bacteria and are the most powerful human poisons.On the other hand,it has been well documented that urokinase-type plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR),making up the uPA system,are overexpressed in a variety of human tumors and tumor cell lines.The expression of uPA system is highly correlated with tumor invasion and metastasis.To exploit these characteristics in the design of tumor cell-selective cytotoxins,two prominent bacterial protein toxins,i.e.,the diphtheria toxin and anthrax toxin are deliberately engineered through placing a sequence targeted specifically by the uPA system to form anticancer recombinant fusion proteins.These uPA system-targeted bacterial protein toxins are activated selectively on the surface of uPA systemexpressing tumor cells,thereby killing these cells.This article provides a review on the latest progress in the exploitation of these recombinant fusion proteins as potent tumoricidal agents.It is perceptible that the strategies for cancer therapy are being innovated by this novel therapeutic approach.

  4. Translation with frameshifting of ribosome along mRNA transcript

    CERN Document Server

    Li, Jingwei

    2015-01-01

    Translation is an important process for prokaryotic and eukaryotic cells to produce necessary proteins for cell growth. Numerious experiments have been performed to explore the translational properties. Diverse models have also been developed to determine the biochemical mechanism of translation. However, to simplify the majority of the existing models, the frameshifting of ribosome along the mRNA transcript is neglected, which actually occurs in real cells and has been extensively experimentally studied. The frameshifting of ribosome evidently influences the efficiency and speed of translation, considering that the peptide chains synthesized by shifted ribosomes will not fold into functional proteins and will degrade rapidly. In this study, a theoretical model is presented to describe the translational process based on the model for totally asymmetric simple exclusion process. In this model, the frameshifting of the ribosome along the mRNA transcript and the attachment/detachment of the ribosome to/from the ...

  5. Lactococcus lactis YfiA is necessary and sufficient for ribosome dimerization

    NARCIS (Netherlands)

    Puri, Pranav; Eckhardt, Thomas H; Franken, Linda E; Fusetti, Fabrizia; Stuart, Marc C A; Boekema, Egbert J; Kuipers, Oscar P; Kok, Jan; Poolman, Berend

    2014-01-01

    Dimerization and inactivation of ribosomes in Escherichia coli is a two-step process that involves the binding of ribosome modulation factor (RMF) and hibernation promotion factor (HPF). Lactococcus lactisMG1363 expresses a protein, YfiA(Ll), which associates with ribosomes in the stationary phase o

  6. Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance

    Directory of Open Access Journals (Sweden)

    Cashman Kathleen A

    2008-06-01

    Full Text Available Abstract Background There is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, recombinant Lassa virus (LASV proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for expression and purification of recombinant LASV nucleoprotein (NP, glycoprotein 1 (GP1, and glycoprotein 2 (GP2. Results Full-length NP and the ectodomains of GP1 and GP2 were generated as maltose-binding protein (MBP fusions in the Rosetta strains of Escherichia coli (E. coli using pMAL-c2x vectors. Average fusion protein yields per liter of culture for MBP-NP, MBP-GP1, and MBP-GP2 were 10 mg, 9 mg, and 9 mg, respectively. Each protein was captured from cell lysates using amylose resin, cleaved with Factor Xa, and purified using size-exclusion chromatography (SEC. Fermentation cultures resulted in average yields per liter of 1.6 mg, 1.5 mg, and 0.7 mg of purified NP, GP1 and GP2, respectively. LASV-specific antibodies in human convalescent sera specifically detected each of the purified recombinant LASV proteins, highlighting their utility in diagnostic applications. In addition, mouse hyperimmune ascitic fluids (MHAF against a panel of Old and New World arenaviruses demonstrated selective cross reactivity with LASV proteins in Western blot and enzyme-linked immunosorbent assay (ELISA. Conclusion These results demonstrate the potential for developing broadly reactive immunological assays that employ all three arenaviral proteins individually and in combination.

  7. Cross-protective effect of a combined L5 plus L3 Leishmania major ribosomal protein based vaccine combined with a Th1 adjuvant in murine cutaneous and visceral leishmaniasis

    OpenAIRE

    Ramírez, Laura; Corvo, Laura; Duarte, Mariana C; Miguel A Chávez-Fumagalli; Diogo G Valadares; Santos, Diego M.; de Oliveira, Camila I.; Escutia, Marta R; Alonso, Carlos; Bonay, Pedro; Carlos A. P. Tavares; Coelho, Eduardo A. F.; Soto, Manuel

    2014-01-01

    Abstract Background Two Leishmania major ribosomal proteins L3 (LmL3) and L5 (LmL5) have been described as protective molecules against cutaneous leishmaniasis due to infection with L. major and Leishmania braziliensis in BALB/c mice when immunized with a Th1 adjuvant (non-methylated CpG-oligodeoxynucleotides; CpG-ODN). In the present study we analyzed the cross-protective efficacy of an LmL3-LmL5-CpG ODN combined vaccine against infection with Leishmania amazonensis and Leishmania chagasi (s...

  8. Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins.

    Science.gov (United States)

    Jin, Lin; Ham, Jong Hyun; Hage, Rosemary; Zhao, Wanying; Soto-Hernández, Jaricelis; Lee, Sang Yeol; Paek, Seung-Mann; Kim, Min Gab; Boone, Charles; Coplin, David L; Mackey, David

    2016-05-01

    Bacterial AvrE-family Type-III effector proteins (T3Es) contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii, we employed a yeast-two-hybrid screen with non-lethal fragments of WtsE and a synthetic genetic array with full-length WtsE. Together these screens indicate that WtsE targets maize protein phosphatase 2A (PP2A) heterotrimeric enzyme complexes via direct interaction with B' regulatory subunits. AvrE1, another AvrE-family T3E from Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), associates with specific PP2A B' subunit proteins from its susceptible host Arabidopsis that are homologous to the maize B' subunits shown to interact with WtsE. Additionally, AvrE1 was observed to associate with the WtsE-interacting maize proteins, indicating that PP2A B' subunits are likely conserved targets of AvrE-family T3Es. Notably, the ability of AvrE1 to promote bacterial growth and/or suppress callose deposition was compromised in Arabidopsis plants with mutations of PP2A genes. Also, chemical inhibition of PP2A activity blocked the virulence activity of both WtsE and AvrE1 in planta. The function of HopM1, a Pto DC3000 T3E that is functionally redundant to AvrE1, was also impaired in specific PP2A mutant lines, although no direct interaction with B' subunits was observed. These results indicate that sub-component specific PP2A complexes are targeted by bacterial T3Es, including direct targeting by members of the widely conserved AvrE-family. PMID:27191168

  9. Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins.

    Directory of Open Access Journals (Sweden)

    Lin Jin

    2016-05-01

    Full Text Available Bacterial AvrE-family Type-III effector proteins (T3Es contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii, we employed a yeast-two-hybrid screen with non-lethal fragments of WtsE and a synthetic genetic array with full-length WtsE. Together these screens indicate that WtsE targets maize protein phosphatase 2A (PP2A heterotrimeric enzyme complexes via direct interaction with B' regulatory subunits. AvrE1, another AvrE-family T3E from Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000, associates with specific PP2A B' subunit proteins from its susceptible host Arabidopsis that are homologous to the maize B' subunits shown to interact with WtsE. Additionally, AvrE1 was observed to associate with the WtsE-interacting maize proteins, indicating that PP2A B' subunits are likely conserved targets of AvrE-family T3Es. Notably, the ability of AvrE1 to promote bacterial growth and/or suppress callose deposition was compromised in Arabidopsis plants with mutations of PP2A genes. Also, chemical inhibition of PP2A activity blocked the virulence activity of both WtsE and AvrE1 in planta. The function of HopM1, a Pto DC3000 T3E that is functionally redundant to AvrE1, was also impaired in specific PP2A mutant lines, although no direct interaction with B' subunits was observed. These results indicate that sub-component specific PP2A complexes are targeted by bacterial T3Es, including direct targeting by members of the widely conserved AvrE-family.

  10. Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins

    Science.gov (United States)

    Jin, Lin; Ham, Jong Hyun; Hage, Rosemary; Zhao, Wanying; Soto-Hernández, Jaricelis; Lee, Sang Yeol; Paek, Seung-Mann; Kim, Min Gab; Boone, Charles; Coplin, David L.; Mackey, David

    2016-01-01

    Bacterial AvrE-family Type-III effector proteins (T3Es) contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii, we employed a yeast-two-hybrid screen with non-lethal fragments of WtsE and a synthetic genetic array with full-length WtsE. Together these screens indicate that WtsE targets maize protein phosphatase 2A (PP2A) heterotrimeric enzyme complexes via direct interaction with B’ regulatory subunits. AvrE1, another AvrE-family T3E from Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), associates with specific PP2A B’ subunit proteins from its susceptible host Arabidopsis that are homologous to the maize B’ subunits shown to interact with WtsE. Additionally, AvrE1 was observed to associate with the WtsE-interacting maize proteins, indicating that PP2A B’ subunits are likely conserved targets of AvrE-family T3Es. Notably, the ability of AvrE1 to promote bacterial growth and/or suppress callose deposition was compromised in Arabidopsis plants with mutations of PP2A genes. Also, chemical inhibition of PP2A activity blocked the virulence activity of both WtsE and AvrE1 in planta. The function of HopM1, a Pto DC3000 T3E that is functionally redundant to AvrE1, was also impaired in specific PP2A mutant lines, although no direct interaction with B’ subunits was observed. These results indicate that sub-component specific PP2A complexes are targeted by bacterial T3Es, including direct targeting by members of the widely conserved AvrE-family. PMID:27191168

  11. Ribosome Mechanics Informs about Mechanism.

    Science.gov (United States)

    Zimmermann, Michael T; Jia, Kejue; Jernigan, Robert L

    2016-02-27

    The essential aspects of the ribosome's mechanism can be extracted from coarse-grained simulations, including the ratchet motion, the movement together of critical bases at the decoding center, and movements of the peptide tunnel lining that assist in the expulsion of the synthesized peptide. Because of its large size, coarse graining helps to simplify and to aid in the understanding of its mechanism. Results presented here utilize coarse-grained elastic network modeling to extract the dynamics, and both RNAs and proteins are coarse grained. We review our previous results, showing the well-known ratchet motions and the motions in the peptide tunnel and in the mRNA tunnel. The motions of the lining of the peptide tunnel appear to assist in the expulsion of the growing peptide chain, and clamps at the ends of the mRNA tunnel with three proteins ensure that the mRNA is held tightly during decoding and essential for the helicase activity at the entrance. The entry clamp may also assist in base recognition to ensure proper selection of the incoming tRNA. The overall precision of the ribosome machine-like motions is remarkable. PMID:26687034

  12. Nucleolar dominance and ribosomal RNA gene silencing

    OpenAIRE

    Tucker, Sarah; Vitins, Alexa; Pikaard, Craig S.

    2010-01-01

    Nucleolar dominance is an epigenetic phenomenon that occurs in genetic hybrids and describes the expression of 45S rRNA genes inherited from one progenitor due to the silencing of the other progenitor’s rRNA genes. Nucleolar dominance is a manifestation of rRNA gene dosage control, which also occurs in non-hybrids, regulating the number of active rRNA genes according to the cellular demand for ribosomes and protein synthesis. Ribosomal RNA gene silencing involves changes in DNA methylation an...

  13. Two Proteins Form a Heteromeric Bacterial Self-Recognition Complex in Which Variable Subdomains Determine Allele-Restricted Binding

    OpenAIRE

    Cardarelli, Lia; Saak, Christina; Gibbs, Karine A

    2015-01-01

    ABSTRACT Self- versus nonself-recognition in bacteria has been described recently through genetic analyses in multiple systems; however, understanding of the biochemical properties and mechanisms of recognition-determinant proteins remains limited. Here we extend the molecular and biochemical understanding of two recognition-determinant proteins in bacteria. We have found that a heterotypic complex is formed between two bacterial self-recognition proteins, IdsD and IdsE, the genes of which ha...

  14. Identification of multiple physicochemical and structural properties associated with soluble expression of eukaryotic proteins in cell-free bacterial extracts

    OpenAIRE

    AlexanderA.Tokmakov

    2014-01-01

    Bacterial extracts are widely used to synthesize recombinant proteins. Vast data volumes have been accumulated in cell-free expression databases, covering a whole range of existing proteins. It makes possible comprehensive bioinformatics analysis and identification of multiple features associated with protein solubility and aggregation. In the present paper, an approach to identify the multiple physicochemical and structural properties of amino acid sequences associated with soluble expressio...

  15. Allosteric effects of chromophore interaction with dimeric near-infrared fluorescent proteins engineered from bacterial phytochromes.

    Science.gov (United States)

    Stepanenko, Olesya V; Baloban, Mikhail; Bublikov, Grigory S; Shcherbakova, Daria M; Stepanenko, Olga V; Turoverov, Konstantin K; Kuznetsova, Irina M; Verkhusha, Vladislav V

    2016-01-01

    Fluorescent proteins (FPs) engineered from bacterial phytochromes attract attention as probes for in vivo imaging due to their near-infrared (NIR) spectra and use of available in mammalian cells biliverdin (BV) as chromophore. We studied spectral properties of the iRFP670, iRFP682 and iRFP713 proteins and their mutants having Cys residues able to bind BV either in both PAS (Cys15) and GAF (Cys256) domains, in one of these domains, or without these Cys residues. We show that the absorption and fluorescence spectra and the chromophore binding depend on the location of the Cys residues. Compared with NIR FPs in which BV covalently binds to Cys15 or those that incorporate BV noncovalently, the proteins with BV covalently bound to Cys256 have blue-shifted spectra and higher quantum yield. In dimeric NIR FPs without Cys15, the covalent binding of BV to Сys256 in one monomer allosterically inhibits the covalent binding of BV to the other monomer, whereas the presence of Cys15 allosterically promotes BV binding to Cys256 in both monomers. The NIR FPs with both Cys residues have the narrowest blue-shifted spectra and the highest quantum yield. Our analysis resulted in the iRFP713/Val256Cys protein with the highest brightness in mammalian cells among available NIR FPs. PMID:26725513

  16. Ribosomal Protein S6 Kinase (RSK-2 as a central effector molecule in RON receptor tyrosine kinase mediated epithelial to mesenchymal transition induced by macrophage-stimulating protein

    Directory of Open Access Journals (Sweden)

    Zhang Rui-Wen

    2011-05-01

    Full Text Available Abstract Background Epithelial to mesenchymal transition (EMT occurs during cancer cell invasion and malignant metastasis. Features of EMT include spindle-like cell morphology, loss of epithelial cellular markers and gain of mesenchymal phenotype. Activation of the RON receptor tyrosine kinase by macrophage-stimulating protein (MSP has been implicated in cellular EMT program; however, the major signaling determinant(s responsible for MSP-induced EMT is unknown. Results The study presented here demonstrates that RSK2, a downstream signaling protein of the Ras-Erk1/2 pathway, is the principal molecule that links MSP-activated RON signaling to complete EMT. Using MDCK cells expressing RON as a model, a spindle-shape based screen was conducted, which identifies RSK2 among various intracellular proteins as a potential signaling molecule responsible for MSP-induced EMT. MSP stimulation dissociated RSK2 with Erk1/2 and promoted RSK2 nuclear translocation. MSP strongly induced RSK2 phosphorylation in a dose-dependent manner. These effects relied on RON and Erk1/2 phosphorylation, which is significantly potentiated by transforming growth factor (TGF-β1, an EMT-inducing cytokine. Specific RSK inhibitor SL0101 completely prevented MSP-induced RSK phosphorylation, which results in inhibition of MSP-induced spindle-like morphology and suppression of cell migration associated with EMT. In HT-29 cancer cells that barely express RSK2, forced RSK2 expression results in EMT-like phenotype upon MSP stimulation. Moreover, specific siRNA-mediated silencing of RSK2 but not RSK1 in L3.6pl pancreatic cancer cells significantly inhibited MSP-induced EMT-like phenotype and cell migration. Conclusions MSP-induced RSK2 activation is a critical determinant linking RON signaling to cellular EMT program. Inhibition of RSK2 activity may provide a therapeutic opportunity for blocking RON-mediated cancer cell migration and subsequent invasion.

  17. Re-evaluation of a bacterial antifreeze protein as an adhesin with ice-binding activity.

    Directory of Open Access Journals (Sweden)

    Shuaiqi Guo

    Full Text Available A novel role for antifreeze proteins (AFPs may reside in an exceptionally large 1.5-MDa adhesin isolated from an Antarctic Gram-negative bacterium, Marinomonas primoryensis. MpAFP was purified from bacterial lysates by ice adsorption and gel electrophoresis. We have previously reported that two highly repetitive sequences, region II (RII and region IV (RIV, divide MpAFP into five distinct regions, all of which require mM Ca(2+ levels for correct folding. Also, the antifreeze activity is confined to the 322-residue RIV, which forms a Ca(2+-bound beta-helix containing thirteen Repeats-In-Toxin (RTX-like repeats. RII accounts for approximately 90% of the mass of MpAFP and is made up of ∼120 tandem 104-residue repeats. Because these repeats are identical in DNA sequence, their number was estimated here by pulsed-field gel electrophoresis. Structural homology analysis by the Protein Homology/analogY Recognition Engine (Phyre2 server indicates that the 104-residue RII repeat adopts an immunoglobulin beta-sandwich fold that is typical of many secreted adhesion proteins. Additional RTX-like repeats in RV may serve as a non-cleavable signal sequence for the type I secretion pathway. Immunodetection shows both repeated regions are uniformly distributed over the cell surface. We suggest that the development of an AFP-like domain within this adhesin attached to the bacterial outer surface serves to transiently bind the host bacteria to ice. This association would keep the bacteria within the upper reaches of the water column where oxygen and nutrients are potentially more abundant. This novel envirotactic role would give AFPs a third function, after freeze avoidance and freeze tolerance: that of transiently binding an organism to ice.

  18. Amicoumacin A induces cancer cell death by targeting the eukaryotic ribosome

    OpenAIRE

    Prokhorova, Irina V.; Akulich, Kseniya A.; Desislava S. Makeeva; Osterman, Ilya A.; Skvortsov, Dmitry A.; Sergiev, Petr V.; Dontsova, Olga A.; Gulnara Yusupova; Yusupov, Marat M; Dmitriev, Sergey E

    2016-01-01

    Amicoumacin A is an antibiotic that was recently shown to target bacterial ribosomes. It affects translocation and provides an additional contact interface between the ribosomal RNA and mRNA. The binding site of amicoumacin A is formed by universally conserved nucleotides of rRNA. In this work, we showed that amicoumacin A inhibits translation in yeast and mammalian systems by affecting translation elongation. We determined the structure of the amicoumacin A complex with yeast ribosomes at a ...

  19. The Ribosome Comes Alive

    OpenAIRE

    Frank, Joachim

    2010-01-01

    This essay is a reflection on the ways the X-ray structures of the ribosome are helping in the interpretation of cryogenic electron microscopy (cryo-EM) density maps showing the translating ribosome in motion. Through advances in classification methods, cryo-EM and single-particle reconstruction methods have recently evolved to the point where they can yield an array of structures from a single sample (“story in a sample”), providing snapshots of an entire subprocess of translation, such as t...

  20. The Ribosome Comes Alive.

    Science.gov (United States)

    Frank, Joachim

    2010-06-18

    This essay is a reflection on the ways the X-ray structures of the ribosome are helping in the interpretation of cryogenic electron microscopy (cryo-EM) density maps showing the translating ribosome in motion. Through advances in classification methods, cryo-EM and single-particle reconstruction methods have recently evolved to the point where they can yield an array of structures from a single sample ("story in a sample"), providing snapshots of an entire subprocess of translation, such as translocation or decoding. PMID:21072331

  1. Stealth Proteins: In Silico Identification of a Novel Protein Family Rendering Bacterial Pathogens Invisible to Host Immune Defense.

    Directory of Open Access Journals (Sweden)

    2005-11-01

    Full Text Available There are a variety of bacterial defense strategies to survive in a hostile environment. Generation of extracellular polysaccharides has proved to be a simple but effective strategy against the host's innate immune system. A comparative genomics approach led us to identify a new protein family termed Stealth, most likely involved in the synthesis of extracellular polysaccharides. This protein family is characterized by a series of domains conserved across phylogeny from bacteria to eukaryotes. In bacteria, Stealth (previously characterized as SacB, XcbA, or WefC is encoded by subsets of strains mainly colonizing multicellular organisms, with evidence for a protective effect against the host innate immune defense. More specifically, integrating all the available information about Stealth proteins in bacteria, we propose that Stealth is a D-hexose-1-phosphoryl transferase involved in the synthesis of polysaccharides. In the animal kingdom, Stealth is strongly conserved across evolution from social amoebas to simple and complex multicellular organisms, such as Dictyostelium discoideum, hydra, and human. Based on the occurrence of Stealth in most Eukaryotes and a subset of Prokaryotes together with its potential role in extracellular polysaccharide synthesis, we propose that metazoan Stealth functions to regulate the innate immune system. Moreover, there is good reason to speculate that the acquisition and spread of Stealth could be responsible for future epidemic outbreaks of infectious diseases caused by a large variety of eubacterial pathogens. Our in silico identification of a homologous protein in the human host will help to elucidate the causes of Stealth-dependent virulence. At a more basic level, the characterization of the molecular and cellular function of Stealth proteins may shed light on fundamental mechanisms of innate immune defense against microbial invasion.

  2. Stealth proteins: in silico identification of a novel protein family rendering bacterial pathogens invisible to host immune defense.

    Directory of Open Access Journals (Sweden)

    Peter Sperisen

    2005-11-01

    Full Text Available There are a variety of bacterial defense strategies to survive in a hostile environment. Generation of extracellular polysaccharides has proved to be a simple but effective strategy against the host's innate immune system. A comparative genomics approach led us to identify a new protein family termed Stealth, most likely involved in the synthesis of extracellular polysaccharides. This protein family is characterized by a series of domains conserved across phylogeny from bacteria to eukaryotes. In bacteria, Stealth (previously characterized as SacB, XcbA, or WefC is encoded by subsets of strains mainly colonizing multicellular organisms, with evidence for a protective effect against the host innate immune defense. More specifically, integrating all the available information about Stealth proteins in bacteria, we propose that Stealth is a D-hexose-1-phosphoryl transferase involved in the synthesis of polysaccharides. In the animal kingdom, Stealth is strongly conserved across evolution from social amoebas to simple and complex multicellular organisms, such as Dictyostelium discoideum, hydra, and human. Based on the occurrence of Stealth in most Eukaryotes and a subset of Prokaryotes together with its potential role in extracellular polysaccharide synthesis, we propose that metazoan Stealth functions to regulate the innate immune system. Moreover, there is good reason to speculate that the acquisition and spread of Stealth could be responsible for future epidemic outbreaks of infectious diseases caused by a large variety of eubacterial pathogens. Our in silico identification of a homologous protein in the human host will help to elucidate the causes of Stealth-dependent virulence. At a more basic level, the characterization of the molecular and cellular function of Stealth proteins may shed light on fundamental mechanisms of innate immune defense against microbial invasion.

  3. LocateP: Genome-scale subcellular-location predictor for bacterial proteins

    Directory of Open Access Journals (Sweden)

    Zhou Miaomiao

    2008-03-01

    current tools especially where the N-terminally anchored and the SPIase-cleaved secreted proteins are concerned. Overall, the accuracy of LocateP was always higher than 90%. LocateP was then used to predict the SCLs of all proteins encoded by completed Gram-positive bacterial genomes. The results are stored in the database LocateP-DB http://www.cmbi.ru.nl/locatep-db1. Conclusion LocateP is by far the most accurate and detailed protein SCL predictor for Gram-positive bacteria currently available.

  4. Novel MntR-Independent Mechanism of Manganese Homeostasis in Escherichia coli by the Ribosome-Associated Protein HflX

    OpenAIRE

    Kaur, Gursharan; Sengupta, Sandeepan; Kumar, Vineet; Kumari, Aruna; Ghosh, Aditi; Parrack, Pradeep; Dutta, Dipak

    2014-01-01

    Manganese is a micronutrient required for activities of several important enzymes under conditions of oxidative stress and iron starvation. In Escherichia coli, the manganese homeostasis network primarily constitutes a manganese importer (MntH) and an exporter (MntP), which are regulated by the MntR dual regulator. In this study, we find that deletion of E. coli hflX, which encodes a ribosome-associated GTPase with unknown function, renders extreme manganese sensitivity characterized by ar...

  5. Communication: Microsecond dynamics of the protein and water affect electron transfer in a bacterial bc{sub 1} complex

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Daniel R.; Matyushov, Dmitry V., E-mail: dmitrym@asu.edu [Department of Physics and Department of Chemistry and Biochemistry, Arizona State University, P.O. Box 871504, Tempe, Arizona 85287 (United States)

    2015-04-28

    Cross-membrane electron transport between cofactors localized in proteins of mitochondrial respiration and bacterial photosynthesis is the source of all biological energy. The statistics and dynamics of nuclear fluctuations in these protein/membrane/water heterogeneous systems are critical for their energetic efficiency. The results of 13 μs of atomistic molecular dynamics simulations of the membrane-bound bc{sub 1} bacterial complex are analyzed here. The reaction is affected by a broad spectrum of nuclear modes, with the slowest dynamics in the range of time-scales ∼0.1-1.6 μs contributing half of the reaction reorganization energy. Two reorganization energies are required to describe protein electron transfer due to dynamical arrest of protein conformations on the observation window. This mechanistic distinction allows significant lowering of activation barriers for reactions in proteins.

  6. The aminoglycoside resistance methyltransferase Sgm impedes RsmF methylation at an adjacent rRNA nucleotide in the ribosomal A site

    DEFF Research Database (Denmark)

    Cubrilo, Sonja; Babić, Fedora; Douthwaite, Stephen;

    2009-01-01

    methylated nucleotides including m(4)Cm1402 and m(5)C1407. Modification at m(5)C1407 by the methyltransferase RsmF is impeded as Sgm gains access to its adjacent G1405 target on the 30S ribosomal subunit. An Sgm mutant (G135A), which is impaired in S-adenosylmethionine binding and confers lower resistance......Ribosome-targeting antibiotics block protein synthesis by binding at functionally important regions of the bacterial rRNA. Resistance is often conferred by addition of a methyl group at the antibiotic binding site within an rRNA region that is already highly modified with several nucleotide...... methylations. In bacterial rRNA, each methylation requires its own specific methyltransferase enzyme, and this raises the question as to how an extra methyltransferase conferring antibiotic resistance can be accommodated and how it can gain access to its nucleotide target within a short and functionally...

  7. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

    Directory of Open Access Journals (Sweden)

    Lei eShi

    2014-09-01

    Full Text Available Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

  8. Antiadhesive Properties of Arabinogalactan Protein from Ribes nigrum Seeds against Bacterial Adhesion of Helicobacter pylori

    Directory of Open Access Journals (Sweden)

    Jutta Messing

    2014-03-01

    Full Text Available Fruit extracts from black currants (Ribes nigrum L. are traditionally used for treatment of gastritis based on seed polysaccharides that inhibit the adhesion of Helicobacter pylori to stomach cells. For detailed investigations an arabinogalactan protein (F2 was isolated from seeds and characterized concerning molecular weight, carbohydrate, amino acid composition, linkage, configuration and reaction with β-glucosyl Yariv. Functional testing of F2 was performed by semiquantitative in situ adhesion assay on sections of human gastric mucosa and by quantitative in vitro adhesion assay with FITC-labled H. pylori strain J99 and human stomach AGS cells. Bacterial adhesins affected were identified by overlay assay with immobilized ligands. 125I-radiolabeled F2 served for binding studies to H. pylori and interaction experiments with BabA and SabA. F2 had no cytotoxic effects against H. pylori and AGS cells; but inhibited bacterial binding to human gastric cells. F2 inhibited the binding of BabA and fibronectin-binding adhesin to its specific ligands. Radiolabeled F2 bound non-specifically to different strains of H. pylori; and to BabA deficient mutant. F2 did not lead to subsequent feedback regulation or increased expression of adhesins or virulence factors. From these data the non-specific interactions between F2 and the H. pylori lead to moderate antiadhesive effects.

  9. Antiadhesive properties of arabinogalactan protein from ribes nigrum seeds against bacterial adhesion of Helicobacter pylori.

    Science.gov (United States)

    Messing, Jutta; Niehues, Michael; Shevtsova, Anna; Borén, Thomas; Hensel, Andreas

    2014-01-01

    Fruit extracts from black currants (Ribes nigrum L.) are traditionally used for treatment of gastritis based on seed polysaccharides that inhibit the adhesion of Helicobacter pylori to stomach cells. For detailed investigations an arabinogalactan protein (F2) was isolated from seeds and characterized concerning molecular weight, carbohydrate, amino acid composition, linkage, configuration and reaction with β-glucosyl Yariv. Functional testing of F2 was performed by semiquantitative in situ adhesion assay on sections of human gastric mucosa and by quantitative in vitro adhesion assay with FITC-labled H. pylori strain J99 and human stomach AGS cells. Bacterial adhesins affected were identified by overlay assay with immobilized ligands. ¹²⁵I-radiolabeled F2 served for binding studies to H. pylori and interaction experiments with BabA and SabA. F2 had no cytotoxic effects against H. pylori and AGS cells; but inhibited bacterial binding to human gastric cells. F2 inhibited the binding of BabA and fibronectin-binding adhesin to its specific ligands. Radiolabeled F2 bound non-specifically to different strains of H. pylori; and to BabA deficient mutant. F2 did not lead to subsequent feedback regulation or increased expression of adhesins or virulence factors. From these data the non-specific interactions between F2 and the H. pylori lead to moderate antiadhesive effects. PMID:24662083

  10. Acute phase proteins in serum and cerebrospinal fluid in the course of bacterial meningitis.

    Science.gov (United States)

    Paradowski, M; Lobos, M; Kuydowicz, J; Krakowiak, M; Kubasiewicz-Ujma, B

    1995-08-01

    We carried out estimations of the following acute phase proteins: C-reactive protein (CRP), alpha-1-antitrypsin (AAT), alpha-1-acid glycoprotein (AAG), alpha-2-ceruloplasmin (CER), and alpha-2-haptoglobin (HPT) in serum and in cerebrospinal fluid (CSF) in patients with bacterial meningitis (BM, n = 30) and viral meningitis (VM, n = 30). We have shown that determinations of concentrations of AAG and CRP in serum and CER in CSF are useful in differentiation between BM and VM. The diagnostic power of these three tests (the areas under their ROC curves equal 0.942, 0.929, and 0.931, respectively) is bigger, though statistically not significantly, than that of traditional parameters of BM in CSF, i.e., total protein concentration and white blood cell count. Determination of AAG, CRP, and AAT in serum is a valuable monitoring marker in the course of BM treatment. Convenience of serum sampling constitutes an advantage over traditional BM parameters in CSF. PMID:8521602

  11. Optimized localization of bacterial infections with technetium-99m labelled human immunoglobulin after protein charge selection

    International Nuclear Information System (INIS)

    To improve the scintigraphic detection of bacterial infections a protein charge-purified fraction of polyclonal human immunoglobulin was applied as a radiopharmaceutical. This purification was achieved by attaching the immunoglobulin to an anion-exchanger column and by obtaining the column-bound fraction with buffer. The binding to bacteria in vitro and the target to non-target ratios of an experimental thigh infection with Staphylococcus aureus or Klebsiella pneumoniae in mice were evaluated to compare the purified and the unpurified immunoglobulin. The percentage of binding to all gram-positive and gram-negative bacteria used in this study was significantly (P99mTc-labelled protein charge-purified polyclonal human immunoglobulin was administered intravenously. At all time intervals the target (infected thighs) to non-target (non-infected thighs) ratios for both infections were significantly higher (P99mTc-labelled protein charge-purified immunoglobulin localizes both a gram-positive and a gram-negative thigh infection more intensely and faster than 99mTc-labelled unpurified immunoglobulin. (orig.)

  12. Symmetry and scale orient Min protein patterns in shaped bacterial sculptures

    Science.gov (United States)

    Wu, Fabai; van Schie, Bas G. C.; Keymer, Juan E.; Dekker, Cees

    2015-08-01

    The boundary of a cell defines the shape and scale of its subcellular organization. However, the effects of the cell's spatial boundaries as well as the geometry sensing and scale adaptation of intracellular molecular networks remain largely unexplored. Here, we show that living bacterial cells can be ‘sculpted’ into defined shapes, such as squares and rectangles, which are used to explore the spatial adaptation of Min proteins that oscillate pole-to-pole in rod-shaped Escherichia coli to assist cell division. In a wide geometric parameter space, ranging from 2 × 1 × 1 to 11 × 6 × 1 μm3, Min proteins exhibit versatile oscillation patterns, sustaining rotational, longitudinal, diagonal, stripe and even transversal modes. These patterns are found to directly capture the symmetry and scale of the cell boundary, and the Min concentration gradients scale with the cell size within a characteristic length range of 3-6 μm. Numerical simulations reveal that local microscopic Turing kinetics of Min proteins can yield global symmetry selection, gradient scaling and an adaptive range, when and only when facilitated by the three-dimensional confinement of the cell boundary. These findings cannot be explained by previous geometry-sensing models based on the longest distance, membrane area or curvature, and reveal that spatial boundaries can facilitate simple molecular interactions to result in far more versatile functions than previously understood.

  13. Structure of the Bacterial Cytoskeleton Protein Bactofilin by NMR Chemical Shifts and Sequence Variation.

    Science.gov (United States)

    Kassem, Maher M; Wang, Yong; Boomsma, Wouter; Lindorff-Larsen, Kresten

    2016-06-01

    Bactofilins constitute a recently discovered class of bacterial proteins that form cytoskeletal filaments. They share a highly conserved domain (DUF583) of which the structure remains unknown, in part due to the large size and noncrystalline nature of the filaments. Here, we describe the atomic structure of a bactofilin domain from Caulobacter crescentus. To determine the structure, we developed an approach that combines a biophysical model for proteins with recently obtained solid-state NMR spectroscopy data and amino acid contacts predicted from a detailed analysis of the evolutionary history of bactofilins. Our structure reveals a triangular β-helical (solenoid) conformation with conserved residues forming the tightly packed core and polar residues lining the surface. The repetitive structure explains the presence of internal repeats as well as strongly conserved positions, and is reminiscent of other fibrillar proteins. Our work provides a structural basis for future studies of bactofilin biology and for designing molecules that target them, as well as a starting point for determining the organization of the entire bactofilin filament. Finally, our approach presents new avenues for determining structures that are difficult to obtain by traditional means. PMID:27276252

  14. EXPRESSION OF BACTERIAL PROTEIN-A IN TOBACCO LEADS TO ENHANCED RESISTANCE TO STRESS CONDITIONS

    Directory of Open Access Journals (Sweden)

    Chaitali Roy

    2014-08-01

    Full Text Available Tobacco is the most commonly used plant for expression of transgenes from a variety of organisms because it can be easily grown and transformed, it provides abundant amounts of fresh tissue and has a well-established cell culture system. As bacterial enzymes can be synthesized in tobacco, here we explore the possibility of in planta expression of staphylococcal protein-A(PA which is an antibody, an important group among biopharmaceuticals. In our study we have shown that the tobacco plants harboring PA gene could combat the crown gall infection and also effective in resisting abiotic stress conditions. Transgenic plants when subjected to interact with wild variety of Agrobacterium shows its enhanced capability to resist the gall formation. And when transgenic tobacco plants were grown in presence of 200mM NaCl and/or MG(Methylglyoxal solution, shows their increased tolerance towards salinity stress and high MG stress. So far transgenic tobacco plants are concerned, improvements in the expression of recombinant proteins and their recovery from tobacco may also enhance production and commercial use of this protein.

  15. Temporal expression of bacterial proteins instructs host CD4 T cell expansion and Th17 development.

    Directory of Open Access Journals (Sweden)

    Seung-Joo Lee

    2012-01-01

    Full Text Available Pathogens can substantially alter gene expression within an infected host depending on metabolic or virulence requirements in different tissues, however, the effect of these alterations on host immunity are unclear. Here we visualized multiple CD4 T cell responses to temporally expressed proteins in Salmonella-infected mice. Flagellin-specific CD4 T cells expanded and contracted early, differentiated into Th1 and Th17 lineages, and were enriched in mucosal tissues after oral infection. In contrast, CD4 T cells responding to Salmonella Type-III Secretion System (TTSS effectors steadily accumulated until bacterial clearance was achieved, primarily differentiated into Th1 cells, and were predominantly detected in systemic tissues. Thus, pathogen regulation of antigen expression plays a major role in orchestrating the expansion, differentiation, and location of antigen-specific CD4 T cells in vivo.

  16. Ribosomal crystallography: peptide bond formation and its inhibition.

    Science.gov (United States)

    Bashan, Anat; Zarivach, Raz; Schluenzen, Frank; Agmon, Ilana; Harms, Joerg; Auerbach, Tamar; Baram, David; Berisio, Rita; Bartels, Heike; Hansen, Harly A S; Fucini, Paola; Wilson, Daniel; Peretz, Moshe; Kessler, Maggie; Yonath, Ada

    2003-09-01

    Ribosomes, the universal cellular organelles catalyzing the translation of genetic code into proteins, are protein/RNA assemblies, of a molecular weight 2.5 mega Daltons or higher. They are built of two subunits that associate for performing protein biosynthesis. The large subunit creates the peptide bond and provides the path for emerging proteins. The small has key roles in initiating the process and controlling its fidelity. Crystallographic studies on complexes of the small and the large eubacterial ribosomal subunits with substrate analogs, antibiotics, and inhibitors confirmed that the ribosomal RNA governs most of its activities, and indicated that the main catalytic contribution of the ribosome is the precise positioning and alignment of its substrates, the tRNA molecules. A symmetry-related region of a significant size, containing about two hundred nucleotides, was revealed in all known structures of the large ribosomal subunit, despite the asymmetric nature of the ribosome. The symmetry rotation axis, identified in the middle of the peptide-bond formation site, coincides with the bond connecting the tRNA double-helical features with its single-stranded 3' end, which is the moiety carrying the amino acids. This thus implies sovereign movements of tRNA features and suggests that tRNA translocation involves a rotatory motion within the ribosomal active site. This motion is guided and anchored by ribosomal nucleotides belonging to the active site walls, and results in geometry suitable for peptide-bond formation with no significant rearrangements. The sole geometrical requirement for this proposed mechanism is that the initial P-site tRNA adopts the flipped orientation. The rotatory motion is the major component of unified machinery for peptide-bond formation, translocation, and nascent protein progression, since its spiral nature ensures the entrance of the nascent peptide into the ribosomal exit tunnel. This tunnel, assumed to be a passive path for the

  17. Up-regulation of ribosomal protein S13 and L23 are associated with multidrug-resistant phenotype of gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Aims:Prevous study using differential display-PCR had found up-regulation of ribosomal protein S13(RPS13) and L23(RPL23) in vincristine-resistant gastric cancer cells.The aim of this study was to explore the association of RPS13 and RPL23 with multidrug-resistant phenotype of gastric cancer cells.Methods:Northern blot analysis was used to determine the expression of RPS13 and RPL23 in vincristine-resistant gastric cancer cells SGC7901/VCR and its parental cells SGC7901.The full-length cDNAs encoding RPS13 and RPL23 were amplified from SGC7901/VCR cells using RT-PCR.Their sense and antisense expression vectors were constructed by DNA recombinant technique,and transferred into SGC7901 cells(sense vectors) or SGC7901/VCR cells(antisense vectors)by means of Lipofactamine.Drug sensitivity of gastric cancer cells was evalu-ated using MTT assay.Cell cycle analysis was performed using flow cytometry and proliferous index(PI) was calculated.Results:As Northem blot analysis indicated,RNA from SGC7902/VCR cells exhibited moderate signals of PRL23 and RPS13,while RNA from SGC7901 cells exhibited no signal of RPL23 and very weak signal of RPS13.RNA dot blot analysis indicated that RPS13 or RPL23 upregulated SGC7901 cells(SGC7901-RPS13,SGC7901-RPL23)and RPS13 or RPL23 down-regulated SGC7901/VCR cells(SGC7901/VCR-anRPS13,SGC7901/VCR-anRPL23) were successfully prepared by gene transduction.The results of MTT assay indicated that,comparing with non-transfected and empty vector transfected cells,SGC7901-RPL23 cells showed significantly increased IC50 values and resistance index(RI) of vincristine(VCR),adriamycin(ADR),5-fludrouracil(5-Fu) and mitomycin(MMC);SGC7901-RPS12 cells showed significantly increased IC50 values and RI of VCR,ADR and 5-Fu;SGC7901/VCR-anRPL23 cells showed significantly decreased IC50 values and RI of MMC and cisplatin(DDP);SGC7901/VCR-anRPS13 cells showed significantly decreased IC50 values and RI of VCR and MMC.Cell cycle analysis indicated that,comparing with

  18. Dependence of Bacterial Protein Adhesins on Toll-Like Receptors for Proinflammatory Cytokine Induction

    Science.gov (United States)

    Hajishengallis, George; Martin, Michael; Sojar, Hakimuddin T.; Sharma, Ashu; Schifferle, Robert E.; DeNardin, Ernesto; Russell, Michael W.; Genco, Robert J.

    2002-01-01

    Toll-like receptors (TLRs) are important signal transducers that mediate inflammatory reactions induced by microbes through pattern recognition of virulence molecules such as lipopolysaccharide (LPS) and lipoproteins. We investigated whether proinflammatory cytokine responses induced by certain bacterial protein adhesins may also depend on TLRs. In differentiated THP-1 mononuclear cells stimulated by LPS-free recombinant fimbrillin (rFimA) from Porphyromonas gingivalis, cytokine release was abrogated by monoclonal antibodies (MAbs) to CD14 and TLR4 but not to TLR2. Similar experiments using anti-β2 integrin MAbs suggested that β2 integrins (CD11/CD18) also play a role in cytokine induction by rFimA or native fimbriae. Minor fimbriae (distinct from the fimA-encoded major fimbriae) of P. gingivalis induced proinflammatory cytokine release in a CD14- and TLR2-dependent mode. Cytokine induction by BspA, a leucine-rich repeat protein from Bacteroides forsythus, depended heavily on CD14 and TLR2. We also found that the ability of the streptococcal protein AgI/II to stimulate cytokine release depended partially on CD14 and TLR4, and the AgI/II segment that possibly interacts with these receptors was identified as its N-terminal saliva-binding region. When THP-1 cells were exposed to rFimA for 24 h, surface expression of CD14 and CD18 was decreased and the cells became hyporesponsive to cytokine induction by a second challenge with rFimA. However, tolerance induction was abolished when the THP-1 cells were pretreated with rFimA in the presence of either anti-CD14 MAb or anti-TLR4 MAb. Induction of cross-tolerance between rFimA and LPS correlated with downregulation of the pattern recognition receptors involved. Our data suggest that the CD14-TLR2/4 system is involved in cytokine production and tolerance induction upon interaction with certain proinflammatory bacterial protein adhesins. PMID:11874886

  19. Visualization of ribosomal RNA operon copy number distribution

    OpenAIRE

    DasGupta Indrani; Wu Martin; Rastogi Rajat; Fox George E

    2009-01-01

    Abstract Background Results of microbial ecology studies using 16S rRNA sequence information can be deceiving due to differences in rRNA operon copy number and genome size of the detected organisms. It therefore will be useful for investigators to have a better understanding of how these two parameters differ in various organism types. In this study, the number of ribosomal operons and genome size were separately mapped onto a Bacterial phylogenetic tree. Results A representative Bacterial tr...

  20. Optimized localization of bacterial infections with technetium-99m labelled human immunoglobulin after protein charge selection

    Energy Technology Data Exchange (ETDEWEB)

    Welling, M. (Dept. of Diagnostic Radiology and Nuclear Medicine, University Hospital, Leiden (Netherlands)); Feitsma, H.I.J. (Dept. of Diagnostic Radiology and Nuclear Medicine, University Hospital, Leiden (Netherlands)); Calame, W. (Dept. of Diagnostic Radiology and Nuclear Medicine, University Hospital, Leiden (Netherlands)); Ensing, G.J. (Mallinckrodt Medical, Petten (Netherlands)); Goedemans, W. (Mallinckrodt Medical, Petten (Netherlands)); Pauwels, E.K.J. (Dept. of Diagnostic Radiology and Nuclear Medicine, University Hospital, Leiden (Netherlands))

    1994-10-01

    To improve the scintigraphic detection of bacterial infections a protein charge-purified fraction of polyclonal human immunoglobulin was applied as a radiopharmaceutical. This purification was achieved by attaching the immunoglobulin to an anion-exchanger column and by obtaining the column-bound fraction with buffer. The binding to bacteria in vitro and the target to non-target ratios of an experimental thigh infection with Staphylococcus aureus or Klebsiella pneumoniae in mice were evaluated to compare the purified and the unpurified immunoglobulin. The percentage of binding to all gram-positive and gram-negative bacteria used in this study was significantly (P<0.03) higher for the purified than for the unpurified immunoglobulin. For the in vivo study, mice were infected in the thigh muscle with Staph. aureus or K. pneumoniae. After 18 h 0.1 mg of technetium-99m labelled polyclonal immunoglobulin or [sup 99m]Tc-labelled protein charge-purified polyclonal human immunoglobulin was administered intravenously. At all time intervals the target (infected thighs) to non-target (non-infected thighs) ratios for both infections were significantly higher (P<0.03) for protein charge-purified polyclonal immunoglobulin than for unpurified polyclonal human immunoglobulin. Already within 1 h the infected tissues could be detected by the purified immunoglobulin. It is concluded that [sup 99m]Tc-labelled protein charge-purified immunoglobulin localizes both a gram-positive and a gram-negative thigh infection more intensely and faster than [sup 99m]Tc-labelled unpurified immunoglobulin. (orig.)