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Sample records for preventing ribosomal assembly

  1. Defective ribosome assembly in Shwachman-Diamond syndrome.

    Science.gov (United States)

    Wong, Chi C; Traynor, David; Basse, Nicolas; Kay, Robert R; Warren, Alan J

    2011-10-20

    Shwachman-Diamond syndrome (SDS), a recessive leukemia predisposition disorder characterized by bone marrow failure, exocrine pancreatic insufficiency, skeletal abnormalities and poor growth, is caused by mutations in the highly conserved SBDS gene. Here, we test the hypothesis that defective ribosome biogenesis underlies the pathogenesis of SDS. We create conditional mutants in the essential SBDS ortholog of the ancient eukaryote Dictyostelium discoideum using temperature-sensitive, self-splicing inteins, showing that mutant cells fail to grow at the restrictive temperature because ribosomal subunit joining is markedly impaired. Remarkably, wild type human SBDS complements the growth and ribosome assembly defects in mutant Dictyostelium cells, but disease-associated human SBDS variants are defective. SBDS directly interacts with the GTPase elongation factor-like 1 (EFL1) on nascent 60S subunits in vivo and together they catalyze eviction of the ribosome antiassociation factor eukaryotic initiation factor 6 (eIF6), a prerequisite for the translational activation of ribosomes. Importantly, lymphoblasts from SDS patients harbor a striking defect in ribosomal subunit joining whose magnitude is inversely proportional to the level of SBDS protein. These findings in Dictyostelium and SDS patient cells provide compelling support for the hypothesis that SDS is a ribosomopathy caused by corruption of an essential cytoplasmic step in 60S subunit maturation.

  2. Ribosomal assembly influenced by growth in the presence of streptomycin.

    Science.gov (United States)

    Garvin, R T; Rosset, R; Gorini, L

    1973-10-01

    Translational leakiness (i.e., nonspecific suppression) of nonsense mutants of bacteriophage T4 is increased in cells of certain streptomycin-resistant strains previously grown in the presence of streptomycin. Concomitantly, ribosomes extracted from these streptomycin-grown cells possess a high level of misreading. Increased suppression ability as well as ribosomes that highly misread accumulate with kinetics expected for a constant differential rate of synthesis of a new product induced by drug action. The misreading ribosomes do not contain appreciable amounts of streptomycin and the misreading property is lost by exposure to high salt concentrations. It is suggested that streptomycin (or dihydrostreptomycin, or paromomycin) induces a reversible modification in 30S subunit assembly without physically participating in the modified structure. The extent of this modification appears dependent upon the strA allele.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

    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

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

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    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.

  5. Structure and RNA recognition of ribosome assembly factor Utp30.

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    Hu, Jianfei; Zhu, Xing; Ye, Keqiong

    2017-12-01

    The 90S preribosomes are gigantic early assembly intermediates of small ribosomal subunits. Cryo-EM structures of 90S were recently determined, but many of its components have not been accurately modeled. Here we determine the crystal structure of yeast Utp30, a ribosomal L1 domain-containing protein in 90S, at 2.65 Å resolution, revealing a classic two-domain fold. The structure of Utp30 fits well into the cryo-EM density of 90S, confirming its previously assigned location. Utp30 binds to the rearranged helix 41 of 18S rRNA and helix 4 of 5' external transcribed spacer in 90S. Comparison of RNA-binding modes of different L1 domains illustrates that they consistently recognize a short RNA duplex with the concaved surface of domain I, but are versatile in RNA recognition outside the core interface. Cic1 is a paralog of Utp30 associating with large subunit preribosomes. Utp30 and Cic1 share similar RNA-binding modes, suggesting that their distinct functions may be executed by a single protein in other organisms. Deletion of Utp30 does not affect the composition of 90S. The nonessential role of Utp30 could be ascribed to its peripheral localization and redundant interactions in 90S. © 2017 Hu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  6. Stepwise assembly of the earliest precursors of large ribosomal subunits in yeast.

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    Chen, Wu; Xie, Zhensheng; Yang, Fuquan; Ye, Keqiong

    2017-06-20

    Small ribosomal subunits are co-transcriptionally assembled on the nascent precursor rRNA in Saccharomyces cerevisiae. It is unknown how the highly intertwined structure of 60S large ribosomal subunits is initially formed. Here, we affinity purified and analyzed a series of pre-60S particles assembled in vivo on plasmid-encoded pre-rRNA fragments of increasing lengths, revealing a spatiotemporal assembly map for 34 trans-acting assembly factors (AFs), 30 ribosomal proteins and 5S rRNA. The gradual association of AFs and ribosomal proteins with the pre-rRNA fragments strongly supports that the pre-60S is co-transcriptionally, rather than post-transcriptionally, assembled. The internal and external transcribed spacers ITS1, ITS2 and 3΄ ETS in pre-rRNA must be processed in pre-60S. We show that the processing machineries for ITS1 and ITS2 are primarily recruited by the 5΄ and 3΄ halves of pre-27S RNA, respectively. Nevertheless, processing of both ITS1 and ITS2 requires a complete 25S region. The 3΄ ETS plays a minor role in ribosome assembly, but is important for efficient rRNA processing and ribosome maturation. We also identified a distinct pre-60S state occurring before ITS2 processing. Our data reveal the elusive co-transcriptional assembly pathway of large ribosomal subunit. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Probing the structure of ribosome assembly intermediates in vivo using DMS and hydroxyl radical footprinting.

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    Hulscher, Ryan M; Bohon, Jen; Rappé, Mollie C; Gupta, Sayan; D'Mello, Rhijuta; Sullivan, Michael; Ralston, Corie Y; Chance, Mark R; Woodson, Sarah A

    2016-07-01

    The assembly of the Escherichia coli ribosome has been widely studied and characterized in vitro. Despite this, ribosome biogenesis in living cells is only partly understood because assembly is coupled with transcription, modification and processing of the pre-ribosomal RNA. We present a method for footprinting and isolating pre-rRNA as it is synthesized in E. coli cells. Pre-rRNA synthesis is synchronized by starvation, followed by nutrient upshift. RNA synthesized during outgrowth is metabolically labeled to facilitate isolation of recent transcripts. Combining this technique with two in vivo RNA probing methods, hydroxyl radical and DMS footprinting, allows the structure of nascent RNA to be probed over time. Together, these can be used to determine changes in the structures of ribosome assembly intermediates as they fold in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Involvement of ribosomal protein L6 in assembly of functional 50S ribosomal subunit in Escherichia coli cells

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    Shigeno, Yuta [Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567 (Japan); Uchiumi, Toshio [Department of Biology, Faculty of Science, Niigata University, Niigata 950-2181 (Japan); Nomura, Takaomi, E-mail: nomurat@shinshu-u.ac.jp [Division of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567 (Japan)

    2016-04-22

    Ribosomal protein L6, an essential component of the large (50S) subunit, primarily binds to helix 97 of 23S rRNA and locates near the sarcin/ricin loop of helix 95 that directly interacts with GTPase translation factors. Although L6 is believed to play important roles in factor-dependent ribosomal function, crucial biochemical evidence for this hypothesis has not been obtained. We constructed and characterized an Escherichia coli mutant bearing a chromosomal L6 gene (rplF) disruption and carrying a plasmid with an arabinose-inducible L6 gene. Although this ΔL6 mutant grew more slowly than its wild-type parent, it proliferated in the presence of arabinose. Interestingly, cell growth in the absence of arabinose was biphasic. Early growth lasted only a few generations (LI-phase) and was followed by a suspension of growth for several hours (S-phase). This suspension was followed by a second growth phase (LII-phase). Cells harvested at both LI- and S-phases contained ribosomes with reduced factor-dependent GTPase activity and accumulated 50S subunit precursors (45S particles). The 45S particles completely lacked L6. Complete 50S subunits containing L6 were observed in all growth phases regardless of the L6-depleted condition, implying that the ΔL6 mutant escaped death because of a leaky expression of L6 from the complementing plasmid. We conclude that L6 is essential for the assembly of functional 50S subunits at the late stage. We thus established conditions for the isolation of L6-depleted 50S subunits, which are essential to study the role of L6 in translation. - Highlights: • We constructed an in vivo functional assay system for Escherichia coli ribosomal protein L6. • Growth of an E. coli ΔL6 mutant was biphasic when L6 levels were depleted. • The ΔL6 mutant accumulated 50S ribosomal subunit precursors that sedimented at 45S. • L6 is a key player in the late stage of E. coli 50S subunit assembly.

  9. Involvement of ribosomal protein L6 in assembly of functional 50S ribosomal subunit in Escherichia coli cells

    International Nuclear Information System (INIS)

    Shigeno, Yuta; Uchiumi, Toshio; Nomura, Takaomi

    2016-01-01

    Ribosomal protein L6, an essential component of the large (50S) subunit, primarily binds to helix 97 of 23S rRNA and locates near the sarcin/ricin loop of helix 95 that directly interacts with GTPase translation factors. Although L6 is believed to play important roles in factor-dependent ribosomal function, crucial biochemical evidence for this hypothesis has not been obtained. We constructed and characterized an Escherichia coli mutant bearing a chromosomal L6 gene (rplF) disruption and carrying a plasmid with an arabinose-inducible L6 gene. Although this ΔL6 mutant grew more slowly than its wild-type parent, it proliferated in the presence of arabinose. Interestingly, cell growth in the absence of arabinose was biphasic. Early growth lasted only a few generations (LI-phase) and was followed by a suspension of growth for several hours (S-phase). This suspension was followed by a second growth phase (LII-phase). Cells harvested at both LI- and S-phases contained ribosomes with reduced factor-dependent GTPase activity and accumulated 50S subunit precursors (45S particles). The 45S particles completely lacked L6. Complete 50S subunits containing L6 were observed in all growth phases regardless of the L6-depleted condition, implying that the ΔL6 mutant escaped death because of a leaky expression of L6 from the complementing plasmid. We conclude that L6 is essential for the assembly of functional 50S subunits at the late stage. We thus established conditions for the isolation of L6-depleted 50S subunits, which are essential to study the role of L6 in translation. - Highlights: • We constructed an in vivo functional assay system for Escherichia coli ribosomal protein L6. • Growth of an E. coli ΔL6 mutant was biphasic when L6 levels were depleted. • The ΔL6 mutant accumulated 50S ribosomal subunit precursors that sedimented at 45S. • L6 is a key player in the late stage of E. coli 50S subunit assembly.

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

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    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.

  11. A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30S subunit assembly in E. coli.

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    Sashital, Dipali G; Greeman, Candacia A; Lyumkis, Dmitry; Potter, Clinton S; Carragher, Bridget; Williamson, James R

    2014-10-14

    Ribosome assembly is a complex process involving the folding and processing of ribosomal RNAs (rRNAs), concomitant binding of ribosomal proteins (r-proteins), and participation of numerous accessory cofactors. Here, we use a quantitative mass spectrometry/electron microscopy hybrid approach to determine the r-protein composition and conformation of 30S ribosome assembly intermediates in Escherichia coli. The relative timing of assembly of the 3' domain and the formation of the central pseudoknot (PK) structure depends on the presence of the assembly factor RimP. The central PK is unstable in the absence of RimP, resulting in the accumulation of intermediates in which the 3'-domain is unanchored and the 5'-domain is depleted for r-proteins S5 and S12 that contact the central PK. Our results reveal the importance of the cofactor RimP in central PK formation, and introduce a broadly applicable method for characterizing macromolecular assembly in cells.

  12. Kinase Mediated Regulation of 40S Ribosome Assembly in Human Breast Cancer

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    2017-02-01

    be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE...month worked: 2 Contribution to Project: Dr. Karbstein supervises the effort in the Karbstein lab. Funding Support: Name: Homa Ghalei Project...Moffitt Cancer Center) seeks to validate targeting 40S ribosome assembly as a therapeutic target for aggressive breast cancer subtypes, including

  13. Organization of Mitochondrial Gene Expression in Two Distinct Ribosome-Containing Assemblies

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    Kirsten Kehrein

    2015-02-01

    Full Text Available Mitochondria contain their own genetic system that provides subunits of the complexes driving oxidative phosphorylation. A quarter of the mitochondrial proteome participates in gene expression, but how all these factors are orchestrated and spatially organized is currently unknown. Here, we established a method to purify and analyze native and intact complexes of mitochondrial ribosomes. Quantitative mass spectrometry revealed extensive interactions of ribosomes with factors involved in all the steps of posttranscriptional gene expression. These interactions result in large expressosome-like assemblies that we termed mitochondrial organization of gene expression (MIOREX complexes. Superresolution microscopy revealed that most MIOREX complexes are evenly distributed throughout the mitochondrial network, whereas a subset is present as nucleoid-MIOREX complexes that unite the whole spectrum of organellar gene expression. Our work therefore provides a conceptual framework for the spatial organization of mitochondrial protein synthesis that likely developed to facilitate gene expression in the organelle.

  14. Cyclization of polyketides and non-ribosomal peptides on and off their assembly lines.

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    Pang, Bo; Wang, Min; Liu, Wen

    2016-02-01

    Modular polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) are multifunctional megaenzymes that serve as templates to program the assembly of short carboxylic acids and amino acids in a primarily co-linear manner. The variation, combination, permutation and evolution of their functional units (e.g., modules, domains and proteins) along with their association with external enzymes have resulted in the generation of numerous versions of templates, the roles of which have not been fully recognized in the structural diversification of polyketides, non-ribosomal peptides and their hybrids present in nature. In this Highlight, we focus on the assembly-line enzymology and associated chemistry by providing examples of some newly characterized cyclization reactions that occur on and off the assembly lines during and after chain elongation for the purpose of elucidating the template effects of PKSs and NRPSs. A fundamental understanding of the underlying biosynthetic logic would facilitate the elucidation of chemical information contained within the PKS or NRPS templates and benefit the development of strategies for genome mining, biosynthesis-inspired chemical synthesis and combinatorial biosynthesis.

  15. Assembly of the central domain of the 30S ribosomal subunit: roles for the primary binding ribosomal proteins S15 and S8.

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    Jagannathan, Indu; Culver, Gloria M

    2003-07-04

    Assembly of the 30S ribosomal subunit occurs in a highly ordered and sequential manner. The ordered addition of ribosomal proteins to the growing ribonucleoprotein particle is initiated by the association of primary binding proteins. These proteins bind specifically and independently to 16S ribosomal RNA (rRNA). Two primary binding proteins, S8 and S15, interact exclusively with the central domain of 16S rRNA. Binding of S15 to the central domain results in a conformational change in the RNA and is followed by the ordered assembly of the S6/S18 dimer, S11 and finally S21 to form the platform of the 30S subunit. In contrast, S8 is not part of this major platform assembly branch. Of the remaining central domain binding proteins, only S21 association is slightly dependent on S8. Thus, although S8 is a primary binding protein that extensively contacts the central domain, its role in assembly of this domain remains unclear. Here, we used directed hydroxyl radical probing from four unique positions on S15 to assess organization of the central domain of 16S rRNA as a consequence of S8 association. Hydroxyl radical probing of Fe(II)-S15/16S rRNA and Fe(II)-S15/S8/16S rRNA ribonucleoprotein particles reveal changes in the 16S rRNA environment of S15 upon addition of S8. These changes occur predominantly in helices 24 and 26 near previously identified S8 binding sites. These S8-dependent conformational changes are consistent with 16S rRNA folding in complete 30S subunits. Thus, while S8 binding is not absolutely required for assembly of the platform, it appears to affect significantly the 16S rRNA environment of S15 by influencing central domain organization.

  16. Plastid ribosome pausing is induced by multiple features and is linked to protein complex assembly

    DEFF Research Database (Denmark)

    Gawroński, Piotr; Jensen, Poul Erik; Karpinski, Stanislaw

    2018-01-01

    Many mRNAs contain pause sites that briefly interrupt the progress of translation. Specific features that induce ribosome pausing have been described; however, their individual contributions to pause-site formation, and the overall biological significance of ribosome pausing, remain largely uncle...

  17. Assembly of the 30S subunit from Escherichia coli ribosomes occurs via two assembly domains which are initiated by S4 and S7

    International Nuclear Information System (INIS)

    Nowotny, V.; Nierhaus, K.H.

    1988-01-01

    A protein which initiates assembly of ribosomes is defined as a protein which binds to the respective rRNA without cooperativity (i.e., without the help of other proteins) during the onset of assembly and is essential for the formation of active ribosomal subunits. The number of proteins binding without cooperativity was determined by monitoring the reconstitution output of active particles at various inputs of 16S rRNA, in the present of constant amounts of 30S-derived proteins (TP30): This showed that only two of the proteins of the 30S subunit are assembly-initiator proteins. These two proteins are still present on a LiCl core particle comprising 16S rRNA and 12 proteins (including minor proteins). The 12 proteins were isolated, and a series of reconstitution experiments at various levels of rRNA excess demonstrated that S4 and S7 are the initiator proteins. Pulse-chase experiments performed during the early assembly with 14 C- and 3 H-labeled TP30 and the determination of the 14 C/ 3 H ratio of the individual proteins within the assembled particles revealed a bilobal structure of the 30S assembly: A group of six proteins headed by S4 (namely, S4, S20, S16, S15, S6, and S18) resisted the chasing most efficiently (S4 assembly domain). None of the proteins depending on S7 during assembly were found in this group but rather in a second group with intermediate chasing stability [S7 assembly domain; consisting of S7, S9, (S8), S19, and S3]. A number of proteins could be fully chased during the early assembly and therefore represent late assembly proteins (S10, S5, S13, S2, S21, S1). These findings fit well with the 30S assembly map. These data, together with the assembly map, imply that S8 and S5 play an important role in the interconnection of the two assembly domains

  18. The impact of recent improvements in cryo-electron microscopy technology on the understanding of bacterial ribosome assembly.

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    Razi, Aida; Britton, Robert A; Ortega, Joaquin

    2017-02-17

    Cryo-electron microscopy (cryo-EM) had played a central role in the study of ribosome structure and the process of translation in bacteria since the development of this technique in the mid 1980s. Until recently cryo-EM structures were limited to ∼10 Å in the best cases. However, the recent advent of direct electron detectors has greatly improved the resolution of cryo-EM structures to the point where atomic resolution is now achievable. This improved resolution will allow cryo-EM to make groundbreaking contributions in essential aspects of ribosome biology, including the assembly process. In this review, we summarize important insights that cryo-EM, in combination with chemical and genetic approaches, has already brought to our current understanding of the ribosomal assembly process in bacteria using previous detector technology. More importantly, we discuss how the higher resolution structures now attainable with direct electron detectors can be leveraged to propose precise testable models regarding this process. These structures will provide an effective platform to develop new antibiotics that target this fundamental cellular process.

  19. Placeholder factors in ribosome biogenesis: please, pave my way

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    Francisco J. Espinar-Marchena

    2017-04-01

    Full Text Available The synthesis of cytoplasmic eukaryotic ribosomes is an extraordinarily energy-demanding cellular activity that occurs progressively from the nucleolus to the cytoplasm. In the nucleolus, precursor rRNAs associate with a myriad of trans-acting factors and some ribosomal proteins to form pre-ribosomal particles. These factors include snoRNPs, nucleases, ATPases, GTPases, RNA helicases, and a vast list of proteins with no predicted enzymatic activity. Their coordinate activity orchestrates in a spatiotemporal manner the modification and processing of precursor rRNAs, the rearrangement reactions required for the formation of productive RNA folding intermediates, the ordered assembly of the ribosomal proteins, and the export of pre-ribosomal particles to the cytoplasm; thus, providing speed, directionality and accuracy to the overall process of formation of translation-competent ribosomes. Here, we review a particular class of trans-acting factors known as “placeholders”. Placeholder factors temporarily bind selected ribosomal sites until these have achieved a structural context that is appropriate for exchanging the placeholder with another site-specific binding factor. By this strategy, placeholders sterically prevent premature recruitment of subsequently binding factors, premature formation of structures, avoid possible folding traps, and act as molecular clocks that supervise the correct progression of pre-ribosomal particles into functional ribosomal subunits. We summarize the current understanding of those factors that delay the assembly of distinct ribosomal proteins or subsequently bind key sites in pre-ribosomal particles. We also discuss recurrent examples of RNA-protein and protein-protein mimicry between rRNAs and/or factors, which have clear functional implications for the ribosome biogenesis pathway.

  20. Dual functions of ribosome recycling factor in protein biosynthesis: disassembling the termination complex and preventing translational errors.

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    Janosi, L; Ricker, R; Kaji, A

    1996-01-01

    We summarize in this communication the data supporting the two functions of ribosome recycling factor (RRF, originally called ribosome releasing factor). The first described role involves the disassembly of the termination complex which consists of mRNA, tRNA and the ribosome bound to the mRNA at the termination codon. This process is catalyzed by two factors, elongation factor G (EF-G) and RRF. RRF stimulated protein synthesis as much as eight-fold in the in vitro lysozyme synthesis system, when ribosomes were limiting. In the absence of RRF, ribosomes remain mRNA-bound at the termination codon and translate downstream codons. In the in vitro system, the site of reinitiation is the triplet codon 3' to the termination codon. RRF is an essential protein for bacterial life. Temperature sensitive (ts) RRF mutants were isolated and in vivo translational reinitiation due to inactivation of ts RRF was demonstrated using the beta-galactosidase reporter gene placed downstream from the termination codon. A second function of RRF involves preventing errors in translation. In polyphenylalanine synthesis programmed by polyuridylic acid, misincorporation of isoleucine, leucine or a mixture of amino acids was stimulated upto 17-fold when RRF was omitted from the in vitro system. RRF did not influence the large error (10-fold increase) induced by streptomycin. This means that RRF participates not only in the disassembly of the termination complex but also in peptide elongation. Extending this concept and its conventional role for releasing ribosomes from mRNA, involvement of RRF in the reinitiation in the 3A' system (a construct using S aureus protein A, a collaborative work with Dr Isaksson), in programmed frame shifting, in trans-translation with 10Sa RNA (collaborative work with Dr Muto), and in the reinitiation downstream from the ORF A of the IS 3 (insertion sequence of a transposon, collaborative work with Dr Sekine) are discussed on the basis of preliminary data to be

  1. The cryo-EM structure of YjeQ bound to the 30S subunit suggests a fidelity checkpoint function for this protein in ribosome assembly

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    Razi, Aida; Guarné, Alba; Ortega, Joaquin

    2017-01-01

    Recent work suggests that bacterial YjeQ (RsgA) participates in the late stages of assembly of the 30S subunit and aids the assembly of the decoding center but also binds the mature 30S subunit with high affinity. To determine the function and mechanisms of YjeQ in the context of the mature subunit, we determined the cryo-EM structure of the fully assembled 30S subunit in complex with YjeQ at 5.8-Å resolution. We found that binding of YjeQ stabilizes helix 44 into a conformation similar to that adopted by the subunit during proofreading. This finding indicates that, along with acting as an assembly factor, YjeQ has a role as a checkpoint protein, consisting of testing the proofreading ability of the 30S subunit. The structure also informs the mechanism by which YjeQ implements the release from the 30S subunit of a second assembly factor, called RbfA. Finally, it reveals how the 30S subunit stimulates YjeQ GTPase activity and leads to release of the protein. Checkpoint functions have been described for eukaryotic ribosome assembly factors; however, this work describes an example of a bacterial assembly factor that tests a specific translation mechanism of the 30S subunit. PMID:28396444

  2. A Matrine Derivative M54 Suppresses Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss by Targeting Ribosomal Protein S5.

    Science.gov (United States)

    Xin, Zhi; Jin, Cui; Chao, Liu; Zheng, Zhang; Liehu, Cao; Panpan, Pan; Weizong, Weng; Xiao, Zhai; Qingjie, Zhao; Honggang, Hu; Longjuan, Qin; Xiao, Chen; Jiacan, Su

    2018-01-01

    Post-menopausal osteoporosis (PMOP) is a metabolic bone disorder characterized by low bone mass and micro-architectural deterioration of bone tissue. The over-activated osteoclastogenesis, which plays an important role in osteoporosis, has become an important therapeutic target. M54 was a bioactive derivative of the Chinese traditional herb matrine. We found that M54 could suppress RANKL-induced osteoclastogenesis in bone marrow mononuclear cells and RAW264.7 cells through suppressing NF-κB, PI3K/AKT, and MAPKs pathways activity in vitro , and prevent ovariectomy-induced bone loss in vivo . Our previous study has proved that ribosomal protein S5 (RPS5) was a direct target of M19, based on which M54 was synthesized. Thus we deduced that M54 also targeted RPS5. During osteoclastogenesis, the RPS5 level in RAW264.7 cells was significantly down-regulated while M54 could maintain its level. After RPS5 was silenced, the inhibitory effects of M54 on osteoclastogenesis were partially compromised, indicating that M54 took effects through targeting RPS5. In summary, M54 was a potential clinical medicine for post-menopause osteoporosis treatment, and RPS5 is a possible key protein in PMOP.

  3. A Matrine Derivative M54 Suppresses Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss by Targeting Ribosomal Protein S5

    Directory of Open Access Journals (Sweden)

    Zhi Xin

    2018-01-01

    Full Text Available Post-menopausal osteoporosis (PMOP is a metabolic bone disorder characterized by low bone mass and micro-architectural deterioration of bone tissue. The over-activated osteoclastogenesis, which plays an important role in osteoporosis, has become an important therapeutic target. M54 was a bioactive derivative of the Chinese traditional herb matrine. We found that M54 could suppress RANKL-induced osteoclastogenesis in bone marrow mononuclear cells and RAW264.7 cells through suppressing NF-κB, PI3K/AKT, and MAPKs pathways activity in vitro, and prevent ovariectomy-induced bone loss in vivo. Our previous study has proved that ribosomal protein S5 (RPS5 was a direct target of M19, based on which M54 was synthesized. Thus we deduced that M54 also targeted RPS5. During osteoclastogenesis, the RPS5 level in RAW264.7 cells was significantly down-regulated while M54 could maintain its level. After RPS5 was silenced, the inhibitory effects of M54 on osteoclastogenesis were partially compromised, indicating that M54 took effects through targeting RPS5. In summary, M54 was a potential clinical medicine for post-menopause osteoporosis treatment, and RPS5 is a possible key protein in PMOP.

  4. Rat RL23a ribosomal protein efficiently competes with its Saccharomyces cerevisiae L25 homologue for assembly into 60S subunits.

    NARCIS (Netherlands)

    Jeeninga, R.E.; Venema, J.; Raué, H.A.

    1996-01-01

    The large subunit protein RL23a from rat liver ribosomes shows 62% sequence identity with the primary rRNA-binding ribosomal protein L25 from Saccharomyces cerevisiae. In vitro binding studies indicated that both r-proteins are able to recognise the L25 binding site on yeast 25 S rRNA and its

  5. Systems and methods for preventing flashback in a combustor assembly

    Science.gov (United States)

    Johnson, Thomas Edward; Ziminsky, Willy Steve; Stevenson, Christian Xavier

    2016-04-05

    Embodiments of the present application include a combustor assembly. The combustor assembly may include a combustion chamber, a first plenum, a second plenum, and one or more elongate air/fuel premixing injection tubes. Each of the elongate air/fuel premixing injection tubes may include a first length at least partially disposed within the first plenum and configured to receive a first fluid from the first plenum. Moreover, each of the elongate air/fuel premixing injection tubes may include a second length disposed downstream of the first length and at least partially disposed within the second plenum. The second length may be formed of a porous wall configured to allow a second fluid from the second plenum to enter the second length and create a boundary layer about the porous wall.

  6. Apparatus and method for preventing the rotation of rods used in nuclear fuel assemblies

    International Nuclear Information System (INIS)

    Pilgrim, L.G. Jr.; Jackson, L.F.

    1985-01-01

    Apparatus and method for preventing the rotation of one or more elongated rods used in nuclear fuel assemblies include an end plug secured to one longitudinal end of such an elongated rod and having an out-of-cavity, non-round structure affixed thereto and configured to mate with a complementary shaped structure in a lower tie plate of a nuclear fuel assembly in such a manner as to prevent the rotation of the rod about its longitudinal axis. In one embodiment, the end plug includes a pair of flats formed on a portion of the end plug and configured to abut against a pair of flats formed on the outer surface of a cylindrical boss or sleeve of the lower tie plate, thereby to prevent the rotation of the rod. In another embodiment, four grooves, disposed 90 0 apart about the periphery of an end plug of a rod form a spline. The grooves are configured to receive four, radially inwardly protruding, key members disposed 90 0 apart about the periphery of a sleeve secured to the lower tie plate, thereby to prevent the rotation of the rod. In a further embodiment, a sleeve is secured to an end plug of a rod and includes four elongated slots disposed 90 0 apart about the periphery of the sleeve and configured in width, depth and spacing to receive and mate with four web portions of the lower tie plate of the nuclear fuel assembly, thereby to secure the rod against rotation about its longitudinal axis

  7. Chloroplast and cytoplasmic ribosomes of Euglena: selective binding of dihydrostreptomycin to chloroplast ribosomes.

    Science.gov (United States)

    Schwartzbach, S D; Schiff, J A

    1974-10-01

    Dihydrostreptomycin binds preferentially to chloroplast ribosomes of wild-type Euglena gracilis Klebs var. bacillaris Pringsheim. The K(diss) for the wild-type chloroplast ribosome-dihydrostreptomycin complex is 2 x 10(-7) M, a value comparable with that found for the Escherichia coli ribosome-dihydrostreptomycin complex. Chloroplast ribosomes isolated from the streptomycin-resistant mutant Sm(1) (r)BNgL and cytoplasmic ribosomes from wild-type have a much lower affinity for the antibiotic. The K(diss) for the chloroplast ribosome-dihydrostreptomycin complex of Sm(1) (r) is 387 x 10(-7) M, and the value for the cytoplasmic ribosome-dihydrostreptomycin complex of the wild type is 1,400 x 10(-7) M. Streptomycin competes with dihydrostreptomycin for the chloroplast ribosome binding site, and preincubation of streptomycin with hydroxylamine prevents the binding of streptomycin to the chloroplast ribosome. These results indicate that the inhibition of chloroplast development and replication in Euglena by streptomycin and dihydrostreptomycin is related to the specific inhibition of protein synthesis on the chloroplast ribosomes of Euglena.

  8. Chloroplast and Cytoplasmic Ribosomes of Euglena: Selective Binding of Dihydrostreptomycin to Chloroplast Ribosomes1

    Science.gov (United States)

    Schwartzbach, Steven D.; Schiff, Jerome A.

    1974-01-01

    Dihydrostreptomycin binds preferentially to chloroplast ribosomes of wild-type Euglena gracilis Klebs var. bacillaris Pringsheim. The Kdiss for the wild-type chloroplast ribosome-dihydrostreptomycin complex is 2 × 10−7 M, a value comparable with that found for the Escherichia coli ribosome-dihydrostreptomycin complex. Chloroplast ribosomes isolated from the streptomycin-resistant mutant Sm1rBNgL and cytoplasmic ribosomes from wild-type have a much lower affinity for the antibiotic. The Kdiss for the chloroplast ribosome-dihydrostreptomycin complex of Sm1r is 387 × 10−7 M, and the value for the cytoplasmic ribosome-dihydrostreptomycin complex of the wild type is 1,400 × 10−7 M. Streptomycin competes with dihydrostreptomycin for the chloroplast ribosome binding site, and preincubation of streptomycin with hydroxylamine prevents the binding of streptomycin to the chloroplast ribosome. These results indicate that the inhibition of chloroplast development and replication in Euglena by streptomycin and dihydrostreptomycin is related to the specific inhibition of protein synthesis on the chloroplast ribosomes of Euglena. PMID:4138802

  9. Illuminating parasite protein production by ribosome profiling

    Science.gov (United States)

    Parsons, Marilyn; Myler, Peter J.

    2016-01-01

    While technologies for global enumeration of transcript abundance are well-developed, those that assess protein abundance require tailoring to penetrate to low abundance proteins. Ribosome profiling circumvents this challenge by measuring global protein production via sequencing small mRNA fragments protected by the assembled ribosome. This powerful approach is now being applied to protozoan parasites, including trypanosomes and Plasmodium. It has been used to identify new protein coding sequences (CDSs) and clarify the boundaries of previously annotated CDSs in Trypanosoma brucei. Ribosome profiling has demonstrated that translation efficiencies vary widely between genes and, for trypanosomes at least, for the same gene across stages. The ribosomal proteins are themselves subjected to translational control, suggesting a means of reinforcing global translational regulation. PMID:27061497

  10. The Modular Adaptive Ribosome.

    Directory of Open Access Journals (Sweden)

    Anupama Yadav

    Full Text Available The ribosome is an ancient machine, performing the same function across organisms. Although functionally unitary, recent experiments suggest specialized roles for some ribosomal proteins. Our central thesis is that ribosomal proteins function in a modular fashion to decode genetic information in a context dependent manner. We show through large data analyses that although many ribosomal proteins are essential with consistent effect on growth in different conditions in yeast and similar expression across cell and tissue types in mice and humans, some ribosomal proteins are used in an environment specific manner. The latter set of variable ribosomal proteins further function in a coordinated manner forming modules, which are adapted to different environmental cues in different organisms. We show that these environment specific modules of ribosomal proteins in yeast have differential genetic interactions with other pathways and their 5'UTRs show differential signatures of selection in yeast strains, presumably to facilitate adaptation. Similarly, we show that in higher metazoans such as mice and humans, different modules of ribosomal proteins are expressed in different cell types and tissues. A clear example is nervous tissue that uses a ribosomal protein module distinct from the rest of the tissues in both mice and humans. Our results suggest a novel stratification of ribosomal proteins that could have played a role in adaptation, presumably to optimize translation for adaptation to diverse ecological niches and tissue microenvironments.

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

    Science.gov (United States)

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

    2008-09-01

    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 human disease with a specific defect in red cell development is unknown. Here, we investigated the role of r-proteins in ribosome biogenesis in order to find out whether those mutated in DBA have any similarities. We depleted HeLa cells using siRNA for several individual r-proteins of the small (RPS6, RPS7, RPS15, RPS16, RPS17, RPS19, RPS24, RPS25, RPS28) or large subunit (RPL5, RPL7, RPL11, RPL14, RPL26, RPL35a) and studied the effect on rRNA processing and ribosome production. Depleting r-proteins in one of the subunits caused, with a few exceptions, a decrease in all r-proteins of the same subunit and a decrease in the corresponding subunit, fully assembled ribosomes, and polysomes. R-protein depletion, with a few exceptions, led to the accumulation of specific rRNA precursors, highlighting their individual roles in rRNA processing. Depletion of r-proteins mutated in DBA always compromised ribosome biogenesis while affecting either subunit and disturbing rRNA processing at different levels, indicating that the rate of ribosome production rather than a specific step in ribosome biogenesis is critical in patients with DBA.

  12. Ribosome Profiling in Maize.

    Science.gov (United States)

    Chotewutmontri, Prakitchai; Stiffler, Nicholas; Watkins, Kenneth P; Barkan, Alice

    2018-01-01

    Ribosome profiling (also known as Ribo-seq) provides a genome-wide, high-resolution, and quantitative accounting of mRNA segments that are occupied by ribosomes in vivo. The method has been used to address numerous questions in bacteria, yeast, and metazoa, but its application to questions in plant biology is just beginning. This chapter provides a detailed protocol for profiling ribosomes in plant leaf tissue. The method was developed and optimized with maize, but it has been used successfully with Arabidopsis and tobacco as well. The method captures ribosome footprints from the chloroplast and cytosol in the same preparation, but it is not optimal for detecting the footprints of mitochondrial ribosomes. The protocol is robust and simpler than many of the methods reported previously for ribosome profiling in plants.

  13. Post-termination Ribosome Intermediate Acts as the Gateway to Ribosome Recycling

    Directory of Open Access Journals (Sweden)

    Arjun Prabhakar

    2017-07-01

    Full Text Available During termination of translation, the nascent peptide is first released from the ribosome, which must be subsequently disassembled into subunits in a process known as ribosome recycling. In bacteria, termination and recycling are mediated by the translation factors RF, RRF, EF-G, and IF3, but their precise roles have remained unclear. Here, we use single-molecule fluorescence to track the conformation and composition of the ribosome in real time during termination and recycling. Our results show that peptide release by RF induces a rotated ribosomal conformation. RRF binds to this rotated intermediate to form the substrate for EF-G that, in turn, catalyzes GTP-dependent subunit disassembly. After the 50S subunit departs, IF3 releases the deacylated tRNA from the 30S subunit, thus preventing reassembly of the 70S ribosome. Our findings reveal the post-termination rotated state as the crucial intermediate in the transition from termination to recycling.

  14. A ribosome without RNA

    Directory of Open Access Journals (Sweden)

    Harold S Bernhardt

    2015-11-01

    Full Text Available It was Francis Crick who first asked why the ribosome contains so much RNA, and discussed the implications of this for the direct flow of genetic information from DNA to protein. Remarkable advances in our understanding of the ribosome and protein synthesis, including the recent publication of two mammalian mitochondrial ribosome structures, have shed new light on this intriguing aspect of evolution in molecular biology. We examine here whether RNA is indispensable for coded protein synthesis, or whether an all-protein ‘ribosome’ (or ‘synthosome’ might be possible, with a protein enzyme catalyzing peptide synthesis, and release factor-like protein adaptors able to read a message composed of deoxyribonucleotides. We also compare the RNA world hypothesis with the alternative ‘proteins first’ hypothesis in terms of their different understandings of the evolution of the ribosome, and whether this might have been preceded by an ancestral form of nonribosomal peptide synthesis catalyzed by protein enzymes.

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

    Directory of Open Access Journals (Sweden)

    Melody G Campbell

    2011-01-01

    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.

  16. 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......-proteins that are not actively transported into the nucleus; moreover, this might imply that the "stalk" constituents are assembled onto the ribosomal particle at the very last step of ribosomal maturation, which takes part in the cell cytoplasm....

  17. Seasonal changes in phosphorus competition and allelopathy of a benthic microbial assembly facilitate prevention of cyanobacterial blooms.

    Science.gov (United States)

    Wu, Yonghong; Wang, Fengwu; Xiao, Xi; Liu, Junzhuo; Wu, Chenxi; Chen, Hong; Kerr, Philip; Shurin, Jonathan

    2017-06-01

    Interactions among microbes determine the prevalence of harmful algal blooms that threaten water quality. These interactions can be indirectly mediated by shared resources or consumers, or through interference by the production of allelochemicals. Allelopathic interactions and resource competition have been shown to occur among algae and associated microbes. However, little work has considered seasonal influences on ecosystem structure and function. Here, we report results of our investigations on seasonal changes in the interactions between benthic microbial assemblies and the bloom forming cyanobacterium Microcystis aeruginosa. We show that phosphorus (P) competition and allelopathy by the microbial assembly vary seasonally and inhibit growth of M. aeruginosa. The interactions per unit biomass of the microbial assembly are stronger under winter than summer conditions and inhibit the recruitment of the cyanobacteria, thereby preventing the reoccurrence of cyanobacterial blooms in the following summer. The seasonality of these interactions correlates with changes in composition, metabolic activity and functional diversity of the microbial assembly. Our findings highlight the importance of competitive and allelopathic interactions in regulating the occurrence of harmful algal blooms. Our results also imply that seasonal variation of competition and allelopathy of the microbial assembly might be beneficial to adjust aquatic ecosystem structure and function. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  18. Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site.

    Science.gov (United States)

    Fuchs, Gabriele; Petrov, Alexey N; Marceau, Caleb D; Popov, Lauren M; Chen, Jin; O'Leary, Seán E; Wang, Richard; Carette, Jan E; Sarnow, Peter; Puglisi, Joseph D

    2015-01-13

    Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors.

  19. Oxidative and nitrosative modifications of tropoelastin prevent elastic fiber assembly in vitro.

    Science.gov (United States)

    Akhtar, Kamal; Broekelmann, Thomas J; Miao, Ming; Keeley, Fred W; Starcher, Barry C; Pierce, Richard A; Mecham, Robert P; Adair-Kirk, Tracy L

    2010-11-26

    Elastic fibers are extracellular structures that provide stretch and recoil properties of tissues, such as lungs, arteries, and skin. Elastin is the predominant component of elastic fibers. Tropoelastin (TE), the precursor of elastin, is synthesized mainly during late fetal and early postnatal stages. The turnover of elastin in normal adult tissues is minimal. However, in several pathological conditions often associated with inflammation and oxidative stress, elastogenesis is re-initiated, but newly synthesized elastic fibers appear abnormal. We sought to determine the effects of reactive oxygen and nitrogen species (ROS/RNS) on the assembly of TE into elastic fibers. Immunoblot analyses showed that TE is oxidatively and nitrosatively modified by peroxynitrite (ONOO(-)) and hypochlorous acid (HOCl) and by activated monocytes and macrophages via release of ONOO(-) and HOCl. In an in vitro elastic fiber assembly model, oxidatively modified TE was unable to form elastic fibers. Oxidation of TE enhanced coacervation, an early step in elastic fiber assembly, but reduced cross-linking and interactions with other proteins required for elastic fiber assembly, including fibulin-4, fibulin-5, and fibrillin-2. These findings establish that ROS/RNS can modify TE and that these modifications affect the assembly of elastic fibers. Thus, we speculate that oxidative stress may contribute to the abnormal structure and function of elastic fibers in pathological conditions.

  20. Participatory ergonomic intervention for prevention of low back pain: assembly line redesign case.

    Science.gov (United States)

    Bernardes, João Marcos; Wanderck, Claudia; Moro, Antônio Renato Pereira

    2012-01-01

    This paper gives an overview of a participatory ergonomic intervention aimed at reducing low back pain cases in the dispatch department of a catalogue and e-commerce retail company. Based on the findings of the ergonomic analysis and design committee, the company's own employees redesigned the assembly line's layout. As a result of these changes two job tasks that involved manual material handling of boxes, identified by the revised NIOSH equation as posing an increased risk for lifting-related low back pain, were totally eliminated, and the employees responsible for moving boxes from the end of the assembly line to pallets on the ground were given more control over their jobs, and these jobs were also enriched with a new, less heavy task. These results demonstrate that participatory ergonomic interventions are a viable and effective strategy to reduce the exposure to work-related physical and psychosocial risk factors for low back pain.

  1. Efficacy of dry-ice blasting in preventive maintenance of auto robotic assemblies

    Science.gov (United States)

    Baluch, Nazim; Mohtar, Shahimi; Abdullah, Che Sobry

    2016-08-01

    Welding robots are extensively applied in the automotive assemblies and `Spot Welding' is the most common welding application found in the auto stamping assembly manufacturing. Every manufacturing process is subject to variations - with resistance welding, these include; part fit up, part thickness variations, misaligned electrodes, variations in coating materials or thickness, sealers, weld force variations, shunting, machine tooling degradation; and slag and spatter damage. All welding gun tips undergo wear; an elemental part of the process. Though adaptive resistance welding control automatically compensates to keep production and quality up to the levels needed as gun tips undergo wear so that the welds remain reliable; the system cannot compensate for deterioration caused by the slag and spatter on the part holding fixtures, sensors, and gun tips. To cleanse welding robots of slag and spatter, dry-ice blasting has proven to be an effective remedy. This paper describes Spot welding process, analyses the slag and spatter formation during robotic welding of stamping assemblies, and concludes that the dry ice blasting process's utility in cleansing of welding robots in auto stamping plant operations is paramount and exigent.

  2. Bioinformatic Analysis of ChIP-seq Data on the Repetitive Ribosomal RNA Gene.

    Science.gov (United States)

    Schwartz, Uwe; Längst, Gernot

    2016-01-01

    Ribosomal RNA genes are highly repetitive and therefore not annotated in genome assemblies. We did recently analyze the epigenetic and architectural regulation of murine ribosomal genes by the Transcription Termination Factor I and made use of genome-wide histone modification ChIP-seq data. This method paper describes how repetitive genomic regions can be integrated into custom genomic assemblies and be used with genome-wide profiling data.

  3. Post-termination Ribosome Intermediate Acts as the Gateway to Ribosome Recycling.

    Science.gov (United States)

    Prabhakar, Arjun; Capece, Mark C; Petrov, Alexey; Choi, Junhong; Puglisi, Joseph D

    2017-07-05

    During termination of translation, the nascent peptide is first released from the ribosome, which must be subsequently disassembled into subunits in a process known as ribosome recycling. In bacteria, termination and recycling are mediated by the translation factors RF, RRF, EF-G, and IF3, but their precise roles have remained unclear. Here, we use single-molecule fluorescence to track the conformation and composition of the ribosome in real time during termination and recycling. Our results show that peptide release by RF induces a rotated ribosomal conformation. RRF binds to this rotated intermediate to form the substrate for EF-G that, in turn, catalyzes GTP-dependent subunit disassembly. After the 50S subunit departs, IF3 releases the deacylated tRNA from the 30S subunit, thus preventing reassembly of the 70S ribosome. Our findings reveal the post-termination rotated state as the crucial intermediate in the transition from termination to recycling. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Driving nucleolar assembly.

    Science.gov (United States)

    McCann, Kathleen L; Baserga, Susan J

    2014-02-01

    In this issue of Genes & Development, Grob and colleagues (pp. 220-230) identify the minimal molecular requirements to assemble a fully functional nucleolus in human cells and demonstrate the importance of the nucleolar transcription factor upstream binding factor (UBF) as a mitotic bookmark at the ribosomal DNA (rDNA).

  5. Functional analysis of transcribed spacers of yeast ribosomal DNA.

    Science.gov (United States)

    Musters, W; Boon, K; van der Sande, C A; van Heerikhuizen, H; Planta, R J

    1990-12-01

    Making use of an rDNA unit, containing oligonucleotide tags in both the 17S and 26S rRNA gene, we have analyzed the effect of various deletions in the External Transcribed Spacer (ETS) and in one of the Internal Transcribed Spacers 1 (ITS1) on the process of ribosome formation in yeast. By following the fate of the tagged transcripts of this rDNA unit in vivo by Northern hybridization we found that deleting various parts of the ETS prevents the accumulation of tagged 17S rRNA and its assembly into 40S subunits, but not the formation of 60S subunits. Deleting the central region of ITS1, including a processing site that is used in an early stage of the maturation process, was also found to prevent the accumulation of functional 49 S subunits, whereas no effect on the formation of 60S subunits was detected. The implications of these findings for yeast pre-rRNA processing are discussed.

  6. Nucleolus: The ribosome factory

    Czech Academy of Sciences Publication Activity Database

    Cmarko, Dušan; Šmigová, J.; Minichová, L.; Popov, Alexey

    2008-01-01

    Roč. 23, č. 10 (2008), s. 1291-1298 ISSN 0213-3911 R&D Projects: GA ČR(CZ) GA304/06/1691 Grant - others:Wellcome Trust(XE) 075834/04/Z; GA MŠk(CZ) LC535; GA ČR(CZ) GA304/06/1662 Program:LC Institutional research plan: CEZ:AV0Z50110509 Keywords : nucleolus * nucleolar architecture * ribosome biogenesis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.194, year: 2008

  7. Sustainable MSD prevention: management for continuous improvement between prevention and production. Ergonomic intervention in two assembly line companies.

    Science.gov (United States)

    Caroly, S; Coutarel, F; Landry, A; Mary-Cheray, I

    2010-07-01

    To increase output and meet customers' needs, companies have turned to the development of production management systems: Kaizen, one piece flow, Kanban, etc. The aim of such systems is to accelerate decisions, react to environmental issues and manage various productions. In the main, this type of management system has led to the continuous improvement of production performance. Consequently, such production management systems can have unexpected negative effects on operators' health and safety. Conversely, regulation and control systems focusing on work-related risks have obliged firms to implement health and safety management systems such as OHSAS 18001. The purpose of this type of system, also based on continuous improvement, is to reduce risks, facilitate work-related activities and identify solutions in terms of equipment and tools. However, the prevention actions introduced through health and safety systems often result in other unexpected and unwanted effects on production. This paper shows how companies can improve the way they are run by taking into account both types of management system. Copyright 2010 Elsevier Ltd. All rights reserved.

  8. Ion Exchange Technology Development in Support of the Urine Processor Assembly Precipitation Prevention Project for the International Space Station

    Science.gov (United States)

    Mitchell, Julie L.; Broyan, James L.; Pickering, Karen D.; Adam, Niklas; Casteel, Michael; Callahan, Michael; Carrier, Chris

    2012-01-01

    In support of the Urine Processor Assembly Precipitation Prevention Project (UPA PPP), multiple technologies were explored to prevent CaSO4 2H2O (gypsum) precipitation during the on-orbit distillation process. Gypsum precipitation currently limits the water recovery rate onboard the International Space Station (ISS) to 70% versus the planned 85% target water recovery rate. Due to its ability to remove calcium cations in pretreated augmented urine (PTAU), ion exchange was selected as one of the technologies for further development by the PPP team. A total of 13 ion exchange resins were evaluated in various equilibrium and dynamic column tests with solutions of dissolved gypsum, urine ersatz, PTAU, and PTAU brine at 85% water recovery. While initial evaluations indicated that the Purolite SST60 resin had the highest calcium capacity in PTAU (0.30 meq/mL average), later tests showed that the Dowex G26 and Amberlite FPC12H resins had the highest capacity (0.5 meq/mL average). Testing at the Marshall Spaceflight Center (MSFC) integrates the ion exchange technology with a UPA ground article under flight-like pulsed flow conditions with PTAU. To date, no gypsum precipitation has taken place in any of the initial evaluations.

  9. Interdependence in the processing of ribosomal RNAs in Schizosaccharomyces pombe.

    Science.gov (United States)

    Good, L; Intine, R V; Nazar, R N

    1997-11-07

    Eukaryotic rRNAs are produced by cleavage of a large 35 to 45 S pre-rRNA transcript which initially must be fully transcribed and assembled into an 80 to 90 S nucleolar ribonucleoprotein particle. Despite this need for a completed transcript, several investigations have reported a split processing scheme for independent maturation of the large and small subunit rRNAs. Here, an efficiently expressed rDNA plasmid was used to quantitatively analyze the effects of mutations in the internal transcribed spacer (ITS) region in the yeast, Schizosaccharomyces pombe. The results show that substitution of ITS regions inhibits the processing of distant external transcribed spacers (ETS) and that deletion of the ITS2 spacer not only prevents the maturation of the large subunit, but severely affects maturation of the small subunit rRNA. This indicates that the processing mechanisms are not fully split and, when taken together with other evidence of interdependences in rRNA maturation, the results suggest that the interdependences act as a quality control mechanism to help ensure that only functional rRNA is incorporated into ribosomes. Copyright 1997 Academic Press Limited

  10. Ribosomes as molecular energy machines.

    Science.gov (United States)

    Kremen, A

    1994-10-07

    The idea that ribosomes operate as biological molecular energy machines offers an alternative description to that based on the assumption that the functionally important motions in ribosomes are only coupled to thermal degrees of freedom. The alternative model assumes that the energy gained in GTP cleavage generates in the ribosomal complex a localized metastable region temporarily not interacting with thermal motions. The metastable region may move along a definite pathway and in distinct sites promote irreversible logical operations involved in polypeptide biosynthesis. The new alternative represents a more complex and advanced algorithm offering advantages in speed, accuracy, more sophisticated control, and better resistance to various kinds of noise.

  11. Transcriptome-wide measurement of ribosomal occupancy by ribosome profiling.

    Science.gov (United States)

    Aeschimann, Florian; Xiong, Jieyi; Arnold, Andreas; Dieterich, Christoph; Grosshans, Helge

    2015-09-01

    Gene expression profiling provides a tool to analyze the internal states of cells or organisms, and their responses to perturbations. While global measurements of mRNA levels have thus been widely used for many years, it is only through the recent development of the ribosome profiling technique that an analogous examination of global mRNA translation programs has become possible. Ribosome profiling reveals which RNAs are being translated to what extent and where the translated open reading frames are located. In addition, different modes of translation regulation can be distinguished and characterized. Here, we present an optimized, step-by-step protocol for ribosome profiling. Although established in Caenorhabditis elegans, our protocol and optimization approaches should be equally usable for other model organisms or cell culture with little adaptation. Next to providing a protocol, we compare two different methods for isolation of single ribosomes and two different library preparations, and describe strategies to optimize the RNase digest and to reduce ribosomal RNA contamination in the libraries. Moreover, we discuss bioinformatic strategies to evaluate the quality of the data and explain how the data can be analyzed for different applications. In sum, this article seeks to facilitate the understanding, execution, and optimization of ribosome profiling experiments. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. GTP hydrolysis by IF2 guides progression of the ribosome into elongation

    Science.gov (United States)

    Marshall, R. Andrew; Aitken, Colin Echeverría; Puglisi, Joseph D.

    2015-01-01

    Summary Recent structural data have revealed two distinct conformations of the ribosome during initiation. We employed single-molecule fluorescence methods to probe the dynamic relation of these ribosomal conformations in real time. In the absence of initiation factors, the ribosome assembles in two distinct conformations. The initiation factors discriminate between these two conformations, guiding assembly of the conformation that can enter the elongation cycle. In particular, IF2 both accelerates the rate of subunit joining and actively promotes the transition to the elongation-competent conformation. Blocking GTP hydrolysis by IF2 results in 70S complexes formed in the conformation unable to enter elongation. We observe that rapid GTP hydrolysis by IF2 drives the transition to the elongation-competent conformation, thus committing the ribosome to enter the elongation cycle. PMID:19595714

  13. Linker 2 of the eukaryotic pre-ribosomal processing factor Mrd1p is an essential interdomain functionally coupled to upstream RNA Binding Domain 2 (RBD2)

    OpenAIRE

    Lackmann, Fredrik; Belikov, Sergey; Wieslander, Lars

    2017-01-01

    Ribosome synthesis is an essential process in all cells. In Sacharomyces cerevisiae, the precursor rRNA, 35S pre-rRNA, is folded and assembled into a 90S pre-ribosomal complex. The 40S ribosomal subunit is processed from the pre-ribosomal complex. This requires concerted action of small nucleolar RNAs, such as U3 snoRNA, and a large number of trans-acting factors. Mrd1p, one of the essential small ribosomal subunit synthesis factors is required for cleavage of the 35S pre-rRNA to generate 18S...

  14. The complete structure of the chloroplast 70S ribosome in complex with translation factor pY.

    Science.gov (United States)

    Bieri, Philipp; Leibundgut, Marc; Saurer, Martin; Boehringer, Daniel; Ban, Nenad

    2017-02-15

    Chloroplasts are cellular organelles of plants and algae that are responsible for energy conversion and carbon fixation by the photosynthetic reaction. As a consequence of their endosymbiotic origin, they still contain their own genome and the machinery for protein biosynthesis. Here, we present the atomic structure of the chloroplast 70S ribosome prepared from spinach leaves and resolved by cryo-EM at 3.4 Å resolution. The complete structure reveals the features of the 4.5S rRNA, which probably evolved by the fragmentation of the 23S rRNA, and all five plastid-specific ribosomal proteins. These proteins, required for proper assembly and function of the chloroplast translation machinery, bind and stabilize rRNA including regions that only exist in the chloroplast ribosome. Furthermore, the structure reveals plastid-specific extensions of ribosomal proteins that extensively remodel the mRNA entry and exit site on the small subunit as well as the polypeptide tunnel exit and the putative binding site of the signal recognition particle on the large subunit. The translation factor pY, involved in light- and temperature-dependent control of protein synthesis, is bound to the mRNA channel of the small subunit and interacts with 16S rRNA nucleotides at the A-site and P-site, where it protects the decoding centre and inhibits translation by preventing tRNA binding. The small subunit is locked by pY in a non-rotated state, in which the intersubunit bridges to the large subunit are stabilized. © 2016 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

  15. Mg2+ dependence of 70 S ribosomal protein flexibility revealed by hydrogen/deuterium exchange and mass spectrometry.

    Science.gov (United States)

    Yamamoto, Tatsuya; Shimizu, Yoshihiro; Ueda, Takuya; Shiro, Yoshitsugu

    2010-02-19

    The ribosome from Escherichia coli requires a specific concentration of Mg(2+) to maintain the 70 S complex formation and allow protein synthesis, and then the structure must be stable and flexible. How does the ribosome acquire these conflicting factors at the same time? Here, we investigated the hydrogen/deuterium exchange of 52 proteins in the 70 S ribosome, which controlled stability and flexibility under various Mg(2+) concentrations, using mass spectrometry. Many proteins exhibited a sigmoidal curve for Mg(2+) concentration dependence, incorporating more deuterium at lower Mg(2+) concentration. By comparing deuterium incorporation with assembly, we have discovered a typical mechanism of complexes for acquiring both stability and flexibility at the same time. In addition, we got information of the localization of flexibility in ribosomal function by the analysis of related proteins with stalk protein, tRNA, mRNA, and nascent peptide, and demonstrate the relationship between structure, assembly, flexibility, and function of the ribosome.

  16. Valve assembly

    International Nuclear Information System (INIS)

    Sandling, M.

    1981-01-01

    An improved valve assembly, used for controlling the flow of radioactive slurry, is described. Radioactive contamination of the air during removal or replacement of the valve is prevented by sucking air from the atmosphere through a portion of the structure above the valve housing. (U.K.)

  17. Prevention of significant deterioration permit application for the Fueled Clad Fabrication System, the Radioisotope Power Systems Facility, and the Fuel Assembly Area

    Energy Technology Data Exchange (ETDEWEB)

    1989-08-01

    This New Source Review'' has been submitted by the US Department of Energy-Richland Operations Office (PO Box 550, Richland, Washington 99352), pursuant to WAC 173-403-050 and in compliance with the Department of Ecology Guide to Processing A Prevention Of Significant Deterioration (PSD) Permit'' for three new sources of radionuclide emissions at the Hanford Site in Washington State. The three new sources, the Fueled Clad Fabrication System (FCFS), the Radioisotope Power Systems Facility (RPSF), and the Fuel Assembly Area (FAA), will be located in one facility, the Fuels and Materials Examination Facility (FMEF) of the 400 Area. The FMEF was originally designed to provide for post-irradiation examination and fabrication of breeder reactor fuels. These FMEF missions were cancelled before the introduction of any fuel materials or any irradiated material. The current plans are to use the facility to fabricate power supplies for use in space applications and to produce Fast Flux Test Facility (FFTF) fuel and target assemblies. The FCFS and the RPSF will produce materials and assemblies for application in space. The FAA project will produce FFTF fuel and target assemblies. The FCFS and the RPSF will share the same building, stack, and, in certain cases, the same floor space. Given this relationship, these systems will be dealt with separately to the extent possible. The FAA is a comparatively independent operation though it will share the FMEF complex.

  18. Streptomycin dependence in Escherichia coli: effects of antibiotic deprivation on ribosomes.

    Science.gov (United States)

    Viceps, D I; Brownstein, B L

    1975-03-01

    The inhibition of cell division and the ultimate loss of viability after removal of streptomycin from growing cultures of streptomycin-dependent bacteria are not the result of "unbalanced growth" or of the breakdown of ribosomes. The streptomycin-dependent strain of Escherichia coli K-12 studied continued to synthesize ribonucleic acid (RNA) and protein during streptomycin starvation. There was no evidence of a gross imbalance in the ratio of RNA to protein synthesized or of selective degradation of either protein or RNA. Using the sedimentation of subunits in sucrose as the criterion, normal ribosomes were synthesized even after 18 h of streptomycin deprivation, although the rates of appearance of mature 30S and 50S subunits decreased with time of deprivation. Once formed, these ribosomes appeared stable, as did those synthesized before the onset of starvation. Ribosomes isolated from starved dependent cells were as "functional" as ribosomes from cells grown with streptomycin in their capacity to bind aminoacyl-transfer RNA in response to polyuridylic acid or natural messenger RNA to interconvert between active and inactive transfer RNA binding states, and to synthesize proteins in cell-free systems. The effects are consistent with an impaired rate of synthesis of ribosomal components or assembly of ribosomes resulting in a continually diminishing rate of protein synthesis. The effect on cell division may be the result of a decreased rate of protein synthesis in general and the requirement for a specific protein(s) in particular.

  19. Fuel assembly

    International Nuclear Information System (INIS)

    Nakatsuka, Masafumi; Matsuzuka, Ryuji.

    1976-01-01

    Object: To provide a fuel assembly which can decrease pressure loss of coolant to uniform temperature. Structure: A sectional area of a flow passage in the vicinity of an inner peripheral surface of a wrapper tube is limited over the entire length to prevent the temperature of a fuel element in the outermost peripheral portion from being excessively decreased to thereby flatten temperature distribution. To this end, a plurality of pincture-frame-like sheet metals constituting a spacer for supporting a fuel assembly, which has a plurality of fuel elements planted lengthwise and in given spaced relation within the wrapper tube, is disposed in longitudinal grooves and in stacked fashion to form a substantially honeycomb-like space in cross section. The fuel elements are inserted and supported in the space to form a fuel assembly. (Kamimura, M.)

  20. Ribosomal targets for antibiotic drug discovery

    Energy Technology Data Exchange (ETDEWEB)

    Blanchard, Scott C.; Feldman, Michael Brian; Wang, Leyi; Doudna Cate, James H.; Pulk, Arto; Altman, Roger B.; Wasserman, Michael R

    2016-09-13

    The present invention relates to methods to identify molecules that binds in the neomycin binding pocket of a bacterial ribosome using structures of an intact bacterial ribosome that reveal how the ribosome binds tRNA in two functionally distinct states, determined by 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 (RRF) and binds tRNA in a highly bent conformation in a hybrid peptidyl/exit (P/E) site. Additionally, the invention relates to various assays, including single-molecule assay for ribosome recycling, and methods to identify compounds that interfere with ribosomal function by detecting newly identified intermediate FRET states using known and novel FRET pairs on the ribosome. The invention also provides vectors and compositions with an N-terminally tagged S13 protein.

  1. Proteomic LC-MS analysis of Arabidopsis cytosolic ribosomes : Identification of ribosomal protein paralogs and re-annotation of the ribosomal protein genes

    NARCIS (Netherlands)

    Hummel, Maureen; Dobrenel, Thomas; Cordewener, Jan J H G; Davanture, Marlène; Meyer, Christian; Smeekens, Sjef J C M; Bailey-Serres, Julia; America, Twan A H P; Hanson, Johannes

    2015-01-01

    UNLABELLED: Arabidopsis thaliana cytosolic ribosomes are large complexes containing eighty-one distinct ribosomal proteins (r-proteins), four ribosomal RNAs (rRNA) and a plethora of associated (non-ribosomal) proteins. In plants, r-proteins of cytosolic ribosomes are each encoded by two to seven

  2. Repurposing ribosomes for synthetic biology.

    Science.gov (United States)

    Liu, Yi; Kim, Do Soon; Jewett, Michael C

    2017-10-01

    The translation system is the cell's factory for protein biosynthesis, stitching together hundreds to thousands of amino acids into proteins, which are required for the structure, function, and regulation of living systems. The extraordinary synthetic capability of this system, which includes the ribosome and its associated factors required for polymerization, has driven extensive efforts to harness it for societal use in areas as diverse as energy, materials, and medicine. A powerful example is recombinant protein production, which has impacted the lives of patients through the synthesis of biopharmaceuticals such as insulin. In nature, however, only limited sets of monomers are utilized, thereby resulting in limited sets of biopolymers (i.e., proteins). Expanding nature's repertoire of ribosomal monomers could yield new classes of enzymes, therapeutics, materials, and chemicals with diverse, genetically encoded chemistry. Here, we discuss recent progress towards engineering ribosomes both in vivo and in vitro. These fundamental and technical breakthroughs open doors for advanced applications in biotechnology and synthetic biology. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. 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. © 2015 Reja et al.; Published by Cold Spring Harbor Laboratory Press.

  4. Control of ribosome formation in rat heart

    International Nuclear Information System (INIS)

    Russo, L.A.

    1987-01-01

    Diabetes of 9 days duration produced a 17% diminution in the rate of total protein synthesis in rat hearts perfused as Langendorff preparations supplied with glucose, plasma levels of amino acids, and 400 μU/ml insulin. This reduction was attributable to a decrease in efficiency of protein synthesis and total RNA content. Total messenger RNA content decreased in diabetic hearts in proportion to the reduction in total RNA. Diabetes also resulted in diminished ribosome content as reflected by the induction in total RNA. Ribosome production was investigated by monitoring incorporation of [ 3 H]phenylalanine into the proteins of cytoplasmic ribosomes. Rates of ribosome formation in diabetic hearts were as fast as control rates in the presence of insulin, and were faster than control rates in the absence of the hormone. These results indicated that ribosome content fell in diabetic hearts despite unchanged or faster rates of ribosome formation

  5. Organization of proteins in mammalian mitochondrial ribosomes: accessibility to lactoperoxidase-catalyzed radioiodination

    International Nuclear Information System (INIS)

    Denslow, N.D.; O'Brien, T.W.

    1984-01-01

    To assess the relative exposure of individual ribosomal proteins (r-proteins) in the large and small subunits of the bovine mitochondrial ribosome, double label iodination technique was used. Regions of r-proteins exposed in purified ribosomal subunits were labeled with 131 I using the lactoperoxidase-catalyzed iodination system, and additional reactive groups available upon denaturing the r-proteins in urea were labeled with 125 I using the chloramine-T mediated reaction. The ratio of 131 I to 125 I incorporated into individual proteins under these conditions is representative of the degree of exposure for each of the proteins in the subunits. In this manner, the r-proteins have been grouped into 3 classes depending on their degree of exposure: high exposure, intermediate exposure, and essentially buried. While both subunits have a few proteins in the highly exposed group, and a large number of proteins in the intermediate exposure group, only the large ribosomal subunit has an appreciable number of proteins which appear essentially buried. The more buried proteins may serve mainly structural roles, perhaps acting as assembly proteins, since many from this group bind to ribosomal RNA. The more superficially disposed proteins may comprise binding sites for macromolecules that interact with ribosomes during protein synthesis, as well as stabilizing the association of the large and small subribosomal particles

  6. Prevention

    Science.gov (United States)

    ... Error processing SSI file About Heart Disease & Stroke Prevention Heart disease and stroke are an epidemic in ... secondhand smoke. Barriers to Effective Heart Disease & Stroke Prevention Many people with key risk factors for heart ...

  7. Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae: Roles in ribosome biogenesis and translation.

    Science.gov (United States)

    Al-Hadid, Qais; White, Jonelle; Clarke, Steven

    2016-02-12

    A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed -1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Binding of Signal Recognition Particle Gives Ribosome/Nascent Chain Complexes a Competitive Advantage in Endoplasmic Reticulum Membrane Interaction

    Science.gov (United States)

    Neuhof, Andrea; Rolls, Melissa M.; Jungnickel, Berit; Kalies, Kai-Uwe; Rapoport, Tom A.

    1998-01-01

    Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC–SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes. PMID:9436994

  9. Trapping the ribosome to control gene expression.

    Science.gov (United States)

    Boehringer, Daniel; Ban, Nenad

    2007-09-21

    Protein synthesis is often regulated by structured mRNAs that interact with ribosomes. In this issue of Cell, Marzi et al. (2007) provide insights into the autoregulation of protein S15 by visualizing the folded repressor mRNA on the ribosome stalled in the preinitiation state. These results have implications for our understanding of the mechanism of translation initiation in general.

  10. Heat transfer enhancement for spent nuclear fuel assembly disposal packages using metallic void fillers: A prevention technique for solidification shrinkage-induced interfacial gaps

    Science.gov (United States)

    Park, Yongsoo; McKrell, Thomas J.; Driscoll, Michael J.

    2017-06-01

    This study considers replacing the externally accessible void spaces inside a disposal package containing a spent nuclear fuel assembly (SNFA) with high heat conducting metal to increase the effective thermal conductivity of the package and simplify the heat transfer mechanism inside the package by reducing it to a conduction dominant problem. The focus of the study is on preventing the gaps adjacent to the walls of the package components, produced by solidification shrinkage of poured liquid metal. We approached the problem by providing a temporary coating layer on the components to avoid direct build-up of thick metal oxides on their surface to promote metallic bonding at the interfaces under a non-inert environment. Laboratory scale experiments without SNFA were performed with Zn coated low carbon steel canisters and Zamak-3 void filler under two different filling temperature conditions - below and above the melting point of Zn (designated BMP and AMP respectively). Gap formation was successfully prevented in both cases while we confirmed an open gap in a control experiment, which used an uncoated canister. Minor growth of Al-Fe intermetallic phases was observed at the canister/filler interface of the sample produced under the BMP condition while their growth was significant and showed irregularly distributed morphology in the sample produced under the AMP condition, which has a potential to mitigate excessive residual stresses caused by shrinkage prevention. A procedure for the full-scale application was specified based on the results.

  11. Differential Stoichiometry among Core Ribosomal Proteins

    Directory of Open Access Journals (Sweden)

    Nikolai Slavov

    2015-11-01

    Full Text Available Understanding the regulation and structure of ribosomes is essential to understanding protein synthesis and its dysregulation in disease. While ribosomes are believed to have a fixed stoichiometry among their core ribosomal proteins (RPs, some experiments suggest a more variable composition. Testing such variability requires direct and precise quantification of RPs. We used mass spectrometry to directly quantify RPs across monosomes and polysomes of mouse embryonic stem cells (ESC and budding yeast. Our data show that the stoichiometry among core RPs in wild-type yeast cells and ESC depends both on the growth conditions and on the number of ribosomes bound per mRNA. Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in polysomes. Together, our findings support the existence of ribosomes with distinct protein composition and physiological function.

  12. 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

    A newly identified class of highly thiostrepton-resistant mutants of the archaeon Halobacterium halobium carry a missense mutation at codon 18 within the gene encoding ribosomal protein L11. In the mutant proteins, a proline, conserved in archaea and bacteria, is converted to either serine...... or threonine. The mutations do not impair either the assembly of the mutant L11 into 70 S ribosomes in vivo or the binding of thiostrepton to ribosomes in vitro. Moreover, the corresponding mutations at proline 22, in a fusion protein of L11 from Escherichia coli with glutathione-S-transferase, did not reduce...... the binding affinities of the mutated L11 fusion proteins for rRNA of of thiostrepton for the mutant L11-rRNA complexes at rRNA concentrations lower than those prevailing in vivo. Probing the structure of the fusion protein of wild-type L11, from E. coli, using a recently developed protein footprinting...

  13. Ribosomal RNA processing in Candida albicans.

    Science.gov (United States)

    Pendrak, Michael L; Roberts, David D

    2011-12-01

    Ribosome assembly begins with conversion of a polycistronic precursor into 18S, 5.8S, and 25S rRNAs. In the ascomycete fungus Candida albicans, rRNA transcription starts 604 nt upstream of the 18S rRNA junction (site A1). One major internal processing site in the 5' external transcribed spacer (A0) occurs 108 nt from site A1. The A0-A1 fragment persists as a stable species during log phase growth and can be used to assess proliferation rates. Separation of the small and large subunit pre-rRNAs occurs at sites A2 and A3 in internal transcribed spacer-1 Saccharomyces cerevisiae pre-rRNA. However, the 5' end of the 5.8S rRNA is represented by only a 5.8S (S) form, and a 7S rRNA precursor of the 5.8S rRNA extends into internal transcribed spacer 1 to site A2, which differs from S. cerevisiae. External transcribed spacer 1 and internal transcribed spacers 1 and 2 show remarkable structural similarity with S. cerevisiae despite low sequence identity. Maturation of C. albicans rRNA resembles other eukaryotes in that processing can occur cotranscriptionally or post-transcriptionally. During rapid proliferation, U3 snoRNA-dependent processing occurs before large and small subunit rRNA separation, consistent with cotranscriptional processing. As cells pass the diauxic transition, the 18S pre-rRNA accumulates into stationary phase as a 23S species, possessing an intact 5' external transcribed spacer extending to site A3. Nutrient addition to starved cells results in the disappearance of the 23S rRNA, indicating a potential role in normal physiology. Therefore, C. albicans reveals new mechanisms that regulate post- versus cotranscriptional rRNA processing.

  14. Hyper-accurate ribosomes inhibit growth.

    Science.gov (United States)

    Ruusala, T; Andersson, D; Ehrenberg, M; Kurland, C G

    1984-11-01

    We have compared both in vivo and in vitro translation by ribosomes from wild-type bacteria with those from streptomycin-resistant (SmR), streptomycin-dependent (SmD) and streptomycin-pseudo-dependent (SmP) mutants. The three mutant bacteria translate more accurately and more slowly in the absence of streptomycin (Sm) than do wild-type bacteria. In particular, the SmP bacteria grow at roughly half the rate of the wild-type in the absence of Sm. The antibiotic stimulates both the growth rate and the translation rate of SmP bacteria by approximately 2-fold, but it simultaneously increases the nonsense suppression rate quite dramatically. Kinetic experiments in vitro show that the greater accuracy and slower translation rates of mutant ribosomes compared with wild-type ribosomes are associated with much more rigorous proofreading activities of SmR, SmD and SmP ribosomes. Sm reduces the proofreading flows of the mutant ribosomes and stimulates their elongation rates. The data suggest that these excessively accurate ribosomes are kinetically less efficient than wild-type ribosomes, and that this inhibits mutant growth rates. The stimulation of the growth of the mutants by Sm results from the enhanced translational efficiency due to the loss of proofreading, which more than offsets the loss of accuracy caused by the antibiotic.

  15. Prevention

    Science.gov (United States)

    ... Contact Aging & Health A to Z Find a Geriatrics Healthcare Professional Medications & Older Adults Making Your Wishes ... Prevention Hearing Loss Heart Attack High Blood Pressure Nutrition Osteoporosis Shingles Skin Cancer Related News Quitting Smoking, ...

  16. A new system for naming ribosomal proteins

    Science.gov (United States)

    Ban, Nenad; Beckmann, Roland; Cate, Jamie HD; Dinman, Jonathan D; Dragon, François; Ellis, Steven R; Lafontaine, Denis LJ; Lindahl, Lasse; Liljas, Anders; Lipton, Jeffrey M; McAlear, Michael A; Moore, Peter B; Noller, Harry F; Ortega, Joaquin; Panse, Vikram Govind; Ramakrishnan, V; Spahn, Christian MT; Steitz, Thomas A; Tchorzewski, Marek; Tollervey, David; Warren, Alan J; Williamson, James R; Wilson, Daniel; Yonath, Ada; Yusupov, Marat

    2015-01-01

    A system for naming ribosomal proteins is described that the authors intend to use in the future. They urge others to adopt it. The objective is to eliminate the confusion caused by the assignment of identical names to ribosomal proteins from different species that are unrelated in structure and function. In the system proposed here, homologous ribosomal proteins are assigned the same name, regardless of species. It is designed so that new names are similar enough to old names to be easily recognized, but are written in a format that unambiguously identifies them as ‘new system’ names. PMID:24524803

  17. Is The Ribosome Targeted By Adaptive Mutations

    DEFF Research Database (Denmark)

    Jimenez Fernandez, Alicia; Molin, Søren; Johansen, Helle Krogh

    2015-01-01

    by the antibiotic treatment of the patient. However, other mutations cannot be directly associated with antibiotic resistance. Conclusions: Clarification of the potential pleiotropic consequences of the specific mutations in ribosomal proteins is important for our understanding of biological evolution......, and will have impacts on the design of new treatment strategies to combat microbial infections....... in specific ribosomal genes. The bacterial phenotypes of the mutated strains will be investigated. Results: Preliminary assays show that mutant strains have reduced growth rate and an altered antibiotic resistance pattern. The selection for mutations in ribosomal protein genes is partly explainable...

  18. Structural insights into methyltransferase KsgA function in 30S ribosomal subunit biogenesis.

    Science.gov (United States)

    Boehringer, Daniel; O'Farrell, Heather C; Rife, Jason P; Ban, Nenad

    2012-03-23

    The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3'-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation.

  19. Structural Insights into Methyltransferase KsgA Function in 30S Ribosomal Subunit Biogenesis*

    Science.gov (United States)

    Boehringer, Daniel; O'Farrell, Heather C.; Rife, Jason P.; Ban, Nenad

    2012-01-01

    The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3′-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation. PMID:22308031

  20. Insertion of the Biogenesis Factor Rei1 Probes the Ribosomal Tunnel during 60S Maturation.

    Science.gov (United States)

    Greber, Basil Johannes; Gerhardy, Stefan; Leitner, Alexander; Leibundgut, Marc; Salem, Michèle; Boehringer, Daniel; Leulliot, Nicolas; Aebersold, Ruedi; Panse, Vikram Govind; Ban, Nenad

    2016-01-14

    Eukaryotic ribosome biogenesis depends on several hundred assembly factors to produce functional 40S and 60S ribosomal subunits. The final phase of 60S subunit biogenesis is cytoplasmic maturation, which includes the proofreading of functional centers of the 60S subunit and the release of several ribosome biogenesis factors. We report the cryo-electron microscopy (cryo-EM) structure of the yeast 60S subunit in complex with the biogenesis factors Rei1, Arx1, and Alb1 at 3.4 Å resolution. In addition to the network of interactions formed by Alb1, the structure reveals a mechanism for ensuring the integrity of the ribosomal polypeptide exit tunnel. Arx1 probes the entire set of inner-ring proteins surrounding the tunnel exit, and the C terminus of Rei1 is deeply inserted into the ribosomal tunnel, where it forms specific contacts along almost its entire length. We provide genetic and biochemical evidence that failure to insert the C terminus of Rei1 precludes subsequent steps of 60S maturation. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Ribosomal RNAs synthesized by isolated squid nerves and ganglia differ from native ribosomal RNAs.

    Science.gov (United States)

    Perrone-Capano, C; Crispino, M; Menichini, E; Kaplan, B B; Giuditta, A

    1999-03-01

    The large rRNA of the squid comprises two chains that may be dissociated by heating at 65 degrees C. A single chain constitutes the small rRNA. Surprisingly, the RNAs synthesized by dissected squid fin nerves and stellate nerves and ganglia differed in size from native rRNAs and did not manifest thermal instability. Nonetheless, they resembled native rRNAs in relative abundance, subcellular distribution, lack of poly(A), and metabolic stability. In addition, newly synthesized RNA was localized in nerve and glial cells, as shown by autoradiographic analysis, and was assembled into 80S ribosomes, which supported the synthesis of neuron-specific neurofilament proteins. Following incubation of nerves and ganglia for >10 h, native rRNAs started to disappear, while two major newly synthesized RNAs progressively accumulated. As a result, after 20 h, native rRNAs were substituted by the two novel RNAs. With use of 32P-cDNA synthesized from the latter RNAs as a probe, the novel RNAs demonstrated a considerable degree of homology with native rRNA in northern analysis. Taken together, the data suggest that in dissected squid nerves and ganglia, the synthesis of native rRNAs is gradually terminated while two novel rRNAs are being synthesized, presumably as a correlate of reactive gliosis and/or neuronal degeneration/regeneration.

  2. The role of ribosomal conformation in protein biosynthesis: the streptomycin-ribosome interaction.

    Science.gov (United States)

    Sherman, M I; Simpson, M V

    1969-12-01

    The role played by ribosomal conformation in codon-anticodon recognition has been studied using streptomycin as a probe, inasmuch as streptomycin is known to cause misreading of the genetic code. Changes in ribosomal structure have been followed by the method of hydrogen-tritium exchange. The results show that streptomycin induces two types of change in the hydrogen exchange pattern. At low molar ratios of streptomycin to ribosomes, a stimulation of the hydrogen exchange rate ("loosening" of ribosomal structure) is observed, with a small inhibition of polypeptide synthesis. As the streptomycin: ribosome ratio is increased, a maximum exchange rate is reached, after which the rate decreases ("tightening" of structure); in this region, inhibition of peptide synthesis increases sharply, and misreading of the code begins. None of these effects is observed with streptomycin-resistant ribosomes.

  3. THE ROLE OF RIBOSOMAL CONFORMATION IN PROTEIN BIOSYNTHESIS: THE STREPTOMYCIN-RIBOSOME INTERACTION*

    Science.gov (United States)

    Sherman, Michael I.; Simpson, Melvin V.

    1969-01-01

    The role played by ribosomal conformation in codon-anticodon recognition has been studied using streptomycin as a probe, inasmuch as streptomycin is known to cause misreading of the genetic code. Changes in ribosomal structure have been followed by the method of hydrogen-tritium exchange. The results show that streptomycin induces two types of change in the hydrogen exchange pattern. At low molar ratios of streptomycin to ribosomes, a stimulation of the hydrogen exchange rate (“loosening” of ribosomal structure) is observed, with a small inhibition of polypeptide synthesis. As the streptomycin: ribosome ratio is increased, a maximum exchange rate is reached, after which the rate decreases (“tightening” of structure); in this region, inhibition of peptide synthesis increases sharply, and misreading of the code begins. None of these effects is observed with streptomycin-resistant ribosomes. PMID:4916926

  4. Prevention

    DEFF Research Database (Denmark)

    Halken, S; Høst, A

    2001-01-01

    , breastfeeding should be encouraged for 4-6 months. In high-risk infants a documented extensively hydrolysed formula is recommended if exclusive breastfeeding is not possible for the first 4 months of life. There is no evidence for preventive dietary intervention neither during pregnancy nor lactation...... populations. These theories remain to be documented in proper, controlled and prospective studies. Breastfeeding and the late introduction of solid foods (>4 months) is associated with a reduced risk of food allergy, atopic dermatitis, and recurrent wheezing and asthma in early childhood. In all infants....... Preventive dietary restrictions after the age of 4-6 months are not scientifically documented....

  5. A recent intermezzo at the Ribosome Club.

    Science.gov (United States)

    Pavlov, Michael Y; Liljas, Anders; Ehrenberg, Måns

    2017-03-19

    Two sets of ribosome structures have recently led to two different interpretations of what limits the accuracy of codon translation by transfer RNAs. In this review, inspired by this intermezzo at the Ribosome Club, we briefly discuss accuracy amplification by energy driven proofreading and its implementation in genetic code translation. We further discuss general ways by which the monitoring bases of 16S rRNA may enhance the ultimate accuracy (d-values) and how the codon translation accuracy is reduced by the actions of Mg 2+ ions and the presence of error inducing aminoglycoside antibiotics. We demonstrate that complete freezing-in of cognate-like tautomeric states of ribosome-bound nucleotide bases in transfer RNA or messenger RNA is not compatible with recent experiments on initial codon selection by transfer RNA in ternary complex with elongation factor Tu and GTP. From these considerations, we suggest that the sets of 30S subunit structures from the Ramakrishnan group and 70S structures from the Yusupov/Yusupova group may, after all, reflect two sides of the same coin and how the structurally based intermezzo at the Ribosome Club may be resolved simply by taking the dynamic aspects of ribosome function into account.This article is part of the themed issue 'Perspectives on the ribosome'. © 2017 The Authors.

  6. Distinct roles for the IIId2 sub-domain in pestivirus and picornavirus internal ribosome entry sites

    DEFF Research Database (Denmark)

    Willcocks, Margaret M.; Zaini, Salmah; Chamond, Nathalie

    2017-01-01

    activity, while the IIId2 sub-domains of two pestiviruses, classical swine fever virus (CSFV) and border disease virus (BDV), are required for 80S ribosomes assembly and IRES activity. Unlike in SVV, the deletion of IIId2 from the CSFV and BDV IRES elements impairs initiation of translation by inhibiting...

  7. Precursors of ribosomal RNA in yeast nucleus : Biosynthesis and relation to cytoplasmic ribosomal RNA

    NARCIS (Netherlands)

    Sillevis Smitt, W.W.; Vlak, J.M.; Schiphof, R.; Rozijn, Th.H.

    In vivo methylated precursors of ribosomal RNA in yeast have been characterized on acrylamide gels. The initial ribosomal precursor in the yeast nucleus is a 37S RNA component, which is processed to a nuclear 28S RNA. Both the 37S and the 28S RNA components are important constituents of the yeast

  8. Distribution of dwell times of a ribosome: effects of infidelity, kinetic proofreading and ribosome crowding

    Science.gov (United States)

    Sharma, Ajeet K.; Chowdhury, Debashish

    2011-04-01

    Ribosome is a molecular machine that polymerizes a protein where the sequence of the amino acid residues, the monomers of the protein, is dictated by the sequence of codons (triplets of nucleotides) on a messenger RNA (mRNA) that serves as the template. The ribosome is a molecular motor that utilizes the template mRNA strand also as the track. Thus, in each step the ribosome moves forward by one codon and, simultaneously, elongates the protein by one amino acid. We present a theoretical model that captures most of the main steps in the mechanochemical cycle of a ribosome. The stochastic movement of the ribosome consists of an alternating sequence of pause and translocation; the sum of the durations of a pause and the following translocation is the time of dwell of the ribosome at the corresponding codon. We derive the analytical expression for the distribution of the dwell times of a ribosome in our model. Wherever experimental data are available, our theoretical predictions are consistent with those results. We suggest appropriate experiments to test the new predictions of our model, particularly the effects of the quality control mechanism of the ribosome and that of their crowding on the mRNA track.

  9. Ribosomal studies on the 70S ribosome of E.coli by means of neutron scattering

    International Nuclear Information System (INIS)

    Burkhardt, N.

    1997-01-01

    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 ( 1 H) for deuterium ( 2 H), 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.) [de

  10. A role for proteins S3 and S14 in the 30 S ribosomal subunit.

    Science.gov (United States)

    Ramakrishnan, V; Graziano, V; Capel, M S

    1986-11-15

    Small ribosomal subunits prepared by the method of Kirillov et al. (Kirillov, S. V., Makhno, V. I., Peshin, N. N., and Semenkov, Yu. P. (1986) Nucleic Acids Res. 5, 4305-4315) are active but fail to reconstitute. The inability to reconstitute is due to a deficiency in proteins S3 and S14. Supplementation of the protein component with pure S3 and S14 leads to an enhancement of the activity of the reconstituted product. Our results provide evidence that these two proteins are involved in assembly but may not be required once the 30 S subunit has been properly assembled.

  11. Harnessing natural product assembly lines: structure, promiscuity, and engineering.

    Science.gov (United States)

    Ladner, Christopher C; Williams, Gavin J

    2016-03-01

    Many therapeutically relevant natural products are biosynthesized by the action of giant mega-enzyme assembly lines. By leveraging the specificity, promiscuity, and modularity of assembly lines, a variety of strategies has been developed that enables the biosynthesis of modified natural products. This review briefly summarizes recent structural advances related to natural product assembly lines, discusses chemical approaches to probing assembly line structures in the absence of traditional biophysical data, and surveys efforts that harness the inherent or engineered promiscuity of assembly lines for the synthesis of non-natural polyketides and non-ribosomal peptide analogues.

  12. Functional Importance of Mobile Ribosomal Proteins

    Directory of Open Access Journals (Sweden)

    Kai-Chun Chang

    2015-01-01

    Full Text Available Although the dynamic motions and peptidyl transferase activity seem to be embedded in the rRNAs, the ribosome contains more than 50 ribosomal proteins (r-proteins, whose functions remain largely elusive. Also, the precise forms of some of these r-proteins, as being part of the ribosome, are not structurally solved due to their high flexibility, which hinders the efforts in their functional elucidation. Owing to recent advances in cryo-electron microscopy, single-molecule techniques, and theoretical modeling, much has been learned about the dynamics of these r-proteins. Surprisingly, allosteric regulations have been found in between spatially separated components as distant as those in the opposite sides of the ribosome. Here, we focus on the functional roles and intricate regulations of the mobile L1 and L12 stalks and L9 and S1 proteins. Conformational flexibility also enables versatile functions for r-proteins beyond translation. The arrangement of r-proteins may be under evolutionary pressure that fine-tunes mass distributions for optimal structural dynamics and catalytic activity of the ribosome.

  13. Eukaryotic Initiation Factor 6, an evolutionarily conserved regulator of ribosome biogenesis and protein translation

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Jianjun [Harvard University; Jin, Zhaoqing [ORNL; Yang, Xiaohan [ORNL; Li, Jian-Feng [Harvard University; Chen, Jay [ORNL

    2011-01-01

    We recently identified Receptor for Activated C Kinase 1 (RACK1) as one of the molecular links between abscisic acid (ABA) signaling and its regulation on protein translation. Moreover, we identified Eukaryotic Initiation Factor 6 (eIF6) as an interacting partner of RACK1. Because the interaction between RACK1 and eIF6 in mammalian cells is known to regulate the ribosome assembly step of protein translation initiation, it was hypothesized that the same process of protein translation in Arabidopsis is also regulated by RACK1 and eIF6. In this article, we analyzed the amino acid sequences of eIF6 in different species from different lineages and discovered some intriguing differences in protein phosphorylation sites that may contribute to its action in ribosome assembly and biogenesis. In addition, we discovered that, distinct from non-plant organisms in which eIF6 is encoded by a single gene, all sequenced plant genomes contain two or more copies of eIF6 genes. While one copy of plant eIF6 is expressed ubiquitously and might possess the conserved function in ribosome biogenesis and protein translation, the other copy seems to be only expressed in specific organs and therefore may have gained some new functions. We proposed some important studies that may help us better understand the function of eIF6 in plants.

  14. Molecular flexibility of Mycobacterium tuberculosis ribosome ...

    Indian Academy of Sciences (India)

    2013-11-06

    Nov 6, 2013 ... Mycobacterium tuberculosis RRF does not complement E. coli for its deficiency of RRF (in the ... [Selvaraj M, Govindan A, Seshadri A, Dubey B, Varshney U and Vijayan M 2013 Molecular flexibility of Mycobacterium tuberculosis ribosome ...... Janosi L, Mottagui-Tabar S, Isaksson LA, Sekine Y, Ohtsubo E,.

  15. Ribosome binding site recognition using neural networks

    Directory of Open Access Journals (Sweden)

    Márcio Ferreira da Silva Oliveira

    2004-01-01

    Full Text Available Pattern recognition is an important process for gene localization in genomes. The ribosome binding sites are signals that can help in the identification of a gene. It is difficult to find these signals in the genome through conventional methods because they are highly degenerated. Artificial Neural Networks is the approach used in this work to address this problem.

  16. Mutant ribosomes can generate dominant kirromycin resistance.

    Science.gov (United States)

    Tubulekas, I; Buckingham, R H; Hughes, D

    1991-01-01

    Mutations in the two genes for EF-Tu in Salmonella typhimurium and Escherichia coli, tufA and tufB, can confer resistance to the antibiotic kirromycin. Kirromycin resistance is a recessive phenotype expressed when both tuf genes are mutant. We describe a new kirromycin-resistant phenotype dominant to the effect of wild-type EF-Tu. Strains carrying a single kirromycin-resistant tuf mutation and an error-restrictive, streptomycin-resistant rpsL mutation are resistant to high levels of kirromycin, even when the other tuf gene is wild type. This phenotype is dependent on error-restrictive mutations and is not expressed with nonrestrictive streptomycin-resistant mutations. Kirromycin resistance is also expressed at a low level in the absence of any mutant EF-Tu. These novel phenotypes exist as a result of differences in the interactions of mutant and wild-type EF-Tu with the mutant ribosomes. The restrictive ribosomes have a relatively poor interaction with wild-type EF-Tu and are thus more easily saturated with mutant kirromycin-resistant EF-Tu. In addition, the mutant ribosomes are inherently kirromycin resistant and support a significantly faster EF-Tu cycle time in the presence of the antibiotic than do wild-type ribosomes. A second phenotype associated with combinations of rpsL and error-prone tuf mutations is a reduction in the level of resistance to streptomycin. PMID:2050625

  17. Ribosome display for improved biotherapeutic molecules.

    Science.gov (United States)

    Rothe, Achim; Hosse, Ralf J; Power, Barbara E

    2006-02-01

    Ribosome display presents an innovative in vitro technology for the rapid isolation and evolution of high-affinity peptides or proteins. Displayed proteins are bound to and recovered from target molecules in multiple rounds of selection in order to enrich for specific binding proteins. No transformation step is necessary, which could lead to a loss of library diversity. A cycle of display and selection can be performed in one day, enabling the existing gene repertoire to be rapidly scanned. Proteins isolated from the panning rounds can be further modified through random or directed molecular evolution for affinity maturation, as well as selected for characteristics such as protein stability, folding and functional activity. Recently, the field of display technologies has become more prominent due to the generation of new scaffolds for ribosome display, isolation of high-affinity human antibodies by phage display, and their implementation in the discovery of novel protein-protein interactions. Applications for this technology extend into the broad field of antibody engineering, proteomics, and synthetic enzymes for diagnostics and therapeutics in cancer, autoimmune and infectious diseases, neurodegenerative diseases and inflammatory disorders. This review highlights the role of ribosome display in drug discovery, discusses advantages and disadvantages of the system, and attempts to predict the future impact of ribosome display technology on the development of novel engineered biopharmaceutical products for biological therapies.

  18. Control of Ribosome Synthesis in Escherichia coli

    DEFF Research Database (Denmark)

    Molin, Søren; Meyenburg, K. von; Måløe, O.

    1977-01-01

    The rate of ribosome synthesis and accumulation in Escherichia coli during the transition after an energy source shift-down was analyzed. The shift was imposed on cultures of stringent and relaxed strains growing in glucose minimal medium by the addition of the glucose analogue {alpha...

  19. Ribosome evolution: Emergence of peptide synthesis machinery

    Indian Academy of Sciences (India)

    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 ...

  20. Unraveling the Dynamics of Ribosome Translocation

    Science.gov (United States)

    Chen, Jin; Tsai, Albert; O’Leary, Seán E.; Petrov, Alexey; Puglisi, Joseph D.

    2013-01-01

    Translocation is one of the key events in translation, requiring large-scale conformational changes in the ribosome, movements of two transfer RNAs (tRNAs) across a distance of more than 20 Å, and the coupled movement of the messenger RNA (mRNA) by one codon, completing one cycle of peptide-chain elongation. Translocation is catalyzed by elongation factor G (EF-G in bacteria), which hydrolyzes GTP in the process. However, how the conformational rearrangements of the ribosome actually drive the movements of the tRNAs and how EF-G GTP hydrolysis plays a role in this process are still unclear. Fluorescence methods, both single-molecule and bulk, have provided a dynamic view of translocation, allowing us to follow the different conformational changes of the ribosome in real-time. The application of electron microscopy has revealed new conformational intermediates during translocation and important structural rearrangements in the ribosome that drive tRNA movement, while computational approaches have added quantitative views of the translational pathway. These recent advances shed light on the process of translocation, providing insight on how to resolve the different descriptions of translocation in the current literature. PMID:23142574

  1. Dataset of the transcribed 45S ribosomal RNA sequence of the tree crop “yerba mate”

    Directory of Open Access Journals (Sweden)

    Patricia M. Aguilera

    2017-06-01

    Full Text Available This contribution contains data related to the research article entitled “The 18S-25S ribosomal RNA unit of yerba mate (Ilex paraguariensis A. St.-Hil.” (Aguilera et al., 2016 [1]. Through a bioinformatic approach involving NGS data, we provide information of the transcribed 45S ribosomal RNA (rRNA sequence of yerba mate, the first reference for the Ilex L. genus. This dataset (Supplementary file 1 comprises information regarding the assembly and annotation of this rRNA unit. The generated data is applicable for comparative analysis and evolutionary studies among Ilex and related taxa. The raw sequencing data used here is available at DDBJ/EMBL/GenBank (NCBI Resource Coordinators, 2016 [2] Sequence Read Archive (SRA under the accession SRP043293 and the consensus 45S ribosomal RNA sequence has been deposited there under the accession GFHV00000000.

  2. Structure of a prehandover mammalian ribosomal SRP·SRP receptor targeting complex.

    Science.gov (United States)

    Kobayashi, Kan; Jomaa, Ahmad; Lee, Jae Ho; Chandrasekar, Sowmya; Boehringer, Daniel; Shan, Shu-Ou; Ban, Nenad

    2018-04-20

    Signal recognition particle (SRP) targets proteins to the endoplasmic reticulum (ER). SRP recognizes the ribosome synthesizing a signal sequence and delivers it to the SRP receptor (SR) on the ER membrane followed by the transfer of the signal sequence to the translocon. Here, we present the cryo-electron microscopy structure of the mammalian translating ribosome in complex with SRP and SR in a conformation preceding signal sequence handover. The structure visualizes all eukaryotic-specific SRP and SR proteins and reveals their roles in stabilizing this conformation by forming a large protein assembly at the distal site of SRP RNA. We provide biochemical evidence that the guanosine triphosphate hydrolysis of SRP·SR is delayed at this stage, possibly to provide a time window for signal sequence handover to the translocon. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  3. 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

    the binding affinities of the mutated L11 fusion proteins for rRNA of of thiostrepton for the mutant L11-rRNA complexes at rRNA concentrations lower than those prevailing in vivo. Probing the structure of the fusion protein of wild-type L11, from E. coli, using a recently developed protein footprinting...... or threonine. The mutations do not impair either the assembly of the mutant L11 into 70 S ribosomes in vivo or the binding of thiostrepton to ribosomes in vitro. Moreover, the corresponding mutations at proline 22, in a fusion protein of L11 from Escherichia coli with glutathione-S-transferase, did not reduce...

  4. Nuclear fuel assembly

    International Nuclear Information System (INIS)

    Anthony, A.J.

    1980-01-01

    A bimetallic spacer means is cooperatively associated with a nuclear fuel assembly and operative to resist the occurrence of in-reactor bowing of the nuclear fuel assembly. The bimetallic spacer means in one embodiment of the invention includes a space grid formed, at least principally, of zircaloy to the external surface of which are attached a plurality of stainless steel strips. In another embodiment the strips are attached to fuel pins. In each of the embodiments, the stainless steel strips during power production expand outwardly to a greater extent than do the members to which the stainless steel strips are attached, thereby forming stiff springs which abut against like bimetallic spacer means with which the other nuclear fuel assemblies are provided in a given nuclear reactor core to thus prevent the occurrence of in-reactor bowing of the nuclear fuel assemblies. (author)

  5. Programmed Ribosomal Frameshifting Mediates Expression of the α-Carboxysome.

    Science.gov (United States)

    Chaijarasphong, Thawatchai; Nichols, Robert J; Kortright, Kaitlyn E; Nixon, Charlotte F; Teng, Poh K; Oltrogge, Luke M; Savage, David F

    2016-01-16

    Many bacteria employ a protein organelle, the carboxysome, to catalyze carbon dioxide fixation in the Calvin Cycle. Only 10 genes from Halothiobacillus neapolitanus are sufficient for heterologous expression of carboxysomes in Escherichia coli, opening the door to detailed mechanistic analysis of the assembly process of this complex (more than 200MDa). One of these genes, csoS2, has been implicated in assembly but ascribing a molecular function is confounded by the observation that the single csoS2 gene yields expression of two gene products and both display an apparent molecular weight incongruent with the predicted amino acid sequence. Here, we elucidate the co-translational mechanism responsible for the expression of the two protein isoforms. Specifically, csoS2 was found to possess -1 frameshifting elements that lead to the production of the full-length protein, CsoS2B, and a truncated protein, CsoS2A, which possesses a C-terminus translated from the alternate frame. The frameshifting elements comprise both a ribosomal slippery sequence and a 3' secondary structure, and ablation of either sequence is sufficient to eliminate the slip. Using these mutants, we investigated the individual roles of CsoS2B and CsoS2A on carboxysome formation. In this in vivo formation assay, cells expressing only the CsoS2B isoform were capable of producing intact carboxysomes, while those with only CsoS2A were not. Thus, we have answered a long-standing question about the nature of CsoS2 in this model microcompartment and demonstrate that CsoS2B is functionally distinct from CsoS2A in the assembly of α-carboxysomes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Regulation of bacterial gene expression by ribosome stalling and rescuing.

    Science.gov (United States)

    Jin, Yongxin; Jin, Shouguang; Wu, Weihui

    2016-05-01

    Ribosome is responsible for protein synthesis and is able to monitor the sequence and structure of the nascent peptide. Such ability plays an important role in determining overall gene expression profile of the bacteria through ribosome stalling and rescuing. In this review, we briefly summarize our current understanding of the regulation of gene expression through ribosome stalling and rescuing in bacteria, as well as mechanisms that modulate ribosome activity. Understanding the mechanisms of how bacteria modulate ribosome activity will provide not only fundamental insights into bacterial gene regulation, but also new candidate targets for the development of novel antimicrobial agents.

  7. Compression test assembly

    Science.gov (United States)

    Kariotis, A. H. (Inventor)

    1973-01-01

    A compression test assembly is described which prevents buckling of small diameter rigid specimens undergoing compression testing and permits attachment of extensometers for strain measurements. The test specimen is automatically aligned and laterally supported when compressive force is applied to the end caps and transmitted to the test specimen during testing.

  8. Distinct roles for the IIId2 sub-domain in pestivirus and picornavirus internal ribosome entry sites.

    Science.gov (United States)

    Willcocks, Margaret M; Zaini, Salmah; Chamond, Nathalie; Ulryck, Nathalie; Allouche, Delphine; Rajagopalan, Noemie; Davids, Nana A; Fahnøe, Ulrik; Hadsbjerg, Johanne; Rasmussen, Thomas Bruun; Roberts, Lisa O; Sargueil, Bruno; Belsham, Graham J; Locker, Nicolas

    2017-12-15

    Viral internal ribosomes entry site (IRES) elements coordinate the recruitment of the host translation machinery to direct the initiation of viral protein synthesis. Within hepatitis C virus (HCV)-like IRES elements, the sub-domain IIId(1) is crucial for recruiting the 40S ribosomal subunit. However, some HCV-like IRES elements possess an additional sub-domain, termed IIId2, whose function remains unclear. Herein, we show that IIId2 sub-domains from divergent viruses have different functions. The IIId2 sub-domain present in Seneca valley virus (SVV), a picornavirus, is dispensable for IRES activity, while the IIId2 sub-domains of two pestiviruses, classical swine fever virus (CSFV) and border disease virus (BDV), are required for 80S ribosomes assembly and IRES activity. Unlike in SVV, the deletion of IIId2 from the CSFV and BDV IRES elements impairs initiation of translation by inhibiting the assembly of 80S ribosomes. Consequently, this negatively affects the replication of CSFV and BDV. Finally, we show that the SVV IIId2 sub-domain is required for efficient viral RNA synthesis and growth of SVV, but not for IRES function. This study sheds light on the molecular evolution of viruses by clearly demonstrating that conserved RNA structures, within distantly related RNA viruses, have acquired different roles in the virus life cycles. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Off-pathway assembly of fimbria subunits is prevented by chaperone CfaA of CFA/I fimbriae from enterotoxigenic E. coli.

    Science.gov (United States)

    Bao, Rui; Liu, Yang; Savarino, Stephen J; Xia, Di

    2016-12-01

    The assembly of the class 5 colonization factor antigen I (CFA/I) fimbriae of enterotoxigenic E. coli was proposed to proceed via the alternate chaperone-usher pathway. Here, we show that in the absence of the chaperone CfaA, CfaB, the major pilin subunit of CFA/I fimbriae, is able to spontaneously refold and polymerize into cyclic trimers. CfaA kinetically traps CfaB to form a metastable complex that can be stabilized by mutations. Crystal structure of the stabilized complex reveals distinctive interactions provided by CfaA to trap CfaB in an assembly competent state through donor-strand complementation (DSC) and cleft-mediated anchorage. Mutagenesis indicated that DSC controls the stability of the chaperone-subunit complex and the cleft-mediated anchorage of the subunit C-terminus additionally assist in subunit refolding. Surprisingly, over-stabilization of the chaperone-subunit complex led to delayed fimbria assembly, whereas destabilizing the complex resulted in no fimbriation. Thus, CfaA acts predominantly as a kinetic trap by stabilizing subunit to avoid its off-pathway self-polymerization that results in energetically favorable trimers and could serve as a driving force for CFA/I pilus assembly, representing an energetic landscape unique to class 5 fimbria assembly. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Molecular Microbiology published by John Wiley & Sons Ltd.

  10. Translational control of ribosomal protein S15.

    Science.gov (United States)

    Portier, C; Philippe, C; Dondon, L; Grunberg-Manago, M; Ebel, J P; Ehresmann, B; Ehresmann, C

    1990-08-27

    The expression of ribosomal protein S15 is shown to be translationally and negatively autocontrolled using a fusion within a reporter gene. Isolation and characterization of several deregulated mutants indicate that the regulatory site (the translational operator site) overlaps the ribosome loading site of the S15 messenger. In this region, three domains, each exhibiting a stem-loop structure, were determined using chemical and enzymatic probes. The most downstream hairpin carries the Shine-Dalgarno sequence and the initiation codon. Genetic and structural data derived from mutants constructed by site-directed mutagenesis show that the operator is a dynamic structure, two domains of which can form a pseudoknot. Binding of S15 to these two domains suggests that the pseudoknot could be stabilized by S15. A model is presented in which two alternative structures would explain the molecular basis of the S15 autocontrol.

  11. RPG: the Ribosomal Protein Gene database.

    Science.gov (United States)

    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 information about orthologs. In addition, users can view and compare the gene structures of the above organisms and make multiple amino acid sequence alignments. RPG also provides information on small nucleolar RNAs (snoRNAs) that are encoded in the introns of RP genes.

  12. Pleiotropic Antibiotic Resistance Mutations Associated with Ribosomes and Ribosomal Subunits in Mycobacterium smegmatis

    Science.gov (United States)

    Yamada, Takeshi; Masuda, Kunitsugu; Shoji, Ko; Hori, Mitsuo

    1974-01-01

    Viomycin-resistant strains isolated from Mycobacterium smegmatis demonstrated pleiotropic resistance to tuberactinomycin-N, capreomycin, streptomycin, and kanamycin as a result of mutational alteration of ribosomes, even though they were selected for resistance to a single antibiotic. The pleiotropic drug resistance of three mutants isolated by stepwise selection for resistance to viomycin was due to alteration of the 30S ribosomal subunit. One mutant, strain A, isolated independently by multiple-step selection to viomycin resistance, was resistant to viomycin, tuberactinomycin-N, and capreomycin through an alteration of the 50S ribosomal subunit, whereas it was sensitive to kanamycin but resistant to streptomycin through an alteration of the 30S ribosomal subunit. Three streptomycin-resistant strains, which were isolated by one-step selection at a high concentration of streptomycin, did not show significant co-resistance to any other antibiotics tested in culture and cell-free systems; streptomycin resistance in these mutants was localized on the 30S ribosomal subunit. PMID:15828170

  13. Fuel assembly reconstitution

    International Nuclear Information System (INIS)

    Morgado, Mario M.; Oliveira, Monica G.N.; Ferreira Junior, Decio B.M.; Santos, Barbara O. dos; Santos, Jorge E. dos

    2009-01-01

    Fuel failures have been happened in Nuclear Power Plants worldwide, without lost of integrity and safety, mainly for the public, environment and power plants workers. The most common causes of these events are corrosion (CRUD), fretting and pellet cladding interaction. These failures are identified by increasing the activity of fission products, verified by chemical analyses of reactor coolant. Through these analyses, during the fourth operation cycle of Angra 2 Nuclear Power Plant, was possible to observe fuel failure indication. This indication was confirmed in the end of the cycle during the unloading of reactor core through leakage tests of fuel assembly, using the equipment called 'In Mast Sipping' and 'Box Sipping'. After confirmed, the fuel assembly reconstitution was scheduled, and happened in April, 2007, where was identified the cause and the fuel rod failure, which was substitute by dummy rods (zircaloy). The cause was fretting by 'debris'. The actions to avoid and prevent fuel assemblies failures are important. The goals of this work are to describe the methodology of fuel assembly reconstitution using the FARE (Fuel Assembly Reconstitution Equipment) system, to describe the results of this task in economic and security factors of the company and show how the fuel assembly failures are identified during operation and during the outage. (author)

  14. Crystal structure of eukaryotic ribosome and its complexes with inhibitors

    Science.gov (United States)

    Yusupova, Gulnara; Yusupov, Marat

    2017-01-01

    A high-resolution structure of the eukaryotic ribosome has been determined and has led to increased interest in studying protein biosynthesis and regulation of biosynthesis in cells. The functional complexes of the ribosome crystals obtained from bacteria and yeast have permitted researchers to identify the precise residue positions in different states of ribosome function. This knowledge, together with electron microscopy studies, enhances our understanding of how basic ribosome processes, including mRNA decoding, peptide bond formation, mRNA, and tRNA translocation and cotranslational transport of the nascent peptide, are regulated. In this review, we discuss the crystal structure of the entire 80S ribosome from yeast, which reveals its eukaryotic-specific features, and application of X-ray crystallography of the 80S ribosome for investigation of the binding mode for distinct compounds known to inhibit or modulate the protein-translation function of the ribosome. We also refer to a challenging aspect of the structural study of ribosomes, from higher eukaryotes, where the structures of major distinctive features of higher eukaryote ribosome—the high-eukaryote–specific long ribosomal RNA segments (about 1MDa)—remain unresolved. Presently, the structures of the major part of these high-eukaryotic expansion ribosomal RNA segments still remain unresolved. This article is part of the themed issue ‘Perspectives on the ribosome’. PMID:28138070

  15. Ribosomal history reveals origins of modern protein synthesis.

    Directory of Open Access Journals (Sweden)

    Ajith Harish

    Full Text Available The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that the ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not link protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal evolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in morphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in hundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than the catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved, starting with interactions between the oldest proteins (S12 and S17 and the oldest substructure (the ribosomal ratchet in the small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components show similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our study therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of molecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA structures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were recruited and could be relics of an ancient ribonucleoprotein world.

  16. Fuel assembly in a reactor

    International Nuclear Information System (INIS)

    Saito, Shozo; Kawahara, Akira.

    1975-01-01

    Object: To provide a fuel assembly in a reactor which can effectively prevent damage of the clad tube caused by mutual interference between pellets and the clad tube. Structure: A clad tube for a fuel element, which is located in the outer peripheral portion, among the fuel elements constituting fuel assemblies arranged in assembled and lattice fashion within a channel box, is increased in thickness by reducing the inside diameter thereof to be smaller than that of fuel elements internally located, thereby preventing damage of the clad tube resulting from rapid rise in output produced when control rods are removed. (Kamimura, M.)

  17. Neutron detector assembly

    International Nuclear Information System (INIS)

    Hanai, Koi; Shirayama, Shinpei.

    1978-01-01

    Purpose: To prevent gamma-ray from leaking externally passing through the inside of a neutron detector assembly. Constitution: In a neutron detector assembly having a protection pipe formed with an enlarged diameter portion which serves also as a spacer, partition plates with predetermined width are disposed at the upper and the lower portions in this expanded portion. A lot of metal particles are filled into spaces formed by the partition plates. In such a structure, the metal particles well-absorb the gamma-rays from above and convert them into heat to provide shielding for the gamma-rays. (Horiuchi, T.)

  18. Evolutionary Conservation of the Ribosomal Biogenesis Factor Rbm19/Mrd1 : Implications for Function

    OpenAIRE

    Kallberg, Yvonne; Segerstolpe, Åsa; Lackman, Fredrik; Persson, Bengt; Wieslander, Lars

    2012-01-01

    Ribosome biogenesis in eukaryotes requires coordinated folding and assembly of a pre-rRNA into sequential pre-rRNA-protein complexes in which chemical modifications and RNA cleavages occur. These processes require many small nucleolar RNAs (snoRNAs) and proteins. Rbm19/Mrd1 is one such protein that is built from multiple RNA-binding domains (RBDs). We find that Rbm19/Mrd1 with five RBDs is present in all branches of the eukaryotic phylogenetic tree, except in animals and Choanoflagellates, th...

  19. A ribosomal ATPase is a target for hygromycin B inhibition on Escherichia coli ribosomes.

    Science.gov (United States)

    Ganoza, M C; Kiel, M C

    2001-10-01

    We demonstrate that the transfer of fully charged aminoacyl-tRNAs into peptides directed by the MS2 RNA template requires both ATP and GTP, initiation factors (IF1, IF2, and IF3), elongation factors (EF-Tu, EF-Ts, and EF-G), and the ribosomal ATPase (RbbA). The nonhydrolyzable analogue AMPPCP inhibits the reactions, suggesting that hydrolysis of ATP is required for synthesis. The RbbA protein occurs bound to ribosomes and stimulates the ATPase activity of Escherichia coli 70S and 30S particles. The gene encoding RbbA harbors four ATP binding domains; the C-terminal half of the protein bears extensive sequence similarity to EF-3, a ribosome-dependent ATPase. Here, we show that the antibiotic hygromycin B selectively inhibits the ATPase activity of RbbA. Other antibiotics with similar effects on miscoding, streptomycin and neomycin, as well as antibiotics that impair peptide bond synthesis and translocation, had little effect on the ATPase activity of RbbA on 70S ribosomes. Immunoblot analysis indicates that at physiological concentrations, hygromycin B selectively releases RbbA from 70S ribosomes. Hygromycin B protects G1494 and A1408 in the decoding region, and RbbA enhances the reactivity of A889 and G890 of the 16S rRNA switch helix region. Cross-linking and X-ray diffraction data have revealed that this helix switch and the decoding region are in close proximity. Mutations in the switch helix (889-890) region affect translational fidelity and translocation. The binding site of hygromycin B and its known dual effect on the fidelity of decoding and translocation suggest a model for the action of this drug on ribosomes.

  20. 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.

  1. Macrolide antibiotics allosterically predispose the ribosome for translation arrest.

    Science.gov (United States)

    Sothiselvam, Shanmugapriya; Liu, Bo; Han, Wei; Ramu, Haripriya; Klepacki, Dorota; Atkinson, Gemma Catherine; Brauer, Age; Remm, Maido; Tenson, Tanel; Schulten, Klaus; Vázquez-Laslop, Nora; Mankin, Alexander S

    2014-07-08

    Translation arrest directed by nascent peptides and small cofactors controls expression of important bacterial and eukaryotic genes, including antibiotic resistance genes, activated by binding of macrolide drugs to the ribosome. Previous studies suggested that specific interactions between the nascent peptide and the antibiotic in the ribosomal exit tunnel play a central role in triggering ribosome stalling. However, here we show that macrolides arrest translation of the truncated ErmDL regulatory peptide when the nascent chain is only three amino acids and therefore is too short to be juxtaposed with the antibiotic. Biochemical probing and molecular dynamics simulations of erythromycin-bound ribosomes showed that the antibiotic in the tunnel allosterically alters the properties of the catalytic center, thereby predisposing the ribosome for halting translation of specific sequences. Our findings offer a new view on the role of small cofactors in the mechanism of translation arrest and reveal an allosteric link between the tunnel and the catalytic center of the ribosome.

  2. Architecture of the large subunit of the mammalian mitochondrial ribosome.

    Science.gov (United States)

    Greber, Basil J; Boehringer, Daniel; Leitner, Alexander; Bieri, Philipp; Voigts-Hoffmann, Felix; Erzberger, Jan P; Leibundgut, Marc; Aebersold, Ruedi; Ban, Nenad

    2014-01-23

    Mitochondrial ribosomes synthesize a number of highly hydrophobic proteins encoded on the genome of mitochondria, the organelles in eukaryotic cells that are responsible for energy conversion by oxidative phosphorylation. The ribosomes in mammalian mitochondria have undergone massive structural changes throughout their evolution, including ribosomal RNA shortening and acquisition of mitochondria-specific ribosomal proteins. Here we present the three-dimensional structure of the 39S large subunit of the porcine mitochondrial ribosome determined by cryo-electron microscopy at 4.9 Å resolution. The structure, combined with data from chemical crosslinking and mass spectrometry experiments, reveals the unique features of the 39S subunit at near-atomic resolution and provides detailed insight into the architecture of the polypeptide exit site. This region of the mitochondrial ribosome has been considerably remodelled compared to its bacterial counterpart, providing a specialized platform for the synthesis and membrane insertion of the highly hydrophobic protein components of the respiratory chain.

  3. The CRM domain: An RNA binding module derived from an ancient ribosome-associated protein

    Science.gov (United States)

    Barkan, Alice; Klipcan, Larik; Ostersetzer, Oren; Kawamura, Tetsuya; Asakura, Yukari; Watkins, Kenneth P.

    2007-01-01

    The CRS1–YhbY domain (also called the CRM domain) is represented as a stand-alone protein in Archaea and Bacteria, and in a family of single- and multidomain proteins in plants. The function of this domain is unknown, but structural data and the presence of the domain in several proteins known to interact with RNA have led to the proposal that it binds RNA. Here we describe a phylogenetic analysis of the domain, its incorporation into diverse proteins in plants, and biochemical properties of a prokaryotic and eukaryotic representative of the domain family. We show that a bacterial member of the family, Escherichia coli YhbY, is associated with pre-50S ribosomal subunits, suggesting that YhbY functions in ribosome assembly. GFP fused to a single-domain CRM protein from maize localizes to the nucleolus, suggesting that an analogous activity may have been retained in plants. We show further that an isolated maize CRM domain has RNA binding activity in vitro, and that a small motif shared with KH RNA binding domains, a conserved “GxxG” loop, contributes to its RNA binding activity. These and other results suggest that the CRM domain evolved in the context of ribosome function prior to the divergence of Archaea and Bacteria, that this function has been maintained in extant prokaryotes, and that the domain was recruited to serve as an RNA binding module during the evolution of plant genomes. PMID:17105995

  4. The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein.

    Science.gov (United States)

    Barkan, Alice; Klipcan, Larik; Ostersetzer, Oren; Kawamura, Tetsuya; Asakura, Yukari; Watkins, Kenneth P

    2007-01-01

    The CRS1-YhbY domain (also called the CRM domain) is represented as a stand-alone protein in Archaea and Bacteria, and in a family of single- and multidomain proteins in plants. The function of this domain is unknown, but structural data and the presence of the domain in several proteins known to interact with RNA have led to the proposal that it binds RNA. Here we describe a phylogenetic analysis of the domain, its incorporation into diverse proteins in plants, and biochemical properties of a prokaryotic and eukaryotic representative of the domain family. We show that a bacterial member of the family, Escherichia coli YhbY, is associated with pre-50S ribosomal subunits, suggesting that YhbY functions in ribosome assembly. GFP fused to a single-domain CRM protein from maize localizes to the nucleolus, suggesting that an analogous activity may have been retained in plants. We show further that an isolated maize CRM domain has RNA binding activity in vitro, and that a small motif shared with KH RNA binding domains, a conserved "GxxG" loop, contributes to its RNA binding activity. These and other results suggest that the CRM domain evolved in the context of ribosome function prior to the divergence of Archaea and Bacteria, that this function has been maintained in extant prokaryotes, and that the domain was recruited to serve as an RNA binding module during the evolution of plant genomes.

  5. Ribosomal DNA polymorphisms in the yeast Geotrichum candidum.

    Science.gov (United States)

    Alper, Iraz; Frenette, Michel; Labrie, Steve

    2011-12-01

    The dimorphic yeast Geotrichum candidum (teleomorph: Galactomyces candidus) is commonly used to inoculate washed-rind and bloomy-rind cheeses. However, little is known about the phylogenetic lineage of this microorganism. We have sequenced the complete 18S, 5.8S, 26S ribosomal RNA genes and their internal transcribed spacers (ITS1) and ITS2 regions (5126 nucleotides) from 18 G. candidum strains from various environmental niches, with a focus on dairy strains. Multiple sequence alignments revealed the presence of 60 polymorphic sites, which is generally unusual for ribosomal DNA (rDNA) within a given species because of the concerted evolution mechanism. This mechanism drives genetic homogenization to prevent the divergent evolution of rDNA copies within individuals. While the polymorphisms observed were mainly substitutions, one insertion/deletion (indel) polymorphism was detected in ITS1. No polymorphic sites were detected downstream from this indel site, that is, in 5.8S and ITS2. More surprisingly, many sequence electrophoregrams generated during the sequencing of the rDNA had dual peaks, suggesting that many individuals exhibited intragenomic rDNA variability. The ITS1-5.8S-ITS2 regions of four strains were cloned. The sequence analysis of 68 clones revealed 32 different ITS1-5.8S-ITS2 variants within these four strains. Depending on the strain, from four to twelve variants were detected, indicating that multiple rDNA copies were present in the genomes of these G. candidum strains. These results contribute to the debate concerning the use of the ITS region for barcoding fungi and suggest that community profiling techniques based on rDNA should be used with caution. Copyright © 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  6. The architecture of mammalian ribosomal protein promoters

    Directory of Open Access Journals (Sweden)

    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.

  7. The extent of ribosome queuing in budding yeast.

    Science.gov (United States)

    Diament, Alon; Feldman, Anna; Schochet, Elisheva; Kupiec, Martin; Arava, Yoav; Tuller, Tamir

    2018-01-01

    Ribosome queuing is a fundamental phenomenon suggested to be related to topics such as genome evolution, synthetic biology, gene expression regulation, intracellular biophysics, and more. However, this phenomenon hasn't been quantified yet at a genomic level. Nevertheless, methodologies for studying translation (e.g. ribosome footprints) are usually calibrated to capture only single ribosome protected footprints (mRPFs) and thus limited in their ability to detect ribosome queuing. On the other hand, most of the models in the field assume and analyze a certain level of queuing. Here we present an experimental-computational approach for studying ribosome queuing based on sequencing of RNA footprints extracted from pairs of ribosomes (dRPFs) using a modified ribosome profiling protocol. We combine our approach with traditional ribosome profiling to generate a detailed profile of ribosome traffic. The data are analyzed using computational models of translation dynamics. The approach was implemented on the Saccharomyces cerevisiae transcriptome. Our data shows that ribosome queuing is more frequent than previously thought: the measured ratio of ribosomes within dRPFs to mRPFs is 0.2-0.35, suggesting that at least one to five translating ribosomes is in a traffic jam; these queued ribosomes cannot be captured by traditional methods. We found that specific regions are enriched with queued ribosomes, such as the 5'-end of ORFs, and regions upstream to mRPF peaks, among others. While queuing is related to higher density of ribosomes on the transcript (characteristic of highly translated genes), we report cases where traffic jams are relatively more severe in lowly expressed genes and possibly even selected for. In addition, our analysis demonstrates that higher adaptation of the coding region to the intracellular tRNA levels is associated with lower queuing levels. Our analysis also suggests that the Saccharomyces cerevisiae transcriptome undergoes selection for eliminating

  8. The extent of ribosome queuing in budding yeast.

    Directory of Open Access Journals (Sweden)

    Alon Diament

    2018-01-01

    Full Text Available Ribosome queuing is a fundamental phenomenon suggested to be related to topics such as genome evolution, synthetic biology, gene expression regulation, intracellular biophysics, and more. However, this phenomenon hasn't been quantified yet at a genomic level. Nevertheless, methodologies for studying translation (e.g. ribosome footprints are usually calibrated to capture only single ribosome protected footprints (mRPFs and thus limited in their ability to detect ribosome queuing. On the other hand, most of the models in the field assume and analyze a certain level of queuing. Here we present an experimental-computational approach for studying ribosome queuing based on sequencing of RNA footprints extracted from pairs of ribosomes (dRPFs using a modified ribosome profiling protocol. We combine our approach with traditional ribosome profiling to generate a detailed profile of ribosome traffic. The data are analyzed using computational models of translation dynamics. The approach was implemented on the Saccharomyces cerevisiae transcriptome. Our data shows that ribosome queuing is more frequent than previously thought: the measured ratio of ribosomes within dRPFs to mRPFs is 0.2-0.35, suggesting that at least one to five translating ribosomes is in a traffic jam; these queued ribosomes cannot be captured by traditional methods. We found that specific regions are enriched with queued ribosomes, such as the 5'-end of ORFs, and regions upstream to mRPF peaks, among others. While queuing is related to higher density of ribosomes on the transcript (characteristic of highly translated genes, we report cases where traffic jams are relatively more severe in lowly expressed genes and possibly even selected for. In addition, our analysis demonstrates that higher adaptation of the coding region to the intracellular tRNA levels is associated with lower queuing levels. Our analysis also suggests that the Saccharomyces cerevisiae transcriptome undergoes selection

  9. Ribosomal Resistance to Streptomycin and Spectinomycin in Neisseria gonorrhoeae

    Science.gov (United States)

    Maness, Michael J.; Foster, Gayle C.; Sparling, P. Frederick

    1974-01-01

    A cell-free protein synthesizing system was used to study the mechanism of resistance to streptomycin (Str) and spectinomycin (Spc) in laboratory mutants and clinical isolates of Neisseria gonorrhoeae. The 70S ribosomes from sensitive strains were sensitive to the effects of Str and Spc on synthesis directed by several synthetic polynucleotide messengers, whereas 70S ribosomes from resistant strains were resistant to these same effects. In each case, the alteration was localized to the 30S ribosomal subunit by studying antibiotic sensitivities of hybrid 70S ribosomes formed by combining subunits from sensitive and resistant strains. No evidence was found for streptomycin- or spectinomycin-inactivating enzymes. PMID:4279906

  10. In Profile: Models of Ribosome Biogenesis Defects and Regulation of Protein Synthesis

    NARCIS (Netherlands)

    Essers, P.B.M.

    2013-01-01

    Ribosomes are the mediators of protein synthesis in the cell and therefore crucial to proper cell function. In addition, ribosomes are highly abundant, with ribosomal RNA making up 80% of the RNA in the cell. A large amount of resources go into maintaining this pool of ribosomes, so ribosome

  11. Molecular dynamics simulation of ribosome jam

    KAUST Repository

    Matsumoto, Shigenori

    2011-09-01

    We propose a coarse-grained molecular dynamics model of ribosome molecules to study the dependence of translation process on environmental parameters. We found the model exhibits traffic jam property, which is consistent with an ASEP model. We estimated the influence of the temperature and concentration of molecules on the hopping probability used in the ASEP model. Our model can also treat environmental effects on the translation process that cannot be explained by such cellular automaton models. © 2010 Elsevier B.V. All rights reserved.

  12. Ribosomal RNA gene functioning in avian oogenesis.

    Science.gov (United States)

    Koshel, Elena; Galkina, Svetlana; Saifitdinova, Alsu; Dyomin, Alexandr; Deryusheva, Svetlana; Gaginskaya, Elena

    2016-12-01

    Despite long-term exploration into ribosomal RNA gene functioning during the oogenesis of various organisms, many intriguing problems remain unsolved. In this review, we describe nucleolus organizer region (NOR) activity in avian oocytes. Whereas oocytes from an adult avian ovary never reveal the formation of the nucleolus in the germinal vesicle (GV), an ovary from juvenile birds possesses both nucleolus-containing and non-nucleolus-containing oocytes. The evolutionary diversity of oocyte NOR functioning and the potential non-rRNA-related functions of the nucleolus in oocytes are also discussed.

  13. Hyper-accurate ribosomes inhibit growth.

    OpenAIRE

    Ruusala, T; Andersson, D; Ehrenberg, M; Kurland, C G

    1984-01-01

    We have compared both in vivo and in vitro translation by ribosomes from wild-type bacteria with those from streptomycin-resistant (SmR), streptomycin-dependent (SmD) and streptomycin-pseudo-dependent (SmP) mutants. The three mutant bacteria translate more accurately and more slowly in the absence of streptomycin (Sm) than do wild-type bacteria. In particular, the SmP bacteria grow at roughly half the rate of the wild-type in the absence of Sm. The antibiotic stimulates both the growth rate a...

  14. A Molecular Titration System Coordinates Ribosomal Protein Gene Transcription with Ribosomal RNA Synthesis.

    Science.gov (United States)

    Albert, Benjamin; Knight, Britta; Merwin, Jason; Martin, Victoria; Ottoz, Diana; Gloor, Yvonne; Bruzzone, Maria Jessica; Rudner, Adam; Shore, David

    2016-11-17

    Cell growth potential is determined by the rate of ribosome biogenesis, a complex process that requires massive and coordinated transcriptional output. In the yeast Saccharomyces cerevisiae, ribosome biogenesis is highly regulated at the transcriptional level. Although evidence for a system that coordinates ribosomal RNA (rRNA) and ribosomal protein gene (RPG) transcription has been described, the molecular mechanisms remain poorly understood. Here we show that an interaction between the RPG transcriptional activator Ifh1 and the rRNA processing factor Utp22 serves to coordinate RPG transcription with that of rRNA. We demonstrate that Ifh1 is rapidly released from RPG promoters by a Utp22-independent mechanism following growth inhibition, but that its long-term dissociation requires Utp22. We present evidence that RNA polymerase I activity inhibits the ability of Utp22 to titrate Ifh1 from RPG promoters and propose that a dynamic Ifh1-Utp22 interaction fine-tunes RPG expression to coordinate RPG and rRNA transcription. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. 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...

  16. Phosphorylation of ribosomal protein S6 mediates compensatory renal hypertrophy

    Science.gov (United States)

    Xu, Jinxian; Chen, Jianchun; Dong, Zheng; Meyuhas, Oded; Chen, Jian-Kang

    2014-01-01

    The molecular mechanism underlying renal hypertrophy and progressive nephron damage remains poorly understood. Here we generated congenic ribosomal protein S6 (rpS6) knockin mice expressing non-phosphorylatable rpS6 and found that uninephrectomy-induced renal hypertrophy was significantly blunted in these knockin mice. Uninephrectomy-induced increases in cyclin D1 and decreases in cyclin E in the remaining kidney were attenuated in the knockin mice compared to their wild-type littermates. Uninephrectomy induced rpS6 phosphorylation in the wild type mice; however, no rpS6 phosphorylation was detected in uninephrectomized or sham-operated knockin mice. Nonetheless, uninephrectomy stimulated comparable 4E-BP1 phosphorylation in both knockin and wild type mice, indicating that mTORC1 was still activated in the knockin mice. Moreover, the mTORC1 inhibitor rapamycin prevented both rpS6 and 4E-BP1 phosphorylation, significantly blunted uninephrectomy-induced renal hypertrophy in wild type mice, but did not prevent residual renal hypertrophy despite inhibiting 4E-BP1 phosphorylation in uninephrectomized knockin mice. Thus, both genetic and pharmacological approaches unequivocally demonstrate that phosphorylated rpS6 is a downstream effector of the mTORC1-S6K1 signaling pathway mediating renal hypertrophy. Hence, rpS6 phosphorylation facilitates the increase in cyclin D1 and decrease in cyclin E1 that underlie the hypertrophic nature of uninephrectomy-induced kidney growth. PMID:25229342

  17. DksA guards elongating RNA polymerase against ribosome-stalling-induced arrest.

    Science.gov (United States)

    Zhang, Yan; Mooney, Rachel A; Grass, Jeffrey A; Sivaramakrishnan, Priya; Herman, Christophe; Landick, Robert; Wang, Jue D

    2014-03-06

    In bacteria, translation-transcription coupling inhibits RNA polymerase (RNAP) stalling. We present evidence suggesting that, upon amino acid starvation, inactive ribosomes promote rather than inhibit RNAP stalling. We developed an algorithm to evaluate genome-wide polymerase progression independently of local noise and used it to reveal that the transcription factor DksA inhibits promoter-proximal pausing and increases RNAP elongation when uncoupled from translation by depletion of charged tRNAs. DksA has minimal effect on RNAP elongation in vitro and on untranslated RNAs in vivo. In these cases, transcripts can form RNA structures that prevent backtracking. Thus, the effect of DksA on transcript elongation may occur primarily upon ribosome slowing/stalling or at promoter-proximal locations that limit the potential for RNA structure. We propose that inactive ribosomes prevent formation of backtrack-blocking mRNA structures and that, in this circumstance, DksA acts as a transcription elongation factor in vivo. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Fuel assembly

    International Nuclear Information System (INIS)

    Abe, Hideaki; Sakai, Takao; Ishida, Tomio; Yokota, Norikatsu.

    1992-01-01

    The lower ends of a plurality of plate-like shape memory alloys are secured at the periphery of the upper inside of the handling head of a fuel assembly. As the shape memory alloy, a Cu-Zn alloy, a Ti-Pd alloy or a Fe-Ni alloy is used. When high temperature coolants flow out to the handling head, the shape memory alloy deforms by warping to the outer side more greatly toward the upper portion thereof with the temperature increase of the coolants. As the result, the shape of the flow channel of the coolants is changed so as to enlarge at the exit of the upper end of the fuel assembly. Then, the pressure loss of the coolants in the fuel assembly is decreased by the enlargement. Accordingly, the flow rate of the coolants in the fuel assembly is increased to lower the temperature of the coolants. Further, high temperature coolants and low temperature coolants are mixed sufficiently just above the fuel assembly. This can suppress the temperature fluctuation of the mixed coolants in the upper portion of the reactor core, thereby enabling to decrease a fatigue and failures of the structural components in the upper portion of the reactor core. (I.N.)

  19. Deciphering the role of the Gag-Pol ribosomal frameshift signal in HIV-1 RNA genome packaging.

    Science.gov (United States)

    Nikolaitchik, Olga A; Hu, Wei-Shau

    2014-04-01

    A key step of retroviral replication is packaging of the viral RNA genome during virus assembly. Specific packaging is mediated by interactions between the viral protein Gag and elements in the viral RNA genome. In HIV-1, similar to most retroviruses, the packaging signal is located within the 5' untranslated region and extends into the gag-coding region. A recent study reported that a region including the Gag-Pol ribosomal frameshift signal plays an important role in HIV-1 RNA packaging; deletions or mutations that affect the RNA structure of this signal lead to drastic decreases (10- to 50-fold) in viral RNA packaging and virus titer. We examined here the role of the ribosomal frameshift signal in HIV-1 RNA packaging by studying the RNA packaging and virus titer in the context of proviruses. Three mutants with altered ribosomal frameshift signal, either through direct deletion of the signal, mutation of the 6U slippery sequence, or alterations of the secondary structure were examined. We found that RNAs from all three mutants were packaged efficiently, and they generate titers similar to that of a virus containing the wild-type ribosomal frameshift signal. We conclude that although the ribosomal frameshift signal plays an important role in regulating the replication cycle, this RNA element is not directly involved in regulating RNA encapsidation. To generate infectious viruses, HIV-1 must package viral RNA genome during virus assembly. The specific HIV-1 genome packaging is mediated by interactions between the structural protein Gag and elements near the 5' end of the viral RNA known as packaging signal. In this study, we examined whether the Gag-Pol ribosomal frameshift signal is important for HIV-1 RNA packaging as recently reported. Our results demonstrated that when Gag/Gag-Pol is supplied in trans, none of the tested ribosomal frameshift signal mutants has defects in RNA packaging or virus titer. These studies provide important information on how HIV-1

  20. Cloning and expression of antiviral/ribosome-inactivating protein ...

    Indian Academy of Sciences (India)

    Madhu urs

    2007-12-16

    Dec 16, 2007 ... The cleaved and purified recombinant. BBAP1 exhibited ribosome-inhibiting rRNA N-glycosidase activity, and imparted a high level of resistance against the tobacco mosaic virus (TMV). [Choudhary N, Kapoor H C and Lodha M L 2008 Cloning and expression of antiviral/ribosome-inactivating protein from ...

  1. The characterization of cytoplasmic ribosomal protein genes in ...

    African Journals Online (AJOL)

    The novel arrangements of 'AAATTT-like signal – CCC/GGG-like motif – transcription start site' are present in the upstream sequences of ribosomal protein genes, and several regulatory elements that may have synergy with introns of ribosomal protein genes for its high transcriptional frequency were detected too.

  2. Mechanism of recycling of post-termination ribosomal complexes in ...

    Indian Academy of Sciences (India)

    Madhu

    all pathway of ribosome recycling in eubacteria with especial reference to the important role of the initiation factor ... [Seshadri A and Varshney U 2006 Mechanism of recycling of post-termination ribosomal complexes in eubacteria: a new role of initiation factor 3 .... RRF binding results in a remarkable conformational change.

  3. Cloning and expression of antiviral/ribosome-inactivating protein ...

    Indian Academy of Sciences (India)

    Madhu urs

    2007-12-16

    Dec 16, 2007 ... Many higher plant species belonging to various taxonomic families are known to produce endogenous, non-stress induced inhibitor proteins called antiviral proteins (AVPs). Many of these AVPs have ribosome-inhibiting rRNA N- glycosidase activity and are known as ribosome-inactivating proteins (RIPs).

  4. Close sequence identity between ribosomal DNA episomes of the ...

    Indian Academy of Sciences (India)

    Unknown

    Entamoeba dispar and Entamoeba histolytica are now recognized as two distinct species – the former being nonpathogenic to humans. We had earlier studied the organization of ribosomal RNA genes in E. histolytica. Here we report the analysis of ribosomal RNA genes in E. dispar. The rRNA genes of E. dispar, like their ...

  5. Association of ribosomal anti-P antibodies with different parameters ...

    African Journals Online (AJOL)

    antibodies with neuropsychiatric lupus manifestations and to find out the relationship of ribosomal anti-P antibodies with other autoimmune parameters of lupus. Ribosomal anti-P antibodies were evaluated in the serum of 41 systemic lupus erythematosus (SLE) patients as well as ANA, dsDNA, anti- Sm, anti-SSA, anti-SSB, ...

  6. Ribosomal resistance of clinical enterococcal to streptomycin isolates.

    OpenAIRE

    Eliopoulos, G M; Farber, B F; Murray, B E; Wennersten, C; Moellering, R C

    1984-01-01

    The mechanism of high-level resistance to streptomycin was studied in 12 clinical isolates of Streptococcus faecalis. Six strains produced streptomycin-modifying enzymes. Each of three enzyme-negative strains tested demonstrated ribosomal resistance to streptomycin. Lack of ribosomal susceptibility is a significant cause of high-level streptomycin resistance among clinical enterococcal isolates.

  7. The Complete Structure of the Mycobacterium smegmatis 70S Ribosome

    Directory of Open Access Journals (Sweden)

    Jendrik Hentschel

    2017-07-01

    Full Text Available The ribosome carries out the synthesis of proteins in every living cell. It consequently represents a frontline target in anti-microbial therapy. Tuberculosis ranks among the leading causes of death worldwide, due in large part to the combination of difficult-to-treat latency and antibiotic resistance. Here, we present the 3.3-Å cryo-EM structure of the 70S ribosome of Mycobacterium smegmatis, a close relative to the human pathogen Mycobacterium tuberculosis. The structure reveals two additional ribosomal proteins and localizes them to the vicinity of drug-target sites in both the catalytic center and the decoding site of the ribosome. Furthermore, we visualized actinobacterium-specific rRNA and protein expansions that extensively remodel the ribosomal surface with implications for polysome organization. Our results provide a foundation for understanding the idiosyncrasies of mycobacterial translation and reveal atomic details of the structure that will facilitate the design of anti-tubercular therapeutics.

  8. Fuel assemblies

    International Nuclear Information System (INIS)

    Nakamura, Mitsuya; Yamashita, Jun-ichi; Mochida, Takaaki.

    1986-01-01

    Purpose: To improve the fuel economy by increasing the reactivity at the latter burning stage of fuel assemblies and thereby increasing the burn-up degree. Constitution: At the later stage of the burning where the infinite multiplication factor of a fuel assembly is lowered, fuel rods are partially discharged to increase the fuel-moderator volume ratio in the fuel assembly. Then, plutonium is positively burnt by bringing the ratio near to an optimum point where the infinite multiplication factor becomes maximum and the reactivity of the fuel assembly is increased by utilizing the spectral shift effect. The number of the fuel rods to be removed is selected so as to approach the fuel-moderator atom number ratio where the infinite multiplication factor is maximum. Further, the positions where the thermal neutron fluxes are low are most effective for removing the rods and those positions between which no fuel rods are present and which are adjacent with neither the channel box nor the water rods are preferred. The rods should be removed at the time when the burning is proceeded at lest for one cycle. The reactivity is thus increased and the burn-up degree of fuels upon taking-out can be improved. (Kamimura, M.)

  9. 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. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  10. Crystal Structures of the uL3 Mutant Ribosome: Illustration of the Importance of Ribosomal Proteins for Translation Efficiency

    Science.gov (United States)

    Mailliot, Justine; de Loubresse, Nicolas Garreau; Yusupova, Gulnara; Meskauskas, Arturas; Dinman, Jonathan D.; Yusupov, Marat

    2017-01-01

    The ribosome has been described as a ribozyme in which ribosomal RNA is responsible for peptidyl-transferase reaction catalysis. The W255C mutation of the universally conserved ribosomal protein uL3 has diverse effects on ribosome function (e.g., increased affinities for transfer RNAs, decreased rates of peptidyl-transfer), and cells harboring this mutation are resistant to peptidyl-transferase inhibitors (e.g., anisomycin). These observations beg the question of how a single amino acid mutation may have such wide ranging consequences. Here, we report the structure of the vacant yeast uL3 W255C mutant ribosome by X-ray crystallography, showing a disruption of the A-site side of the peptidyl-transferase center (PTC). An additional X-ray crystallographic structure of the anisomycin-containing mutant ribosome shows that high concentrations of this inhibitor restore a “WT-like” configuration to this region of the PTC, providing insight into the resistance mechanism of the mutant. Globally, our data demonstrate that ribosomal protein uL3 is structurally essential to ensure an optimal and catalytically efficient organization of the PTC, highlighting the importance of proteins in the RNA-centered ribosome. PMID:26906928

  11. 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 polyacryl...... polyacrylamide gel analysis of the purified acidic proteins and 80-S particles showed identical phosphoproteins in the 16 000 dalton region....

  12. Fuel assembly for a nuclear reactor

    International Nuclear Information System (INIS)

    Gjertsen, R.K.

    1982-01-01

    A fuel assembly in a nuclear reactor comprises a locking mechanism that is capable of locking the fuel assembly to the core plate of a nuclear reactor to prevent inadvertent movement of the fuel assembly. The locking mechanism comprises a ratchet mechanism 108 that allows the fuel assembly to be easily locked to the core plate but prevents unlocking except when the ratchet is disengaged. The ratchet mechanism is coupled to the locking mechanism by a rotatable guide tube for a control rod or water displacer rod. (author)

  13. Fuel assemblies

    International Nuclear Information System (INIS)

    Echigoya, Hironori; Nomata, Terumitsu.

    1983-01-01

    Purpose: To render the axial distribution relatively flat. Constitution: First nuclear element comprises a fuel can made of zircalloy i.e., the metal with less neutron absorption, which is filled with a plurality of UO 2 pellets and sealed by using a lower end plug, a plenum spring and an upper end plug by means of welding. Second fuel element is formed by substituting a part of the UO 2 pellets with a water tube which is sealed with water and has a space for allowing the heat expansion. The nuclear fuel assembly is constituted by using the first and second fuel elements together. In such a structure, since water reflects neutrons and decrease their leakage to increase the temperature, reactivity is added at the upper portion of the fuel assembly to thereby flatten the axial power distribution. Accordingly, stable operation is possible only by means of deep control rods while requiring no shallow control rods. (Sekiya, K.)

  14. Assembling consumption

    DEFF Research Database (Denmark)

    Assembling Consumption marks a definitive step in the institutionalisation of qualitative business research. By gathering leading scholars and educators who study markets, marketing and consumption through the lenses of philosophy, sociology and anthropology, this book clarifies and applies...... the investigative tools offered by assemblage theory, actor-network theory and non-representational theory. Clear theoretical explanation and methodological innovation, alongside empirical applications of these emerging frameworks will offer readers new and refreshing perspectives on consumer culture and market...

  15. Molecular paleontology: a biochemical model of the ancestral ribosome.

    Science.gov (United States)

    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; Petrov, Anton S; Wartell, Roger M; Harvey, Stephen C; Hud, Nicholas V; Williams, Loren Dean

    2013-03-01

    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 experiments support formation of predicted secondary and tertiary structure. Gel shift, spectroscopic and yeast three-hybrid assays show specific interactions between ancestral rRNA and ribosomal protein fragments, independent of other, more recent, components of the ribosome. This robustness suggests that the catalytic core of the ribosome is an ancient construct that has survived billions of years of evolution without major changes in structure. Collectively, the data here support a model in which ancestors of the large and small subunits originated and evolved independently of each other, with autonomous functionalities.

  16. On the control of ribosomal protein biosynthesis in Escherichia coli

    International Nuclear Information System (INIS)

    Pichon, J.; Marvaldi, J.; Coeroli, C.; Cozzone, A.; Marchis-Mouren, G.

    1977-01-01

    The rate of individual ribosomal protein synthesis relative to total protein synthesis has been determined in Escherichia coli rel + and rel - cells, under valyl-tRNA deprivation. These strains have a temperature-sensitive valyl-tRNA synthetase. Starvation was obtained following transfer of the cells to non-permissive temperature. Ribosomal proteins were obtained by treatment of either total lysates of freeze-thawed lysozyme spheroplasts or ammonium sulphate precipitate of ribosomes, with acetic acid. Differential labelling of the ribosomal proteins was observed in both strains: proteins from the rel + strain appear more labelled than those from the rel - strain, the rate of labelling of individual proteins being about the same in both strains. Moreover ribosomal proteins were found as stable during starvation as total protein. It is thus concluded that in starving cells individual ribosomal proteins are not synthesized at equal rates. This indicates that the synthesis of ribosomal proteins is not only under the control of the rel gene

  17. General Assembly

    CERN Multimedia

    Staff Association

    2016-01-01

    5th April, 2016 – Ordinary General Assembly of the Staff Association! In the first semester of each year, the Staff Association (SA) invites its members to attend and participate in the Ordinary General Assembly (OGA). This year the OGA will be held on Tuesday, April 5th 2016 from 11:00 to 12:00 in BE Auditorium, Meyrin (6-2-024). During the Ordinary General Assembly, the activity and financial reports of the SA are presented and submitted for approval to the members. This is the occasion to get a global view on the activities of the SA, its financial management, and an opportunity to express one’s opinion, including taking part in the votes. Other points are listed on the agenda, as proposed by the Staff Council. Who can vote? Only “ordinary” members (MPE) of the SA can vote. Associated members (MPA) of the SA and/or affiliated pensioners have a right to vote on those topics that are of direct interest to them. Who can give his/her opinion? The Ordinary General Asse...

  18. Fuel assembly

    International Nuclear Information System (INIS)

    Ueda, Sei; Ando, Ryohei; Mitsutake, Toru.

    1995-01-01

    The present invention concerns a fuel assembly suitable to a BWR-type reactor and improved especially with the nuclear characteristic, heat performance, hydraulic performance, dismantling or assembling performance and economical property. A part of poison rods are formed as a large-diameter/multi-region poison rods having a larger diameter than a fuel rod. A large number of fuel rods are disposed surrounding a large diameter water rod and a group of the large-diameter/multi-region poison rods in adjacent with the water rod. The large-diameter water rod has a burnable poison at the tube wall portion. At least a portion of the large-diameter poison rods has a coolant circulation portion allowing coolants to circulate therethrough. Since the large-diameter poison rods are disposed at a position of high neutron fluxes, a large neutron multiplication factor suppression effect can be provided, thereby enabling to reduce the number of burnable poison rods relative to fuels. As a result, power peaking in the fuel assembly is moderated and a greater amount of plutonium can be loaded. In addition the flow of cooling water which tends to gather around the large diameter water rod can be controlled to improve cooling performance of fuels. (N.H.)

  19. Phyllanthus emblica Fruit Extract Activates Spindle Assembly Checkpoint, Prevents Mitotic Aberrations and Genomic Instability in Human Colon Epithelial NCM460 Cells.

    Science.gov (United States)

    Guo, Xihan; Wang, Xu

    2016-09-03

    The fruit of Phyllanthus emblica Linn. (PE) has been widely consumed as a functional food and folk medicine in Southeast Asia due to its remarkable nutritional and pharmacological effects. Previous research showed PE delays mitotic progress and increases genomic instability (GIN) in human colorectal cancer cells. This study aimed to investigate the similar effects of PE by the biomarkers related to spindle assembly checkpoint (SAC), mitotic aberrations and GIN in human NCM460 normal colon epithelial cells. Cells were treated with PE and harvested differently according to the biomarkers observed. Frequencies of micronuclei (MN), nucleoplasmic bridge (NPB) and nuclear bud (NB) in cytokinesis-block micronucleus assay were used as indicators of GIN. Mitotic aberrations were assessed by the biomarkers of chromosome misalignment, multipolar division, chromosome lagging and chromatin bridge. SAC activity was determined by anaphase-to- metaphase ratio (AMR) and the expression of core SAC gene budding uninhibited by benzimidazoles related 1 (BubR1). Compared with the control, PE-treated cells showed (1) decreased incidences of MN, NPB and NB (p genomic damages partially by enhancing the function of SAC.

  20. Phyllanthus emblica Fruit Extract Activates Spindle Assembly Checkpoint, Prevents Mitotic Aberrations and Genomic Instability in Human Colon Epithelial NCM460 Cells

    Directory of Open Access Journals (Sweden)

    Xihan Guo

    2016-09-01

    Full Text Available The fruit of Phyllanthus emblica Linn. (PE has been widely consumed as a functional food and folk medicine in Southeast Asia due to its remarkable nutritional and pharmacological effects. Previous research showed PE delays mitotic progress and increases genomic instability (GIN in human colorectal cancer cells. This study aimed to investigate the similar effects of PE by the biomarkers related to spindle assembly checkpoint (SAC, mitotic aberrations and GIN in human NCM460 normal colon epithelial cells. Cells were treated with PE and harvested differently according to the biomarkers observed. Frequencies of micronuclei (MN, nucleoplasmic bridge (NPB and nuclear bud (NB in cytokinesis-block micronucleus assay were used as indicators of GIN. Mitotic aberrations were assessed by the biomarkers of chromosome misalignment, multipolar division, chromosome lagging and chromatin bridge. SAC activity was determined by anaphase-to- metaphase ratio (AMR and the expression of core SAC gene budding uninhibited by benzimidazoles related 1 (BubR1. Compared with the control, PE-treated cells showed (1 decreased incidences of MN, NPB and NB (p < 0.01; (2 decreased frequencies of all mitotic aberration biomarkers (p < 0.01; and (3 decreased AMR (p < 0.01 and increased BubR1 expression (p < 0.001. The results revealed PE has the potential to protect human normal colon epithelial cells from mitotic and genomic damages partially by enhancing the function of SAC.

  1. A Multidisciplinary Workshop: Self-Assembling Peptide Systems in Biology, Medicine and Engineering, Crete, Greece, July 1-6, 1999

    Science.gov (United States)

    1999-07-06

    Nanoparticles on Self- Assembled Biolipid Tubular Templates." Chem. Commun., 1996, 321-322. 4. S. L. Burkett, S. D. Sims, and S. Mann, "Synthesis of...exploitation of biocatalysis, ribosomal and non-ribosomal biosynthesis to enhance permissiveness toward elaboration of useful biomolecular structures and...1999).L. Bromberg, I. Lewin, H. Gottlieb and A. Warshawsky Interaction of mercury (II) and silver (I) with bis[di(2-ethylhexyloxy)- thiophosphoryl

  2. Nuclear fuel assembly

    International Nuclear Information System (INIS)

    Takeda, Tadashi; Sato, Kenji; Goto, Masakazu.

    1984-01-01

    Purpose: To facilitate identification of a fuel assembly upon fuel exchange in BWR type reactors. Constitution: Fluorescent material is coated or metal plating is applied to the impressed portion of a upper tie plate handle of a fuel assembly, and the fluorescent material or the metal plating surface is covered with a protective membrane made of transparent material. This enables to distinguish the impressed surface from a distant place and chemical reaction between the impressed surface and the reactor water can be prevented. Furthermore, since the protective membrane is formed such that it protrudes toward the upper side relative to the impressed surface, there is no risk of depositions of claddings thereover. (Moriyama, K.)

  3. Ribosomal function and its inhibition by antibiotics in prokaryotes

    Science.gov (United States)

    Nierhaus, K. H.; Wittmann, H. G.

    1980-05-01

    Most of the known antibiotics act at the level of protein biosynthesis probably due to the extraordinary complexity of the translational machinery which can be interfered with at many points. At first a survey is given of our present knowledge covering the structure and function of the prokaryotic ribosome. The most important antibiotics acting at the translational level are integrated into this network of data. The binding sites and the inhibition mechanisms of the drugs, together with the ribosomal components altered in resistant mutants are described. Finally, the points of interference with the translational machinery are indicated in an extended scheme of ribosomal functions.

  4. Multiple RNA interactions position Mrd1 at the site of the small subunit pseudoknot within the 90S pre-ribosome.

    Science.gov (United States)

    Segerstolpe, Åsa; Granneman, Sander; Björk, Petra; de Lima Alves, Flavia; Rappsilber, Juri; Andersson, Charlotta; Högbom, Martin; Tollervey, David; Wieslander, Lars

    2013-01-01

    Ribosomal subunit biogenesis in eukaryotes is a complex multistep process. Mrd1 is an essential and conserved small (40S) ribosomal subunit synthesis factor that is required for early cleavages in the 35S pre-ribosomal RNA (rRNA). Yeast Mrd1 contains five RNA-binding domains (RBDs), all of which are necessary for optimal function of the protein. Proteomic data showed that Mrd1 is part of the early pre-ribosomal complexes, and deletion of individual RBDs perturbs the pre-ribosomal structure. In vivo ultraviolet cross-linking showed that Mrd1 binds to the pre-rRNA at two sites within the 18S region, in helix 27 (h27) and helix 28. The major binding site lies in h27, and mutational analyses shows that this interaction requires the RBD1-3 region of Mrd1. RBD2 plays the dominant role in h27 binding, but other RBDs also contribute directly. h27 and helix 28 are located close to the sequences that form the central pseudoknot, a key structural feature of the mature 40S subunit. We speculate that the modular structure of Mrd1 coordinates pseudoknot formation with pre-rRNA processing and subunit assembly.

  5. Phosphorylation of ribosomal protein S6 mediates compensatory renal hypertrophy.

    Science.gov (United States)

    Xu, Jinxian; Chen, Jianchun; Dong, Zheng; Meyuhas, Oded; Chen, Jian-Kang

    2015-03-01

    The molecular mechanism underlying renal hypertrophy and progressive nephron damage remains poorly understood. Here we generated congenic ribosomal protein S6 (rpS6) knock-in mice expressing nonphosphorylatable rpS6 and found that uninephrectomy-induced renal hypertrophy was significantly blunted in these knock-in mice. Uninephrectomy-induced increases in cyclin D1 and decreases in cyclin E in the remaining kidney were attenuated in the knock-in mice compared with their wild-type littermates. Uninephrectomy induced rpS6 phosphorylation in the wild-type mice; however, no rpS6 phosphorylation was detected in uninephrectomized or sham-operated knock-in mice. Nonetheless, uninephrectomy stimulated comparable 4E-BP1 phosphorylation in both knock-in and wild-type mice, indicating that mTORC1 was still activated in the knock-in mice. Moreover, the mTORC1 inhibitor rapamycin prevented both rpS6 and 4E-BP1 phosphorylation, significantly blunted uninephrectomy-induced renal hypertrophy in wild-type mice, but did not prevent residual renal hypertrophy despite inhibiting 4E-BP1 phosphorylation in uninephrectomized knock-in mice. Thus, both genetic and pharmacological approaches unequivocally demonstrate that phosphorylated rpS6 is a downstream effector of the mTORC1-S6K1 signaling pathway mediating renal hypertrophy. Hence, rpS6 phosphorylation facilitates the increase in cyclin D1 and decrease in cyclin E1 that underlie the hypertrophic nature of uninephrectomy-induced kidney growth.

  6. Intracoronary delivery of self-assembling heart-derived microtissues (cardiospheres) for prevention of adverse remodeling in a pig model of convalescent myocardial infarction.

    Science.gov (United States)

    Gallet, Romain; Tseliou, Eleni; Dawkins, James; Middleton, Ryan; Valle, Jackelyn; Angert, David; Reich, Heidi; Luthringer, Daniel; Kreke, Michelle; Smith, Rachel; Marbán, Linda; Marbán, Eduardo

    2015-05-01

    Preclinical studies in rodents and pigs indicate that the self-assembling microtissues known as cardiospheres may be more effective than dispersed cardiosphere-derived cells. However, the more desirable intracoronary route has been assumed to be unsafe for cardiosphere delivery: Cardiospheres are large (30-150 μm), raising concerns about likely microembolization. We questioned these negative assumptions by evaluating the safety and efficacy of optimized intracoronary delivery of cardiospheres in a porcine model of convalescent myocardial infarction. First, we standardized the size of cardiospheres by modifying culture conditions. Then, dosage was determined by infusing escalating doses of cardiospheres in the left anterior descending artery of naive pigs, looking for acute adverse effects. Finally, in a randomized efficacy study, 14 minipigs received allogeneic cardiospheres (1.3 × 10(6)) or vehicle 1 month after myocardial infarction. Animals underwent magnetic resonance imaging before infusion and 1 month later to assess left ventricular ejection fraction, scar mass, and viable mass. In the dosing study, we did not observe any evidence of microembolization after cardiosphere infusion. In the post-myocardial infarction study, cardiospheres preserved LV function, reduced scar mass and increased viable mass, whereas placebo did not. Moreover, cardiosphere decreased collagen content, and increased vessel densities and myocardial perfusion. Importantly, intracoronary cardiospheres decreased left ventricular end-diastolic pressure and increased cardiac output. Intracoronary delivery of cardiospheres is safe. Intracoronary cardiospheres are also remarkably effective in decreasing scar, halting adverse remodeling, increasing myocardial perfusion, and improving hemodynamic status after myocardial infarction in pigs. Thus, cardiospheres may be viable therapeutic candidates for intracoronary infusion in selected myocardial disorders. © 2015 American Heart Association

  7. The use of a P. falciparum specific coiled-coil domain to construct a self-assembling protein nanoparticle vaccine to prevent malaria.

    Science.gov (United States)

    Karch, Christopher P; Doll, Tais A P F; Paulillo, Sara M; Nebie, Issa; Lanar, David E; Corradin, Giampietro; Burkhard, Peter

    2017-09-06

    The parasitic disease malaria remains a major global public health concern and no truly effective vaccine exists. One approach to the development of a malaria vaccine is to target the asexual blood stage that results in clinical symptoms. Most attempts have failed. New antigens such as P27A and P27 have emerged as potential new vaccine candidates. Multiple studies have demonstrated that antigens are more immunogenic and are better correlated with protection when presented on particulate delivery systems. One such particulate delivery system is the self-assembling protein nanoparticle (SAPN) that relies on coiled-coil domains of proteins to form stable nanoparticles. In the past we have used de novo designed amino acid domains to drive the formation of the coiled-coil scaffolds which present the antigenic epitopes on the particle surface. Here we use naturally occurring domains found in the tex1 protein to form the coiled-coil scaffolding of the nanoparticle. Thus, by engineering P27A and a new extended form of the coiled-coil domain P27 onto the N and C terminus of the SAPN protein monomer we have developed a particulate delivery system that effectively displays both antigens on a single particle that uses malaria tex1 sequences to form the nanoparticle scaffold. These particles are immunogenic in a murine model and induce immune responses similar to the ones observed in seropositive individuals in malaria endemic regions. We demonstrate that our P27/P27A-SAPNs induce an immune response akin to the one in seropositive individuals in Burkina Faso. Since P27 is highly conserved among different Plasmodium species, these novel SAPNs may even provide cross-protection between Plasmodium falciparum and Plasmodium vivax the two major human malaria pathogens. As the SAPNs are also easy to manufacture and store they can be delivered to the population in need without complication thus providing a low cost malaria vaccine.

  8. Shingle assembly

    Science.gov (United States)

    Dinwoodie, Thomas L.

    2007-02-20

    A barrier, such as a PV module, is secured to a base by a support to create a shingle assembly with a venting region defined between the barrier and base for temperature regulation. The first edge of one base may be interengageable with the second edge of an adjacent base to be capable of resisting first and second disengaging forces oriented perpendicular to the edges and along planes oriented parallel to and perpendicular to the base. A deflector may be used to help reduce wind uplift forces.

  9. Heater assembly

    International Nuclear Information System (INIS)

    Lee, K.; Ueng, Tzoushin.

    1991-01-01

    An electrical resistance heater, installed in the H1 borehole, is used to thermally perturb the rock mass through a controlled heating and cooling cycle. Heater power levels are controlled by a Variac power transformer and are measured by wattmeters. Temperatures are measured by thermocouples on the borehole wall and on the heater assembly. Power and temperature values are recorded by the DAS described in Chapter 12. The heater assembly consists of a 3.55-m (11.6-ft) long by 20.3-cm (8-in.) O.D., Type 304 stainless steel pipe, containing a tubular hairpin heating element. The element has a heated length of 3 m (9.84 ft). The power rating of the element is 10 kW; however, we plan to operate the unit at a maximum power of only 3 kW. The heater is positioned with its midpoint directly below the axis of the P2 borehole, as shown in the borehole configuration diagram. This heater midpoint position corresponds to a distance of approximately 8.5 m (27.9 ft) from the H1 borehole collar. A schematic of the heater assembly in the borehole is shown. The distance from the borehole collar to the closest point on the assembly (the front end) is 6.5 m (21.3 ft). A high-temperature inflatable packer, used to seal the borehole for moisture collection, is positioned 50 cm (19.7 in.) ahead of the heater front end. The heater is supported and centralized within the borehole by two skids, fabricated from 25-mm (1-in.) O.D. stainless steel pipe. Thermocouples are installed at a number of locations in the H1 borehole. Four thermocouples that are attached to the heater skin monitor temperatures on the outer surface of the can, while three thermocouples that are held in place by rock sections monitor borehole wall temperatures beneath the heater. Temperatures are also monitored at the heater terminal and on the packer hardware

  10. Assembling consumption

    DEFF Research Database (Denmark)

    Assembling Consumption marks a definitive step in the institutionalisation of qualitative business research. By gathering leading scholars and educators who study markets, marketing and consumption through the lenses of philosophy, sociology and anthropology, this book clarifies and applies the i...... societies. This is an essential reading for both seasoned scholars and advanced students of markets, economies and social forms of consumption....... the investigative tools offered by assemblage theory, actor-network theory and non-representational theory. Clear theoretical explanation and methodological innovation, alongside empirical applications of these emerging frameworks will offer readers new and refreshing perspectives on consumer culture and market...

  11. Partial inhibition of polysomal ribosomes of Escherichia coli by streptomycin.

    Science.gov (United States)

    Wallace, B J; Tai, P C; Herzog, E L; Davis, B D

    1973-04-01

    With purified initiation-free polysomes of E. coli, whether endogenous or formed in vitro on phage R17 RNA, streptomycin causes partial inhibition of chain elongation. The degree of inhibition is constant over a broad range of streptomycin concentration and decreases markedly with increasing Mg(++) concentration. Hence, streptomycin evidently complexes readily with polysomal ribosomes, causing a partial block in chain elongation. Streptomycin has already been shown to cause a complete block of chain elongation by free ribosomes forming initiation complexes in its presence. The production of a different effect on polysomal ribosomes explains how streptomycin can exert two mutually exclusive effects in cells: phenotypic suppression at low concentrations and irreversible inhibition of protein synthesis at higher concentrations. It also becomes possible to understand why killing by streptomycin is antagonized by agents (such as chloramphenicol) that cause a stable blockade of the ribosomes in polysomes.

  12. Cotranslational Protein Folding inside the Ribosome Exit Tunnel

    Directory of Open Access Journals (Sweden)

    Ola B. Nilsson

    2015-09-01

    Full Text Available At what point during translation do proteins fold? It is well established that proteins can fold cotranslationally outside the ribosome exit tunnel, whereas studies of folding inside the exit tunnel have so far detected only the formation of helical secondary structure and collapsed or partially structured folding intermediates. Here, using a combination of cotranslational nascent chain force measurements, inter-subunit fluorescence resonance energy transfer studies on single translating ribosomes, molecular dynamics simulations, and cryoelectron microscopy, we show that a small zinc-finger domain protein can fold deep inside the vestibule of the ribosome exit tunnel. Thus, for small protein domains, the ribosome itself can provide the kind of sheltered folding environment that chaperones provide for larger proteins.

  13. Sequence-Dependent Elongation Dynamics on Macrolide-Bound Ribosomes

    Directory of Open Access Journals (Sweden)

    Magnus Johansson

    2014-06-01

    Full Text Available The traditional view of macrolide antibiotics as plugs inside the ribosomal nascent peptide exit tunnel (NPET has lately been challenged in favor of a more complex, heterogeneous mechanism, where drug-peptide interactions determine the fate of a translating ribosome. To investigate these highly dynamic processes, we applied single-molecule tracking of elongating ribosomes during inhibition of elongation by erythromycin of several nascent chains, including ErmCL and H-NS, which were shown to be, respectively, sensitive and resistant to erythromycin. Peptide sequence-specific changes were observed in translation elongation dynamics in the presence of a macrolide-obstructed NPET. Elongation rates were not severely inhibited in general by the presence of the drug; instead, stalls or pauses were observed as abrupt events. The dynamic pathways of nascent-chain-dependent elongation pausing in the presence of macrolides determine the fate of the translating ribosome stalling or readthrough.

  14. Structure of plant nuclear and ribosomal DNA containing chromatin.

    Science.gov (United States)

    Leber, B; Hemleben, V

    1979-11-10

    Digestion of plant chromatin from Brassica pekinensis and Matthiola incana with staphylococcus nuclease leads to a DNA repeat of 175 plus or minus 8 and a core size of 140 base pairs. DNase I digestion results in multiples of 10 bases. Ribosomal RNN genes were studied as a model system for active plant chromatin because of their great redundancy and their high transcriptional activity in growing and differentiating tissues. The actively transcribed genes were identified by nascent RNA of ribosomal origin still attached to its matrix DNA. Hybridization techniques were used to demonstrate that even transcriptionally active gene sequences are present in nuclease generated chromatin subunits. Comparison of the DNase I kinetics of chromatin digestion with the amount of ribosomal RNA genes which is available for hybridization at the given times indicated that ribosomal RNA genes are digested, but not preferentially degraded by DNase I.

  15. Improvement and efficient display of Bacillus thuringiensis toxins on M13 phages and ribosomes.

    Science.gov (United States)

    Pacheco, Sabino; Cantón, Emiliano; Zuñiga-Navarrete, Fernando; Pecorari, Frédéric; Bravo, Alejandra; Soberón, Mario

    2015-12-01

    Bacillus thuringiensis (Bt) produces insecticidal proteins that have been used worldwide in the control of insect-pests in crops and vectors of human diseases. However, different insect species are poorly controlled by the available Bt toxins or have evolved resistance to these toxins. Evolution of Bt toxicity could provide novel toxins to control insect pests. To this aim, efficient display systems to select toxins with increased binding to insect membranes or midgut proteins involved in toxicity are likely to be helpful. Here we describe two display systems, phage display and ribosome display, that allow the efficient display of two non-structurally related Bt toxins, Cry1Ac and Cyt1Aa. Improved display of Cry1Ac and Cyt1Aa on M13 phages was achieved by changing the commonly used peptide leader sequence of the coat pIII-fusion protein, that relies on the Sec translocation pathway, for a peptide leader sequence that relies on the signal recognition particle pathway (SRP) and by using a modified M13 helper phage (Phaberge) that has an amber mutation in its pIII genomic sequence and preferentially assembles using the pIII-fusion protein. Also, both Cry1Ac and Cyt1Aa were efficiently displayed on ribosomes, which could allow the construction of large libraries of variants. Furthermore, Cry1Ac or Cyt1Aa displayed on M13 phages or ribosomes were specifically selected from a mixture of both toxins depending on which antigen was immobilized for binding selection. These improved systems may allow the selection of Cry toxin variants with improved insecticidal activities that could counter insect resistances.

  16. Cisplatin Targeting of Bacterial Ribosomal RNA Hairpins

    Directory of Open Access Journals (Sweden)

    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.

  17. Aminoglycoside interactions and impacts on the eukaryotic ribosome

    Science.gov (United States)

    Prokhorova, Irina; Altman, Roger B.; Djumagulov, Muminjon; Shrestha, Jaya P.; Urzhumtsev, Alexandre; Ferguson, Angelica; Chang, Cheng-Wei Tom; Yusupov, Marat; Blanchard, Scott C.; Yusupova, Gulnara

    2017-01-01

    Aminoglycosides are chemically diverse, broad-spectrum antibiotics that target functional centers within the bacterial ribosome to impact all four principle stages (initiation, elongation, termination, and recycling) of the translation mechanism. The propensity of aminoglycosides to induce miscoding errors that suppress the termination of protein synthesis supports their potential as therapeutic interventions in human diseases associated with premature termination codons (PTCs). However, the sites of interaction of aminoglycosides with the eukaryotic ribosome and their modes of action in eukaryotic translation remain largely unexplored. Here, we use the combination of X-ray crystallography and single-molecule FRET analysis to reveal the interactions of distinct classes of aminoglycosides with the 80S eukaryotic ribosome. Crystal structures of the 80S ribosome in complex with paromomycin, geneticin (G418), gentamicin, and TC007, solved at 3.3- to 3.7-Å resolution, reveal multiple aminoglycoside-binding sites within the large and small subunits, wherein the 6′-hydroxyl substituent in ring I serves as a key determinant of binding to the canonical eukaryotic ribosomal decoding center. Multivalent binding interactions with the human ribosome are also evidenced through their capacity to affect large-scale conformational dynamics within the pretranslocation complex that contribute to multiple aspects of the translation mechanism. The distinct impacts of the aminoglycosides examined suggest that their chemical composition and distinct modes of interaction with the ribosome influence PTC read-through efficiency. These findings provide structural and functional insights into aminoglycoside-induced impacts on the eukaryotic ribosome and implicate pleiotropic mechanisms of action beyond decoding. PMID:29208708

  18. REPARATION : ribosome profiling assisted (re-)annotation of bacterial genomes

    OpenAIRE

    Ndah, Elvis; Jonckheere, Veronique; Giess, Adam; Valen, Eivind; Menschaert, Gerben; Van Damme, Petra

    2017-01-01

    Prokaryotic genome annotation is highly dependent on automated methods, as manual curation cannot keep up with the exponential growth of sequenced genomes. Current automated methods depend heavily on sequence composition and often underestimate the complexity of the proteome. We developed RibosomeE Profiling Assisted (re-)AnnotaTION (REPARATION), a de novo machine learning algorithm that takes advantage of experimental protein synthesis evidence from ribosome profiling (Ribo-seq) to delineate...

  19. REPARATION: ribosome profiling assisted (re-)annotation of bacterial genomes

    OpenAIRE

    Ndah, Elvis; Jonckheere, Veronique; Giess, Adam; Valen, Eivind; Menschaert, Gerben; Van Damme, Petra

    2017-01-01

    Abstract Prokaryotic genome annotation is highly dependent on automated methods, as manual curation cannot keep up with the exponential growth of sequenced genomes. Current automated methods depend heavily on sequence composition and often underestimate the complexity of the proteome. We developed RibosomeE Profiling Assisted (re-)AnnotaTION (REPARATION), a de novo machine learning algorithm that takes advantage of experimental protein synthesis evidence from ribosome profiling (Ribo-seq) to ...

  20. Ribosome slowed by mutation to streptomycin resistance. [Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Galas, D.J.; Branscomb, E.W.

    1976-08-12

    The effect of mutation to streptomycin resistance on the speed of polypeptide elongation in Escherichia coli was investigated. Translation speed was determined by measuring the time required for the first newly synthesized ..beta..-galactosidase molecules to appear after induction of the lactose operon. The results showed that ribosome speed is not a fixed parameter inherent to the protein synthetic apparatus, but a variable determined by the kinetics of translation and ultimately by the structure of the ribosome. (HLW)

  1. Simple and inexpensive ribosome profiling analysis of mRNA translation.

    Science.gov (United States)

    Reid, David W; Shenolikar, Shirish; Nicchitta, Christopher V

    2015-12-01

    The development and application of ribosome profiling has markedly advanced our understanding of ribosomes and mRNA translation. The experimental approach, which relies on deep sequencing of ribosome-protected mRNA fragments generated by treatment of polyribosomes with exogenous nucleases, provides a transcriptome-wide assessment of translation. The broad application of ribosome profiling has been slowed by the complexity and expense of the protocol. Here, we provide a simplified ribosome profiling method that uses micrococcal nuclease to generate ribosome footprints in crude cellular extracts, which are then purified simply by size selection via polyacrylamide gel electrophoresis. This simplification removes the laborious or expensive purification of ribosomes that has typically been used. This direct extraction method generates gene-level ribosome profiling data that are similar to a method that includes ribosome purification. This protocol should significantly ease the barrier to entry for research groups interested in employing ribosome profiling. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Fuel assembly

    International Nuclear Information System (INIS)

    Chaki, Masao; Nishida, Koji; Karasawa, Hidetoshi; Kanazawa, Toru; Orii, Akihito; Nagayoshi, Takuji; Kashiwai, Shin-ichi; Masuhara, Yasuhiro

    1998-01-01

    The present invention concerns a fuel assembly, for a BWR type nuclear reactor, comprising fuel rods in 9 x 9 matrix. The inner width of the channel box is about 132mm and the length of the fuel rods which are not short fuel rods is about 4m. Two water rods having a circular cross section are arranged on a diagonal line in a portion of 3 x 3 matrix at the center of the fuel assembly, and two fuel rods are disposed at vacant spaces, and the number of fuel rods is 74. Eight fuel rods are determined as short fuel rods among 74 fuel rods. Assuming the fuel inventory in the short fuel rod as X(kg), and the fuel inventory in the fuel rods other than the short fuel rods as Y(kg), X and Y satisfy the relation: X + Y ≥ 173m, Y ≤ - 9.7X + 292, Y ≤ - 0.3X + 203 and X > 0. Then, even when the short fuel rods are used, the fuel inventory is increased and fuel economy can be improved. (I.N.)

  3. General Assembly

    CERN Multimedia

    Staff Association

    2016-01-01

    Mardi 5 avril à 11 h 00 BE Auditorium Meyrin (6-2-024) Conformément aux statuts de l’Association du personnel, une Assemblée générale ordinaire est organisée une fois par année (article IV.2.1). Projet d’ordre du jour : Adoption de l’ordre du jour. Approbation du procès-verbal de l’Assemblée générale ordinaire du 5 mai 2015. Présentation et approbation du rapport d’activités 2015. Présentation et approbation du rapport financier 2015. Présentation et approbation du rapport des vérificateurs aux comptes pour 2015. Programme de travail 2016. Présentation et approbation du projet de budget 2016 Approbation du taux de cotisation pour 2017. Modifications aux Statuts de l'Association du personnel proposée. Élections des membres de la Commissio...

  4. General Assembly

    CERN Multimedia

    Staff Association

    2017-01-01

    Conformément aux statuts de l’Association du personnel, une Assemblée générale ordinaire est organisée une fois par année (article IV.2.1). Projet d’ordre du jour : Adoption de l’ordre du jour. Approbation du procès-verbal de l’Assemblée générale ordinaire du 5 avril 2016. Présentation et approbation du rapport d’activités 2016. Présentation et approbation du rapport financier 2016. Présentation et approbation du rapport des vérificateurs aux comptes pour 2016. Programme de travail 2017. Présentation et approbation du projet de budget 2017 Approbation du taux de cotisation pour 2018. Modifications aux Statuts de l'Association du personnel proposées. Élections des membres de la Commission électorale. Élections des vérifica...

  5. General Assembly

    CERN Multimedia

    Staff Association

    2015-01-01

    Mardi 5 mai à 11 h 00 Salle 13-2-005 Conformément aux statuts de l’Association du personnel, une Assemblée générale ordinaire est organisée une fois par année (article IV.2.1). Projet d’ordre du jour : 1- Adoption de l’ordre du jour. 2- Approbation du procès-verbal de l’Assemblée générale ordinaire du 22 mai 2014. 3- Présentation et approbation du rapport d’activités 2014. 4- Présentation et approbation du rapport financier 2014. 5- Présentation et approbation du rapport des vérificateurs aux comptes pour 2014. 6- Programme 2015. 7- Présentation et approbation du projet de budget 2015 et taux de cotisation pour 2015. 8- Pas de modifications aux Statuts de l'Association du personnel proposée. 9- Élections des membres de la Commission é...

  6. General assembly

    CERN Multimedia

    Staff Association

    2015-01-01

    Mardi 5 mai à 11 h 00 Salle 13-2-005 Conformément aux statuts de l’Association du personnel, une Assemblée générale ordinaire est organisée une fois par année (article IV.2.1). Projet d’ordre du jour : Adoption de l’ordre du jour. Approbation du procès-verbal de l’Assemblée générale ordinaire du 22 mai 2014. Présentation et approbation du rapport d’activités 2014. Présentation et approbation du rapport financier 2014. Présentation et approbation du rapport des vérificateurs aux comptes pour 2014. Programme 2015. Présentation et approbation du projet de budget 2015 et taux de cotisation pour 2015. Pas de modifications aux Statuts de l'Association du personnel proposée. Élections des membres de la Commission électorale. &am...

  7. Fuel assembly

    International Nuclear Information System (INIS)

    Nomata, Terumitsu.

    1993-01-01

    Among fuel pellets to be loaded to fuel cans of a fuel assembly, fuel pellets having a small thermal power are charged in a region from the end of each of spacers up to about 50mm on the upstream of coolants that flow vertically at the periphery of fuel rods. Coolants at the periphery of fuel rods are heated by the heat generation, to result in voids. However, since cooling effect on the upstream of the spacers is low due to influences of the spacers. Further, since the fuel pellets disposed in the upstream region have small thermal power, a void coefficient is not increased. Even if a thermal power exceeding cooling performance should be generated, there is no worry of causing burnout in the upstream region. Even if burnout should be caused, safety margin and reliability relative to burnout are improved, to increase an allowable thermal power, thereby enabling to improve integrity and reliability of fuel rods and fuel assemblies. (N.H.)

  8. The dynamic assembly of distinct RNA polymerase I complexes modulates rDNA transcription.

    Science.gov (United States)

    Torreira, Eva; Louro, Jaime Alegrio; Pazos, Irene; González-Polo, Noelia; Gil-Carton, David; Duran, Ana Garcia; Tosi, Sébastien; Gallego, Oriol; Calvo, Olga; Fernández-Tornero, Carlos

    2017-03-06

    Cell growth requires synthesis of ribosomal RNA by RNA polymerase I (Pol I). Binding of initiation factor Rrn3 activates Pol I, fostering recruitment to ribosomal DNA promoters. This fundamental process must be precisely regulated to satisfy cell needs at any time. We present in vivo evidence that, when growth is arrested by nutrient deprivation, cells induce rapid clearance of Pol I-Rrn3 complexes, followed by the assembly of inactive Pol I homodimers. This dual repressive mechanism reverts upon nutrient addition, thus restoring cell growth. Moreover, Pol I dimers also form after inhibition of either ribosome biogenesis or protein synthesis. Our mutational analysis, based on the electron cryomicroscopy structures of monomeric Pol I alone and in complex with Rrn3, underscores the central role of subunits A43 and A14 in the regulation of differential Pol I complexes assembly and subsequent promoter association.

  9. Single-step rapid assembly of DNA origami nanostructures for addressable nanoscale bioreactors

    DEFF Research Database (Denmark)

    Fu, Yanming; Zeng, Dongdong; Chao, Jie

    2013-01-01

    Self-assembled DNA origami nanostructures have shown great promise for bottom-up construction of complex objects with nanoscale addressability. Here we show that DNA origami-based 1D nanoribbons and nanotubes are one-pot assembled with controllable sizes and nanoscale addressability with high speed...... (within only 10-20 min), exhibiting extraordinarily high cooperativity that is often observed in assembly of natural molecular machines in cells (e.g. ribosome). By exploiting the high specificity of DNA-based self-assembly, we can precisely anchor proteins on these DNA origami nanostructures with sub-10...

  10. Fluorescently labeled ribosomes as a tool for analyzing antibiotic binding.

    Science.gov (United States)

    Llano-Sotelo, Beatriz; Hickerson, Robyn P; Lancaster, Laura; Noller, Harry F; Mankin, Alexander S

    2009-08-01

    Measuring the binding of antibiotics and other small-molecular-weight ligands to the 2.5 MDa ribosome often presents formidable challenges. Here, we describe a general method for studying binding of ligands to ribosomes that carry a site-specific fluorescent label covalently attached to one of the ribosomal proteins. As a proof of principle, an environment-sensitive fluorescent group was placed at several specific sites within the ribosomal protein S12. Small ribosomal subunits were reconstituted from native 16S rRNA, individually purified small subunit proteins, and fluorescently labeled S12. The fluorescence characteristics of the reconstituted subunits were affected by several antibiotics, including streptomycin and neomycin, which bind in the vicinity of protein S12. The equilibrium dissociation constants of the drugs obtained using a conventional fluorometer were in good agreement with those observed using previously published methods and with measurements based on the use of radiolabeled streptomycin. The newly developed method is rapid and sensitive, and can be used for determining thermodynamic and kinetic binding characteristics of antibiotics and other small ribosomal ligands. The method can readily be adapted for use in high-throughput screening assays.

  11. Dual effect of chloramphenicol peptides on ribosome inhibition.

    Science.gov (United States)

    Bougas, Anthony; Vlachogiannis, Ioannis A; Gatos, Dimitrios; Arenz, Stefan; Dinos, George P

    2017-05-01

    Chloramphenicol peptides were recently established as useful tools for probing nascent polypeptide chain interaction with the ribosome, either biochemically, or structurally. Here, we present a new 10mer chloramphenicol peptide, which exerts a dual inhibition effect on the ribosome function affecting two distinct areas of the ribosome, namely the peptidyl transferase center and the polypeptide exit tunnel. According to our data, the chloramphenicol peptide bound on the chloramphenicol binding site inhibits the formation of both acetyl-phenylalanine-puromycin and acetyl-lysine-puromycin, showing, however, a decreased peptidyl transferase inhibition compared to chloramphenicol-mediated inhibition per se. Additionally, we found that the same compound is a strong inhibitor of green fluorescent protein synthesis in a coupled in vitro transcription-translation assay as well as a potent inhibitor of lysine polymerization in a poly(A)-programmed ribosome, showing that an additional inhibitory effect may exist. Since chemical protection data supported the interaction of the antibiotic with bases A2058 and A2059 near the entrance of the tunnel, we concluded that the extra inhibition effect on the synthesis of longer peptides is coming from interactions of the peptide moiety of the drug with residues comprising the ribosomal tunnel, and by filling up the tunnel and blocking nascent chain progression through the restricted tunnel. Therefore, the dual interaction of the chloramphenicol peptide with the ribosome increases its inhibitory effect and opens a new window for improving the antimicrobial potency of classical antibiotics or designing new ones.

  12. Recognition of Ribosomal Protein L11 by the Protein Trimethyltransferase PrmA

    Energy Technology Data Exchange (ETDEWEB)

    Demirci,H.; Gregory, S.; Dahlberg, A.; Jogl, G.

    2007-01-01

    Bacterial ribosomal protein L11 is post-translationally trimethylated at multiple residues by a single methyltransferase, PrmA. Here, we describe four structures of PrmA from the extreme thermophile Thermus thermophilus. Two apo-PrmA structures at 1.59 and 2.3 {angstrom} resolution and a third with bound cofactor S-adenosyl-L-methionine at 1.75 {angstrom} each exhibit distinct relative positions of the substrate recognition and catalytic domains, revealing how PrmA can position the L11 substrate for multiple, consecutive side-chain methylation reactions. The fourth structure, the PrmA-L11 enzyme-substrate complex at 2.4 {angstrom} resolution, illustrates the highly specific interaction of the N-terminal domain with its substrate and places Lys39 in the PrmA active site. The presence of a unique flexible loop in the cofactor-binding site suggests how exchange of AdoMet with the reaction product S-adenosyl-L-homocysteine can occur without necessitating the dissociation of PrmA from L11. Finally, the mode of interaction of PrmA with L11 explains its observed preference for L11 as substrate before its assembly into the 50S ribosomal subunit.

  13. 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.

  14. Roles of Transcriptional and Translational Control Mechanisms in Regulation of Ribosomal Protein Synthesis in Escherichia coli.

    Science.gov (United States)

    Burgos, Hector L; O'Connor, Kevin; Sanchez-Vazquez, Patricia; Gourse, Richard L

    2017-11-01

    Bacterial ribosome biogenesis is tightly regulated to match nutritional conditions and to prevent formation of defective ribosomal particles. In Escherichia coli , most ribosomal protein (r-protein) synthesis is coordinated with rRNA synthesis by a translational feedback mechanism: when r-proteins exceed rRNAs, specific r-proteins bind to their own mRNAs and inhibit expression of the operon. It was recently discovered that the second messenger nucleotide guanosine tetra and pentaphosphate (ppGpp), which directly regulates rRNA promoters, is also capable of regulating many r-protein promoters. To examine the relative contributions of the translational and transcriptional control mechanisms to the regulation of r-protein synthesis, we devised a reporter system that enabled us to genetically separate the cis -acting sequences responsible for the two mechanisms and to quantify their relative contributions to regulation under the same conditions. We show that the synthesis of r-proteins from the S20 and S10 operons is regulated by ppGpp following shifts in nutritional conditions, but most of the effect of ppGpp required the 5' region of the r-protein mRNA containing the target site for translational feedback regulation and not the promoter. These results suggest that most regulation of the S20 and S10 operons by ppGpp following nutritional shifts is indirect and occurs in response to changes in rRNA synthesis. In contrast, we found that the promoters for the S20 operon were regulated during outgrowth, likely in response to increasing nucleoside triphosphate (NTP) levels. Thus, r-protein synthesis is dynamic, with different mechanisms acting at different times. IMPORTANCE Bacterial cells have evolved complex and seemingly redundant strategies to regulate many high-energy-consuming processes. In E. coli , synthesis of ribosomal components is tightly regulated with respect to nutritional conditions by mechanisms that act at both the transcription and translation steps. In

  15. Human C4orf14 interacts with the mitochondrial nucleoid and is involved in the biogenesis of the small mitochondrial ribosomal subunit.

    Science.gov (United States)

    He, J; Cooper, H M; Reyes, A; Di Re, M; Kazak, L; Wood, S R; Mao, C C; Fearnley, I M; Walker, J E; Holt, I J

    2012-07-01

    The bacterial homologue of C4orf14, YqeH, has been linked to assembly of the small ribosomal subunit. Here, recombinant C4orf14 isolated from human cells, co-purified with the small, 28S subunit of the mitochondrial ribosome and the endogenous protein co-fractionated with the 28S subunit in sucrose gradients. Gene silencing of C4orf14 specifically affected components of the small subunit, leading to decreased protein synthesis in the organelle. The GTPase of C4orf14 was critical to its interaction with the 28S subunit, as was GTP. Therefore, we propose that C4orf14, with bound GTP, binds to components of the 28S subunit facilitating its assembly, and GTP hydrolysis acts as the release mechanism. C4orf14 was also found to be associated with human mitochondrial nucleoids, and C4orf14 gene silencing caused mitochondrial DNA depletion. In vitro C4orf14 is capable of binding to DNA. The association of C4orf14 with mitochondrial translation factors and the mitochondrial nucleoid suggests that the 28S subunit is assembled at the mitochondrial nucleoid, enabling the direct transfer of messenger RNA from the nucleoid to the ribosome in the organelle.

  16. An optimized single chain TCR scaffold relying on the assembly with the native CD3-complex prevents residual mispairing with endogenous TCRs in human T-cells

    Science.gov (United States)

    Knies, Diana; Klobuch, Sebastian; Xue, Shao-An; Birtel, Matthias; Echchannaoui, Hakim; Yildiz, Oezlem; Omokoko, Tana; Guillaume, Philippe; Romero, Pedro; Stauss, Hans; Sahin, Ugur; Herr, Wolfgang; Theobald, Matthias; Thomas, Simone; Voss, Ralf-Holger

    2016-01-01

    Immunotherapy of cancer envisions the adoptive transfer of T-cells genetically engineered with tumor-specific heterodimeric α/β T-cell receptors (TCRα/β). However, potential mispairing of introduced TCRα/β-chains with endogenous β/α-ones may evoke unpredictable autoimmune reactivities. A novel single chain (sc)TCR format relies on the fusion of the Vα-Linker-Vβ-fragment to the TCR Cβ-domain and coexpression of the TCR Cα-domain capable of recruiting the natural CD3-complex for full and hence, native T-cell signaling. Here, we tested whether such a gp100(280-288)- or p53(264-272) tumor antigen-specific scTCR is still prone to mispairing with TCRα. In a human Jurkat-76 T-cell line lacking endogenous TCRs, surface expression and function of a scTCR could be reconstituted by any cointroduced TCRα-chain indicating mispairing to take place on a molecular basis. In contrast, transduction into human TCRα/β-positive T-cells revealed that mispairing is largely reduced. Competition experiments in Jurkat-76 confirmed the preference of dcTCR to selfpair and to spare scTCR. This also allowed for the generation of dc/scTCR-modified cytomegalovirus/tumor antigen-bispecific T-cells to augment T-cell activation in CMV-infected tumor patients. Residual mispairing was prevented by strenghtening the Vα-Li-Vβ-fragment through the design of a novel disulfide bond between a Vα- and a linker-resident residue close to Vβ. Multimer-stainings, and cytotoxicity-, IFNγ-secretion-, and CFSE-proliferation-assays, the latter towards dendritic cells endogenously processing RNA-electroporated gp100 antigen proved the absence of hybrid scTCR/TCRα-formation without impairing avidity of scTCR/Cα in T-cells. Moreover, a fragile cytomegalovirus pp65(495-503)-specific scTCR modified this way acquired enhanced cytotoxicity. Thus, optimized scTCR/Cα inhibits residual TCR mispairing to accomplish safe adoptive immunotherapy for bulk endogenous TCRα/β-positive T-cells. PMID:27028870

  17. Structure of ERA in Complex with the 3 End of 16s rRNBA Implications for Ribosome Biogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Tu, C.; Zhou, X; Tropea, J; Austin, B; Waugh, D; Court, D; Ji, X

    2009-01-01

    ERA, composed of an N-terminal GTPase domain followed by an RNA-binding KH domain, is essential for bacterial cell viability. It binds to 16S rRNA and the 30S ribosomal subunit. However, its RNA-binding site, the functional relationship between the two domains, and its role in ribosome biogenesis remain unclear. We have determined two crystal structures of ERA, a binary complex with GDP and a ternary complex with a GTP-analog and the 1531AUCACCUCCUUA1542 sequence at the 3? end of 16S rRNA. In the ternary complex, the first nine of the 12 nucleotides are recognized by the protein. We show that GTP binding is a prerequisite for RNA recognition by ERA and that RNA recognition stimulates its GTP-hydrolyzing activity. Based on these and other data, we propose a functional cycle of ERA, suggesting that the protein serves as a chaperone for processing and maturation of 16S rRNA and a checkpoint for assembly of the 30S ribosomal subunit. The AUCA sequence is highly conserved among bacteria, archaea, and eukaryotes, whereas the CCUCC, known as the anti-Shine-Dalgarno sequence, is conserved in noneukaryotes only. Therefore, these data suggest a common mechanism for a highly conserved ERA function in all three kingdoms of life by recognizing the AUCA, with a 'twist' for noneukaryotic ERA proteins by also recognizing the CCUCC.

  18. Structure of ERA in complex with the 3′ end of 16S rRNA: Implications for ribosome biogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Tu, Chao; Zhou, Xiaomei; Tropea, Joseph E.; Austin, Brian P.; Waugh, David S.; Court, Donald L.; Ji, Xinhua; (NCI)

    2009-10-09

    ERA, composed of an N-terminal GTPase domain followed by an RNA-binding KH domain, is essential for bacterial cell viability. It binds to 16S rRNA and the 30S ribosomal subunit. However, its RNA-binding site, the functional relationship between the two domains, and its role in ribosome biogenesis remain unclear. We have determined two crystal structures of ERA, a binary complex with GDP and a ternary complex with a GTP-analog and the {sub 1531}AUCACCUCCUUA{sub 1542} sequence at the 3' end of 16S rRNA. In the ternary complex, the first nine of the 12 nucleotides are recognized by the protein. We show that GTP binding is a prerequisite for RNA recognition by ERA and that RNA recognition stimulates its GTP-hydrolyzing activity. Based on these and other data, we propose a functional cycle of ERA, suggesting that the protein serves as a chaperone for processing and maturation of 16S rRNA and a checkpoint for assembly of the 30S ribosomal subunit. The AUCA sequence is highly conserved among bacteria, archaea, and eukaryotes, whereas the CCUCC, known as the anti-Shine-Dalgarno sequence, is conserved in noneukaryotes only. Therefore, these data suggest a common mechanism for a highly conserved ERA function in all three kingdoms of life by recognizing the AUCA, with a 'twist' for noneukaryotic ERA proteins by also recognizing the CCUCC.

  19. Structural Studies of RNA Helicases Involved in Eukaryotic Pre-mRNA Splicing, Ribosome Biogenesis, and Translation Initiation

    DEFF Research Database (Denmark)

    He, Yangzi

    and ligates the neighbouring exons to generate mature mRNAs. Prp43 is an RNA helicase of the DEAH/RHA family. In yeast, once mRNAs are released, Prp43 catalyzes the disassembly of spliceosomes. The 18S, 5.8S and 25S rRNAs are transcribed as a single polycistronic transcript—the 35S pre......-rRNA. It is nucleolytically cleaved and chemically modified to generate mature rRNAs, which assemble with ribosomal proteins to form the ribosome. Prp43 is required for the processing of the 18S rRNA. Using X-ray crystallography, I determined a high resolution structure of Prp43 bound to ADP, the first structure of a DEAH....../RHA helicase. It defined the conserved structural features of all DEAH/RHA helicases, and unveiled a novel nucleotide binding site. Additionally a preliminary low resolution structure of a ternary complex comprising Prp43, a non-hydrolyzable ATP analogue, and a single-stranded RNA, was obtained. The ribosome...

  20. Structure of the hepatitis C virus IRES bound to the human 80S ribosome: remodeling of the HCV IRES.

    Science.gov (United States)

    Boehringer, Daniel; Thermann, Rolf; Ostareck-Lederer, Antje; Lewis, Joe D; Stark, Holger

    2005-11-01

    Initiation of translation of the hepatitis C virus (HCV) polyprotein is driven by an internal ribosome entry site (IRES) RNA that bypasses much of the eukaryotic translation initiation machinery. Here, single-particle electron cryomicroscopy has been used to study the mechanism of HCV IRES-mediated initiation. A HeLa in vitro translation system was used to assemble human IRES-80S ribosome complexes under near physiological conditions; these were stalled before elongation. Domain 2 of the HCV IRES is bound to the tRNA exit site, touching the L1 stalk of the 60S subunit, suggesting a mechanism for the removal of the HCV IRES in the progression to elongation. Domain 3 of the HCV IRES positions the initiation codon in the ribosomal mRNA binding cleft by binding helix 28 at the head of the 40S subunit. The comparison with the previously published binary 40S-HCV IRES complex reveals structural rearrangements in the two pseudoknot structures of the HCV IRES in translation initiation.

  1. Fuel assemblies

    International Nuclear Information System (INIS)

    Mukai, Hideyuki

    1987-01-01

    Purpose: To prevent bending of fuel rods caused by the difference of irradiation growth between coupling fuel rods and standards fuel rods thereby maintain the fuel rod integrity. Constitution: The f value for a fuel can (the ratio of pole of zirconium crystals in the entire crystals along the axial direction of the fuel can) of a coupling fuel rod secured by upper and lower tie plates is made smaller than the f value for the fuel can of a standard fuel rod not secured by the upper and the lower tie plates. This can make the irradiation growth of the fuel can of the coupling fuel rod greater than the irradiation growth of the fuel can of the standard fuel rod and, accordingly, since the elongation of the standard fuel rod can always by made greater, bending of the standard fuel rod can be prevented. (Yoshihara, M.)

  2. Fuel assemblies

    International Nuclear Information System (INIS)

    Sadaoka, Noriyuki.

    1986-01-01

    Purpose: To maintain a satisfactory integrity by preventing the increase of corrosion at the outer surface of a fuel can near the point of contact between the fuel can and the spacer due to the use of fuel pellets incorporated with burnable poisons. Constitution: Since reactor coolants are at high temperature and high pressure, zirconium and water are brought into reaction to proceed oxidation at the outer surface of a fuel can to form uniform oxidation layers. However, abrasion corrosion is additionally formed at the contact portion between the spacer and the fuel can, by which the corrosion is increased by about 25 %. For preventing such nodular corrosion, fuel pellets not incorporated with burnable poisons are charged at a portion of the fuel rod where the spacer is supported and fuel pellets incorporated with burnable poisons are charged at the positions other than about to thereby suppress the amount of the corrosion at the portion where the corrosion of the fuel can is most liable to be increased to thereby improve the fuel integrity. That is, radiolysis of coolants due to gamma-rays produced from gadolinium is lowered to reduce the oxygen concentration near the outer surface thereby preventing the corrosion. (Kawakami, Y.)

  3. Loss of ribosomal protein L11 affects zebrafish embryonic development through a p53-dependent apoptotic response.

    Directory of Open Access Journals (Sweden)

    Anirban Chakraborty

    Full Text Available Ribosome is responsible for protein synthesis in all organisms and ribosomal proteins (RPs play important roles in the formation of a functional ribosome. L11 was recently shown to regulate p53 activity through a direct binding with MDM2 and abrogating the MDM2-induced p53 degradation in response to ribosomal stress. However, the studies were performed in cell lines and the significance of this tumor suppressor function of L11 has yet to be explored in animal models. To investigate the effects of the deletion of L11 and its physiological relevance to p53 activity, we knocked down the rpl11 gene in zebrafish and analyzed the p53 response. Contrary to the cell line-based results, our data indicate that an L11 deficiency in a model organism activates the p53 pathway. The L11-deficient embryos (morphants displayed developmental abnormalities primarily in the brain, leading to embryonic lethality within 6-7 days post fertilization. Extensive apoptosis was observed in the head region of the morphants, thus correlating the morphological defects with apparent cell death. A decrease in total abundance of genes involved in neural patterning of the brain was observed in the morphants, suggesting a reduction in neural progenitor cells. Upregulation of the genes involved in the p53 pathway were observed in the morphants. Simultaneous knockdown of the p53 gene rescued the developmental defects and apoptosis in the morphants. These results suggest that ribosomal dysfunction due to the loss of L11 activates a p53-dependent checkpoint response to prevent improper embryonic development.

  4. Fuel assembly

    International Nuclear Information System (INIS)

    Bessho, Yasunori; Ishii, Yoshihiko; Sadaoka, Noriyuki.

    1990-01-01

    Burnable poisons are disposed in the lower portions of a water rod, a channel box and a control rod guide pipe in a fuel assembly, and the amount for each of them is set to burn out in one operation cycle. Since the inner side of the water rod and the control rod guide pipe and gaps are filled with steams at the initial and the intermediate stages of the operation cycle, moderation of neutrons is delayed to harden the spectrum. On the other hand, since the burnable poisons are burnt out in the final stage of the operation cycle, γ-ray heating is not expected and since the insides of the water rod and the control rod guide pipe and the gaps are filled with water of great moderation effect, the neutron spectrum arae softened. In view of the above, void coefficient is increased to promote conversion from U-235 to Pu-239 by utilizing exothermic reaction of burnable poisons at the initial and the intermediate stages in the operation cycle and generation of voids are eliminated at the final stage where the burnable poisons are burnt out, thereby enabling effective burning of Pu-239. (N.H.)

  5. Fuel assembly

    International Nuclear Information System (INIS)

    Yamazaki, Hajime.

    1995-01-01

    In a fuel assembly having fuel rods of different length, fuel pellets of mixed oxides of uranium and plutonium are loaded to a short fuel rod. The volume ratio of a pellet-loaded portion to a plenum portion of the short fuel rod is made greater than the volume ratio of a fuel rod to which uranium fuel pellets are loaded. In addition, the volume of the plenum portion of the short fuel rod is set greater depending on the plutonium content in the loaded fuel pellets. MOX fuel pellets are loaded on the short fuel rods having a greater degree of freedom relevant to the setting for the volume of the plenum portion compared with that of a long rod fuel, and the volume of the plenum portion is ensured greater depending on the plutonium content. Even if a large amount of FP gas and He gas are discharged from the MOX fuels compared with that from the uranium fuels, the internal pressure of the MOX fuel rod during operation is maintained substantially identical with that of the uranium fuel rod, so that a risk of generating excess stresses applied to the fuel cladding tubes and rupture of fuels are greatly reduced. (N.H.)

  6. Fuel assembly

    International Nuclear Information System (INIS)

    Nakajima, Akiyoshi; Bessho, Yasunori; Aoyama, Motoo; Koyama, Jun-ichi; Hirakawa, Hiromasa; Yamashita, Jun-ichi; Hayashi, Tatsuo

    1998-01-01

    In a fuel assembly of a BWR type reactor in which a water rod of a large diameter is disposed at the central portion, the cross sectional area perpendicular to the axial direction comprises a region a of a fuel rod group facing to a wide gap water region to which a control rod is inserted, a region b of a fuel rod group disposed on the side of the wide gap water region other than the region a, a region d of a fuel rod group facing to a narrow gap water region and a region c of a fuel rod group disposed on the side of the narrow gap water region other than the region d. When comparing an amount of fission products contained in the four regions relative to that in the entire regions and average enrichment degrees of fuel rods for the four regions, the relative amount and the average enrichment degree of the fuel rod group of the region a is minimized, and the relative amount and the average enrichment degree of the fuel rod group in the region b is maximized. Then, reactor shut down margin during cold operation can be improved while flattening the power in the cross section perpendicular to the axial direction. (N.H.)

  7. Kinetic impairment of restrictive streptomycin-resistant ribosomes.

    Science.gov (United States)

    Bohman, K; Ruusala, T; Jelenc, P C; Kurland, C G

    1984-01-01

    Comparisons in vivo and in vitro of wild-type and otherwise isogenic bacteria with five different mutant alleles of the gene (rpsL) specifying ribosomal protein S12, all resistant to high levels of streptomycin, show that the streptomycin-resistant (Smr) phenotype can be subdivided into major groups: restrictive and non-restrictive. The restrictive bacteria have a characteristically lower frequency of nonsense suppression in vivo, and are also slower than the wild type in their rate of protein synthesis. Non-restrictive Smr bacteria on the other hand do not differ significantly from the wild type either in nonsense suppression frequencies or in the rate of translation. A complementary pattern is seen in vitro, where ribosomes from the restrictive Smr bacteria translate poly(U) with a significantly lower missense error frequency than wild-type ribosomes, and also show an increased Michaelis constant (KM) with respect to their substrate, i.e. ternary complexes. Both effects are correlated with the more aggressive proofreading function that is characteristic of these restrictive ribosomes. In contrast, ribosomes isolated from the non-restrictive Smr bacteria do not show any major difference in either proofreading or missense error in vitro when compared to the wild type.

  8. Engineered Ribosomes for Basic Science and Synthetic Biology.

    Science.gov (United States)

    d'Aquino, Anne E; Kim, Do Soon; Jewett, Michael C

    2018-03-28

    The ribosome is the cell's factory for protein synthesis. With protein synthesis rates of up to 20 amino acids per second and at an accuracy of 99.99%, the extraordinary catalytic capacity of the bacterial translation machinery has attracted extensive efforts to engineer, reconstruct, and repurpose it for biochemical studies and novel functions. Despite these efforts, the potential for harnessing the translation apparatus to manufacture bio-based products beyond natural limits remains underexploited, and fundamental constraints on the chemistry that the ribosome's RNA-based active site can carry out are unknown. This review aims to cover the past and present advances in ribosome design and engineering to understand the fundamental biology of the ribosome to facilitate the construction of synthetic manufacturing machines. The prospects for the development of engineered, or designer, ribosomes for novel polymer synthesis are reviewed, future challenges are considered, and promising advances in a variety of applications are discusse Expected final online publication date for the Annual Review of Chemical and Biomolecular Engineering Volume 9 is June 7, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  9. Ribosomes slide on lysine-encoding homopolymeric A stretches

    Science.gov (United States)

    Koutmou, Kristin S; Schuller, Anthony P; Brunelle, Julie L; Radhakrishnan, Aditya; Djuranovic, Sergej; Green, Rachel

    2015-01-01

    Protein output from synonymous codons is thought to be equivalent if appropriate tRNAs are sufficiently abundant. Here we show that mRNAs encoding iterated lysine codons, AAA or AAG, differentially impact protein synthesis: insertion of iterated AAA codons into an ORF diminishes protein expression more than insertion of synonymous AAG codons. Kinetic studies in E. coli reveal that differential protein production results from pausing on consecutive AAA-lysines followed by ribosome sliding on homopolymeric A sequence. Translation in a cell-free expression system demonstrates that diminished output from AAA-codon-containing reporters results from premature translation termination on out of frame stop codons following ribosome sliding. In eukaryotes, these premature termination events target the mRNAs for Nonsense-Mediated-Decay (NMD). The finding that ribosomes slide on homopolymeric A sequences explains bioinformatic analyses indicating that consecutive AAA codons are under-represented in gene-coding sequences. Ribosome ‘sliding’ represents an unexpected type of ribosome movement possible during translation. DOI: http://dx.doi.org/10.7554/eLife.05534.001 PMID:25695637

  10. The Complete Structure of the Mycobacterium smegmatis 70S Ribosome.

    Science.gov (United States)

    Hentschel, Jendrik; Burnside, Chloe; Mignot, Ingrid; Leibundgut, Marc; Boehringer, Daniel; Ban, Nenad

    2017-07-05

    The ribosome carries out the synthesis of proteins in every living cell. It consequently represents a frontline target in anti-microbial therapy. Tuberculosis ranks among the leading causes of death worldwide, due in large part to the combination of difficult-to-treat latency and antibiotic resistance. Here, we present the 3.3-Å cryo-EM structure of the 70S ribosome of Mycobacterium smegmatis, a close relative to the human pathogen Mycobacterium tuberculosis. The structure reveals two additional ribosomal proteins and localizes them to the vicinity of drug-target sites in both the catalytic center and the decoding site of the ribosome. Furthermore, we visualized actinobacterium-specific rRNA and protein expansions that extensively remodel the ribosomal surface with implications for polysome organization. Our results provide a foundation for understanding the idiosyncrasies of mycobacterial translation and reveal atomic details of the structure that will facilitate the design of anti-tubercular therapeutics. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  11. Class of cyclic ribosomal peptide synthetic genes in filamentous fungi.

    Science.gov (United States)

    Nagano, Nozomi; Umemura, Myco; Izumikawa, Miho; Kawano, Jin; Ishii, Tomoko; Kikuchi, Moto; Tomii, Kentaro; Kumagai, Toshitaka; Yoshimi, Akira; Machida, Masayuki; Abe, Keietsu; Shin-Ya, Kazuo; Asai, Kiyoshi

    2016-01-01

    Ustiloxins were found recently to be the first example of cyclic peptidyl secondary metabolites that are ribosomally synthesized in filamentous fungi. In this work, two function-unknown genes (ustYa/ustYb) in the gene cluster for ustiloxins from Aspergillus flavus were found experimentally to be involved in cyclization of the peptide. Their homologous genes are observed mainly in filamentous fungi and mushrooms. They have two "HXXHC" motifs that might form active sites. Computational genome analyses showed that these genes are frequently located near candidate genes for ribosomal peptide precursors, which have signal peptides at the N-termini and repeated sequences with core peptides for the cyclic portions, in the genomes of filamentous fungi, particularly Aspergilli, as observed in the ustiloxin gene cluster. Based on the combination of the ustYa/ustYb homologous genes and the nearby ribosomal peptide precursor candidate genes, 94 ribosomal peptide precursor candidates that were identified computationally from Aspergilli genome sequences were classified into more than 40 types including a wide variety of core peptide sequences. A set of the predicted ribosomal peptide biosynthetic genes was experimentally verified to synthesize a new cyclic peptide compound, designated as asperipin-2a, which comprises the amino acid sequence in the corresponding precursor gene, distinct from the ustiloxin precursors. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Ribosome. The complete structure of the 55S mammalian mitochondrial ribosome.

    Science.gov (United States)

    Greber, Basil J; Bieri, Philipp; Leibundgut, Marc; Leitner, Alexander; Aebersold, Ruedi; Boehringer, Daniel; Ban, Nenad

    2015-04-17

    Mammalian mitochondrial ribosomes (mitoribosomes) synthesize mitochondrially encoded membrane proteins that are critical for mitochondrial function. Here we present the complete atomic structure of the porcine 55S mitoribosome at 3.8 angstrom resolution by cryo-electron microscopy and chemical cross-linking/mass spectrometry. The structure of the 28S subunit in the complex was resolved at 3.6 angstrom resolution by focused alignment, which allowed building of a detailed atomic structure including all of its 15 mitoribosomal-specific proteins. The structure reveals the intersubunit contacts in the 55S mitoribosome, the molecular architecture of the mitoribosomal messenger RNA (mRNA) binding channel and its interaction with transfer RNAs, and provides insight into the highly specialized mechanism of mRNA recruitment to the 28S subunit. Furthermore, the structure contributes to a mechanistic understanding of aminoglycoside ototoxicity. Copyright © 2015, American Association for the Advancement of Science.

  13. Site-specific labeling of Saccharomyces cerevisiae ribosomes for single-molecule manipulations

    Science.gov (United States)

    Petrov, Alexey; Puglisi, Joseph D.

    2010-01-01

    Site-specific labeling of Escherichia coli ribosomes has allowed application of single-molecule fluorescence spectroscopy and force methods to probe the mechanism of translation. To apply these approaches to eukaryotic translation, eukaryotic ribosomes must be specifically labeled with fluorescent labels and molecular handles. Here, we describe preparation and labeling of the small and large yeast ribosomal subunits. Phylogenetically variable hairpin loops in ribosomal RNA are mutated to allow hybridization of oligonucleotides to mutant ribosomes. We demonstrate specific labeling of the ribosomal subunits, and their use in single-molecule fluorescence and force experiments. PMID:20501598

  14. Kinase-Mediated Regulation of 40S Ribosome Assembly in Human Breast Cancer

    Science.gov (United States)

    2017-02-01

    Katrin Karbstein CONTRACTING ORGANIZATION: The Scripps Research Institute Jupiter , FL 33458 REPORT DATE: February 2017 TYPE OF REPORT: Annual...16-1-0008 5c. PROGRAM ELEMENT NUMBER 5. AUTHOR(S) Katrin Karbstein 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 6. PERFORMING...ORGANIZATION NAME(S) AND ADDRESS(ES) The Scripps Research Institute Jupiter , FL 33458 8. PERFORMING ORGANIZATION REPORT NUMBER 9. SPONSORING/MONITORING AGENCY

  15. Interaction of tRNA with Eukaryotic Ribosome

    Directory of Open Access Journals (Sweden)

    Dmitri Graifer

    2015-03-01

    Full Text Available This paper is a review of currently available data concerning interactions of tRNAs with the eukaryotic ribosome at various stages of translation. These data include the results obtained by means of cryo-electron microscopy and X-ray crystallography applied to various model ribosomal complexes, site-directed cross-linking with the use of tRNA derivatives bearing chemically or photochemically reactive groups in the CCA-terminal fragment and chemical probing of 28S rRNA in the region of the peptidyl transferase center. Similarities and differences in the interactions of tRNAs with prokaryotic and eukaryotic ribosomes are discussed with concomitant consideration of the extent of resemblance between molecular mechanisms of translation in eukaryotes and bacteria.

  16. mRNA pseudoknot structures can act as ribosomal roadblocks

    DEFF Research Database (Denmark)

    Hansen, Jesper Tholstrup; Oddershede, Lene Broeng; Sørensen, Michael Askvad

    2012-01-01

    Several viruses utilize programmed ribosomal frameshifting mediated by mRNA pseudoknots in combination with a slippery sequence to produce a well defined stochiometric ratio of the upstream encoded to the downstream-encoded protein. A correlation between the mechanical strength of mRNA pseudoknots...... and frameshifting efficiency has previously been found; however, the physical mechanism behind frameshifting still remains to be fully understood. In this study, we utilized synthetic sequences predicted to form mRNA pseudoknot-like structures. Surprisingly, the structures predicted to be strongest lead only...... to limited frameshifting. Two-dimensional gel electrophoresis of pulse labelled proteins revealed that a significant fraction of the ribosomes were frameshifted but unable to pass the pseudoknot-like structures. Hence, pseudoknots can act as ribosomal roadblocks, prohibiting a significant fraction...

  17. A streamlined ribosome profiling protocol for the characterization of microorganisms

    DEFF Research Database (Denmark)

    Latif, Haythem; Szubin, Richard; Tan, Justin

    2015-01-01

    in the microbial research community. Here we present a streamlined ribosome profiling protocol with reduced barriers to entry for microbial characterization studies. Our approach provides simplified alternatives during harvest, lysis, and recovery of monosomes and also eliminates several time-consuming steps......Ribosome profiling is a powerful tool for characterizing in vivo protein translation at the genome scale, with multiple applications ranging from detailed molecular mechanisms to systems-level predictive modeling. Though highly effective, this intricate technique has yet to become widely used...

  18. A functional pseudoknot in 16S ribosomal RNA.

    OpenAIRE

    Powers, T; Noller, H F

    1991-01-01

    Several lines of evidence indicate that the universally conserved 530 loop of 16S ribosomal RNA plays a crucial role in translation, related to the binding of tRNA to the ribosomal A site. Based upon limited phylogenetic sequence variation, Woese and Gutell (1989) have proposed that residues 524-526 in the 530 hairpin loop are base paired with residues 505-507 in an adjoining bulge loop, suggesting that this region of 16S rRNA folds into a pseudoknot structure. Here, we demonstrate that Watso...

  19. Evidence of novel type of ribosome in eukaryotic intermediate flatworm

    OpenAIRE

    Haoran H. Wang; Mary X. Gao; Sato Kenzo; Kazumitsu Hirai

    2008-01-01

    In all organisms, messenger-directed protein synthesis is catalyzed by ribonucleoprotein particles called ribosomes. A ribosome is typically composed of one small and one large subunit which contain one short (18S) and one long (28S) rRNAs, respectively. Surprisingly, in this study, three similar size rRNAs (18-21S) were revealed in the electrophoresis profile of the total RNAs of tapeworm _Spirometra erinaceiuropaei_. Northern blot analysis shows that one of the three bands belongs to 18S rR...

  20. Initial bridges between two ribosomal subunits are formed within 9.4 milliseconds, as studied by time-resolved cryo-EM.

    Science.gov (United States)

    Shaikh, Tanvir R; Yassin, Aymen S; Lu, Zonghuan; Barnard, David; Meng, Xing; Lu, Toh-Ming; Wagenknecht, Terence; Agrawal, Rajendra K

    2014-07-08

    Association of the two ribosomal subunits during the process of translation initiation is a crucial step of protein synthesis. The two subunits (30S and 50S) of the bacterial 70S ribosome are held together by 12 dynamic bridges involving RNA-RNA, RNA-protein, and protein-protein interactions. The process of bridge formation, such as whether all these bridges are formed simultaneously or in a sequential order, is poorly understood. To understand such processes, we have developed and implemented a class of microfluidic devices that mix two components to completion within 0.4 ms and spray the mixture in the form of microdroplets onto an electron microscopy grid, yielding a minimum reaction time of 9.4 ms before cryofixation. Using these devices, we have obtained cryo-EM data corresponding to reaction times of 9.4 and 43 ms and have determined 3D structures of ribosomal subunit association intermediates. Molecular analyses of the cryo-EM maps reveal that eight intersubunit bridges (bridges B1a, B1b, B2a, B2b, B3, B7a, B7b, and B8) form within 9.4 ms, whereas the remaining four bridges (bridges B2c, B4, B5, and B6) take longer than 43 ms to form, suggesting that bridges are formed in a stepwise fashion. Our approach can be used to characterize sequences of various dynamic functional events on complex macromolecular assemblies such as ribosomes.

  1. Isolation, crystallization, and investigation of ribosomal protein S8 complexed with specific fragments of rRNA of bacterial or archaeal origin.

    Science.gov (United States)

    Tishchenko, S V; Vassilieva, J M; Platonova, O B; Serganov, A A; Fomenkova, N P; Mudrik, E S; Piendl, W; Ehresmann, C; Ehresmann, B; Garber, M B

    2001-09-01

    The core ribosomal protein S8 binds to the central domain of 16S rRNA independently of other ribosomal proteins and is required for assembling the 30S subunit. It has been shown with E. coli ribosomes that a short rRNA fragment restricted by nucleotides 588-602 and 636-651 is sufficient for strong and specific protein S8 binding. In this work, we studied the complexes formed by ribosomal protein S8 from Thermus thermophilus and Methanococcus jannaschii with short rRNA fragments isolated from the same organisms. The dissociation constants of the complexes of protein S8 with rRNA fragments were determined. Based on the results of binding experiments, rRNA fragments of different length were designed and synthesized in preparative amounts in vitro using T7 RNA-polymerase. Stable S8-RNA complexes were crystallized. Crystals were obtained both for homologous bacterial and archaeal complexes and for hybrid complexes of archaeal protein with bacterial rRNA. Crystals of the complex of protein S8 from M. jannaschii with the 37-nucleotide rRNA fragment from the same organism suitable for X-ray analysis were obtained.

  2. Crystal structure of ribosomal protein S8 from Thermus thermophilus reveals a high degree of structural conservation of a specific RNA binding site.

    Science.gov (United States)

    Nevskaya, N; Tishchenko, S; Nikulin, A; al-Karadaghi, S; Liljas, A; Ehresmann, B; Ehresmann, C; Garber, M; Nikonov, S

    1998-05-29

    S8 is one of the core ribosomal proteins. It binds to 16 S RNA with high affinity and independently of other ribosomal proteins. It also acts as a translational repressor in Escherichia coli by binding to its own mRNA. The structure of Thermus thermophilus S8 has been determined by the method of multiple isomorphous replacement at 2.9 A resolution and refined to a crystallographic R-factor of 16.2% (Rfree 27.5%). The two domains of the structure have an alpha/beta fold and are connected by a long protruding loop. The two molecules in the asymmetric unit of the crystal interact through an extensive hydrophobic core and form a tightly associated dimer, while symmetry-related molecules form a joint beta-sheet of mixed type. This type of protein-protein interaction could be realized within the ribosomal assembly. A comparison of the structures of T. thermophilus and Bacillus stearothermophilus S8 shows that the interdomain loop is eight residues longer in the former and reveals high structural conservation of an extensive region, located in the C-terminal domain. From mutational studies this region was proposed earlier to be involved in specific interaction with RNA. On the basis of these data and on the comparison of the two structures of S8, it is proposed that the three-dimensional structure of specific RNA binding sites in ribosomal proteins is highly conserved among different species.

  3. Combined Effect of the Cfr Methyltransferase and Ribosomal Protein L3 Mutations on Resistance to Ribosome-Targeting Antibiotics

    DEFF Research Database (Denmark)

    Pakula, Kevin K; Hansen, Lykke H; Vester, Birte

    2017-01-01

    Several groups of antibiotics inhibit bacterial growth by binding to bacterial ribosomes. Mutations in ribosomal protein L3 have been associated with resistance to linezolid and tiamulin, which both bind at the peptidyl transferase center in the ribosome. Resistance to these and other antibiotics....... The presence of Cfr has a very minor influence on the growth rate. The resistance of the transformants to linezolid, tiamulin, florfenicol, and Synercid (a combination of quinupristin and dalfopristin [Q-D]) was measured by MIC assays. The resistance from Cfr was, in all cases, stronger than the effects...... of the L3 mutations, but various effects were obtained with the combinations of Cfr and L3 mutations ranging from a synergistic to an antagonistic effect. Linezolid and tiamulin susceptibility varied greatly among the L3 mutations, while no significant effects on florfenicol and Q-D susceptibility were...

  4. Mutations in the 915 region of Escherichia coli 16S ribosomal RNA reduce the binding of streptomycin to the ribosome.

    Science.gov (United States)

    Leclerc, D; Melançon, P; Brakier-Gingras, L

    1991-01-01

    The nine possible single-base substitutions were produced at positions 913 to 915 of the 16S ribosomal RNA of Escherichia coli, a region known to be protected by streptomycin [Moazed, D. and Noller, H.F. (1987) Nature, 327, 389-394]. When the mutations were introduced into the expression vector pKK3535, only two of them (913A----G and 915A----G) permitted recovery of viable transformants. Ribosomes were isolated from the transformed bacteria and were assayed for their response to streptomycin in poly(U)- and MS2 RNA-directed assays. They were resistant to the stimulation of misreading and to the inhibition of protein synthesis by streptomycin, and this correlated with a decreased binding of the drug. These results therefore demonstrate that, in line with the footprinting studies of Moazed and Noller, mutations in the 915 region alter the interaction between the ribosome and streptomycin. PMID:1713666

  5. Nuclear fuel assembly

    International Nuclear Information System (INIS)

    Domoto, Noboru; Masuda, Hiroyuki

    1989-01-01

    In a nuclear fuel assembly loaded with a plurality of fuel rods, the inside of a fuel rod disposed at a high neutron flux region is divided into an inner region and an outer region, and more burnable poisons are mixed in the inner region than in the outer region. Alternatively, the central portion of a pellet disposed in a high neutron flux region is made hollow, in which burnable poisons are charged. This can prevent neutron infinite multiplication factor from decreasing extremely at the initial burning stage. Further, the burnable poisons are not rapidly burnt completely and local peaking coefficient can be controlled. Accordingly, in a case of suppressing a predetermined excess reactivity by using a fuel rod incorporated with the burnable poison, the fuel economy can be improved more and the reactor core controllability can also be improved as compared with the usual case. (T.M.)

  6. Cryo-EM Structure of the Archaeal 50S Ribosomal Subunit in Complex with Initiation Factor 6 and Implications for Ribosome Evolution

    Science.gov (United States)

    Greber, Basil J.; Boehringer, Daniel; Godinic-Mikulcic, Vlatka; Crnkovic, Ana; Ibba, Michael; Weygand-Durasevic, Ivana; Ban, Nenad

    2013-01-01

    Translation of mRNA into proteins by the ribosome is universally conserved in all cellular life. The composition and complexity of the translation machinery differ markedly between the three domains of life. Organisms from the domain Archaea show an intermediate level of complexity, sharing several additional components of the translation machinery with eukaryotes that are absent in bacteria. One of these translation factors is initiation factor 6 (IF6), which associates with the large ribosomal subunit. We have reconstructed the 50S ribosomal subunit from the archaeon Methanothermobacter thermautotrophicus in complex with archaeal IF6 at 6.6 Å resolution using cryo-electron microscopy (EM). The structure provides detailed architectural insights into the 50S ribosomal subunit from a methanogenic archaeon through identification of the rRNA expansion segments and ribosomal proteins that are shared between this archaeal ribosome and eukaryotic ribosomes but are mostly absent in bacteria and in some archaeal lineages. Furthermore, the structure reveals that, in spite of highly divergent evolutionary trajectories of the ribosomal particle and the acquisition of novel functions of IF6 in eukaryotes, the molecular binding of IF6 on the ribosome is conserved between eukaryotes and archaea. The structure also provides a snapshot of the reductive evolution of the archaeal ribosome and offers new insights into the evolution of the translation system in archaea. PMID:22306461

  7. Ribosome Reinitiation Directs Gene-specific Translation and Regulates the Integrated Stress Response*

    Science.gov (United States)

    Young, Sara K.; Willy, Jeffrey A.; Wu, Cheng; Sachs, Matthew S.; Wek, Ronald C.

    2015-01-01

    In the integrated stress response, phosphorylation of eIF2α (eIF2α-P) reduces protein synthesis to conserve resources and facilitate preferential translation of transcripts that promote stress adaptation. Preferentially translated GADD34 (PPP1R15A) and constitutively expressed CReP (PPP1R15B) function to dephosphorylate eIF2α-P and restore protein synthesis. The 5′-leaders of GADD34 and CReP contain two upstream ORFs (uORFs). Using biochemical and genetic approaches we show that features of these uORFs are central for their differential expression. In the absence of stress, translation of an inhibitory uORF in GADD34 acts as a barrier that prevents reinitiation at the GADD34 coding region. Enhanced eIF2α-P during stress directs ribosome bypass of the uORF, facilitating translation of the GADD34 coding region. CReP expression occurs independent of eIF2α-P via an uORF that allows for translation reinitiation at the CReP coding region independent of stress. Importantly, alterations in the GADD34 uORF affect the status of eIF2α-P, translational control, and cell adaptation to stress. These results show that properties of uORFs that permit ribosome reinitiation are critical for directing gene-specific translational control in the integrated stress response. PMID:26446796

  8. Mechanism of recycling of post-termination ribosomal complexes in ...

    Indian Academy of Sciences (India)

    We discuss the overall pathway of ribosome recycling in eubacteria with especial reference to the important role of the initiation factor 3 (IF3) in this process. Depending on the step(s) at which IF3 function is implicated, three models have been proposed. In model 1, RRF and EFG dissociate the post-TCs into the 50S and ...

  9. The evolution and utility of ribosomal ITS sequences in Bambusinae ...

    Indian Academy of Sciences (India)

    Ribosomal internal transcribed spacer (ITS) sequences are commonly used for phylogenetic reconstruction because they are highly reiterated as components of rDNA repeats, and hence are often subject to rapid homogenization through concerted evolution. Concerted evolution leads to intragenomic uniformity of repeats ...

  10. Non-ribosomal peptide synthetases: Identifying the cryptic gene ...

    Indian Academy of Sciences (India)

    2017-01-19

    Jan 19, 2017 ... Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) present in bacteria and fungi are the major multi-modular enzyme complexes which synthesize secondary metabolites like the pharmacologically impor- tant antibiotics and siderophores. Each of the multiple modules of an ...

  11. The evolution and utility of ribosomal ITS sequences in Bambusinae ...

    Indian Academy of Sciences (India)

    2012-08-09

    Aug 9, 2012 ... both direct sequencing and cloning of PCR products were used for ITS analysis, with only one clone for ... ITS sequence was typically cloned to represent the mul- tiple copies of each plant ribosomal ...... Muir G. and Schlötterer C. 1999 Limitations to the phylogenetic use of ITS sequences in closely related ...

  12. (Brassicaceae) based on nuclear ribosomal ITS DNA sequences

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Genetics; Volume 93; Issue 2. Phylogeny and biogeography of Alyssum (Brassicaceae) based on nuclear ribosomal ITS DNA sequences. Yan Li Yan Kong Zhe Zhang Yanqiang Yin Bin Liu Guanghui Lv Xiyong Wang. Research Article Volume 93 Issue 2 August 2014 pp 313-323 ...

  13. Non-ribosomal peptide synthetases: Identifying the cryptic gene ...

    Indian Academy of Sciences (India)

    Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) present in bacteria and fungi are themajor multi-modular enzyme complexes which synthesize secondary metabolites like the pharmacologically importantantibiotics and siderophores. Each of the multiple modules of an NRPS activates a ...

  14. Reverse translocation of tRNA in the ribosome.

    Science.gov (United States)

    Shoji, Shinichiro; Walker, Sarah E; Fredrick, Kurt

    2006-12-28

    A widely held view is that directional movement of tRNA in the ribosome is determined by an intrinsic mechanism and driven thermodynamically by transpeptidation. Here, we show that, in certain ribosomal complexes, the pretranslocation (PRE) state is thermodynamically favored over the posttranslocation (POST) state. Spontaneous and efficient conversion from the POST to PRE state is observed when EF-G is depleted from ribosomes in the POST state or when tRNA is added to the E site of ribosomes containing P-site tRNA. In the latter assay, the rate of tRNA movement is increased by streptomycin and neomycin, decreased by tetracycline, and not affected by the acylation state of the tRNA. In one case, we provide evidence that complex conversion occurs by reverse translocation (i.e., direct movement of the tRNAs from the E and P sites to the P and A sites, respectively). These findings have important implications for the energetics of translocation.

  15. PCR primers for metazoan mitochondrial 12S ribosomal DNA sequences.

    Directory of Open Access Journals (Sweden)

    Ryuji J Machida

    Full Text Available BACKGROUND: Assessment of the biodiversity of communities of small organisms is most readily done using PCR-based analysis of environmental samples consisting of mixtures of individuals. Known as metagenetics, this approach has transformed understanding of microbial communities and is beginning to be applied to metazoans as well. Unlike microbial studies, where analysis of the 16S ribosomal DNA sequence is standard, the best gene for metazoan metagenetics is less clear. In this study we designed a set of PCR primers for the mitochondrial 12S ribosomal DNA sequence based on 64 complete mitochondrial genomes and then tested their efficacy. METHODOLOGY/PRINCIPAL FINDINGS: A total of the 64 complete mitochondrial genome sequences representing all metazoan classes available in GenBank were downloaded using the NCBI Taxonomy Browser. Alignment of sequences was performed for the excised mitochondrial 12S ribosomal DNA sequences, and conserved regions were identified for all 64 mitochondrial genomes. These regions were used to design a primer pair that flanks a more variable region in the gene. Then all of the complete metazoan mitochondrial genomes available in NCBI's Organelle Genome Resources database were used to determine the percentage of taxa that would likely be amplified using these primers. Results suggest that these primers will amplify target sequences for many metazoans. CONCLUSIONS/SIGNIFICANCE: Newly designed 12S ribosomal DNA primers have considerable potential for metazoan metagenetic analysis because of their ability to amplify sequences from many metazoans.

  16. The Ribosome and the 2009 Nobel Prize in Chemistry

    Indian Academy of Sciences (India)

    The 2009 Nobel Prize in Chemistry was awarded jointly to. Venkatraman Ramakrishnan, Thomas Steitz and Ada Yonath. “for studies on the structure and function of the ribosome”. The ribosome is a massive molecular machine comprising proteins and RNA and is responsible for the synthesis of proteins, often termed the ...

  17. Expression of a ribosome inactivating protein (curcin 2) in Jatropha ...

    Indian Academy of Sciences (India)

    Expression of a ribosome inactivating protein (curcin 2) in Jatropha curcas is induced by stress ... Curcin 2; Jatropha curcas; protein induction; stress ... College of Light Industry and Food Engineering Sciences, Sichuan University, Chengdu 610 065, PR China; College of Life Sciences, Sichuan University, Chengdu 610 065 ...

  18. cDNA, genomic sequence cloning and overexpression of ribosomal ...

    African Journals Online (AJOL)

    RPS16 of eukaryote is a component of the 40S small ribosomal subunit encoded by RPS16 gene and is also a homolog of prokaryotic RPS9. The cDNA and genomic sequence of RPS16 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using reverse transcription-polymerase chain ...

  19. Sequence-dependent elongation dynamics on macrolide-bound ribosomes.

    Science.gov (United States)

    Johansson, Magnus; Chen, Jin; Tsai, Albert; Kornberg, Guy; Puglisi, Joseph D

    2014-06-12

    The traditional view of macrolide antibiotics as plugs inside the ribosomal nascent peptide exit tunnel (NPET) has lately been challenged in favor of a more complex, heterogeneous mechanism, where drug-peptide interactions determine the fate of a translating ribosome. To investigate these highly dynamic processes, we applied single-molecule tracking of elongating ribosomes during inhibition of elongation by erythromycin of several nascent chains, including ErmCL and H-NS, which were shown to be, respectively, sensitive and resistant to erythromycin. Peptide sequence-specific changes were observed in translation elongation dynamics in the presence of a macrolide-obstructed NPET. Elongation rates were not severely inhibited in general by the presence of the drug; instead, stalls or pauses were observed as abrupt events. The dynamic pathways of nascent-chain-dependent elongation pausing in the presence of macrolides determine the fate of the translating ribosome stalling or readthrough. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Nuclear ribosomal DNA diversity of a cotton pest ( Rotylenchulus ...

    African Journals Online (AJOL)

    The reniform nematode (Rotylenchulus reniformis) has emerged as a major cotton pest in the United States. A recent analysis of over 20 amphimictic populations of this pest from the US and three other countries has shown no sequence variation at the nuclear ribosomal internal transcribed spacer (ITS) despite the region's ...

  1. The 'rare allele phenomenon' in a ribosomal spacer

    NARCIS (Netherlands)

    Schilthuizen, M.; Hoekstra, R.F.; Gittenberger, E.

    2001-01-01

    We describe the increased frequency of a particular length variant of the internal transcribed spacer 1 (ITS-1) of the ribosomal DNA in a hybrid zone of the land snail Albinaria hippolyti. The phenomenon that normally rare alleles or other markers can increase in frequency in the centre of hybrid

  2. The fluctuating ribosome: thermal molecular dynamics characterized by neutron scattering

    Science.gov (United States)

    Zaccai, Giuseppe; Natali, Francesca; Peters, Judith; Řihová, Martina; Zimmerman, Ella; Ollivier, J.; Combet, J.; Maurel, Marie-Christine; Bashan, Anat; Yonath, Ada

    2016-11-01

    Conformational changes associated with ribosome function have been identified by X-ray crystallography and cryo-electron microscopy. These methods, however, inform poorly on timescales. Neutron scattering is well adapted for direct measurements of thermal molecular dynamics, the ‘lubricant’ for the conformational fluctuations required for biological activity. The method was applied to compare water dynamics and conformational fluctuations in the 30 S and 50 S ribosomal subunits from Haloarcula marismortui, under high salt, stable conditions. Similar free and hydration water diffusion parameters are found for both subunits. With respect to the 50 S subunit, the 30 S is characterized by a softer force constant and larger mean square displacements (MSD), which would facilitate conformational adjustments required for messenger and transfer RNA binding. It has been shown previously that systems from mesophiles and extremophiles are adapted to have similar MSD under their respective physiological conditions. This suggests that the results presented are not specific to halophiles in high salt but a general property of ribosome dynamics under corresponding, active conditions. The current study opens new perspectives for neutron scattering characterization of component functional molecular dynamics within the ribosome.

  3. The characterization of cytoplasmic ribosomal protein genes in ...

    African Journals Online (AJOL)

    USER

    2012-04-17

    Apr 17, 2012 ... ribosomal protein genes of N. bombycis were located in syntenic blocks, indicating that their gene order was conserved ...... Figure 7. Syntenic maps of RPL3 flanking regions on chromosomes/superscaffolds in five microsporidians. ..... promoter annotation in the Drosophila melanogaster genome. Comput.

  4. Sequence analysis of mitochondrial 16S ribosomal RNA gene ...

    Indian Academy of Sciences (India)

    Unknown

    Sequence analysis of mitochondrial 16S ribosomal RNA gene fragment from seven mosquito species. YOGESH S SHOUCHE* and MILIND S PATOLE. National Center for Cell Science, Pune University Campus, Pune 411 007, India. *Corresponding author (Fax, 91-20-5672259; Email, yogesh@nccs.res.in). Mosquitoes are ...

  5. Ribosome-inactivating proteins: potent poisons and molecular tools.

    Science.gov (United States)

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

    2013-11-15

    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 other potent toxins that abolish protein synthesis: the fungal ribotoxins which directly cleave the 28S rRNA and the newly discovered Burkholderia lethal factor 1 (BLF1). BLF1 presents additional challenges to the current classification system since, like the ribotoxins, it does not possess RNA N-glycosidase activity but does irreversibly inactivate ribosomes. We further discuss whether the RIP classification should be broadened to include toxins achieving irreversible ribosome inactivation with similar turnovers to RIPs, but through different enzymatic mechanisms.

  6. Architecture of the E.coli 70S ribosome

    DEFF Research Database (Denmark)

    Burkhardt, N.; Diedrich, G.; Nierhaus, K.H.

    1997-01-01

    The 70S ribosome from E.coli was analysed by neutron scattering focusing on the shape and the internal protein-RNA-distribution of the complex. Measurements on selectively deuterated 70S particles and free 30S and 50S subunits applying conventional contrast variation and proton-spin contrast...

  7. Newnes electronics assembly handbook

    CERN Document Server

    Brindley, Keith

    2013-01-01

    Newnes Electronics Assembly Handbook: Techniques, Standards and Quality Assurance focuses on the aspects of electronic assembling. The handbook first looks at the printed circuit board (PCB). Base materials, basic mechanical properties, cleaning of assemblies, design, and PCB manufacturing processes are then explained. The text also discusses surface mounted assemblies and packaging of electromechanical assemblies, as well as the soldering process. Requirements for the soldering process; solderability and protective coatings; cleaning of PCBs; and mass solder/component reflow soldering are des

  8. Structure based hypothesis of a mitochondrial ribosome rescue mechanism

    Directory of Open Access Journals (Sweden)

    Huynen Martijn A

    2012-05-01

    Full Text Available Abstract Background mtRF1 is a vertebrate mitochondrial protein with an unknown function that arose from a duplication of the mitochondrial release factor mtRF1a. To elucidate the function of mtRF1, we determined the positions that are conserved among mtRF1 sequences but that are different in their mtRF1a paralogs. We subsequently modeled the 3D structure of mtRF1a and mtRF1 bound to the ribosome, highlighting the structural implications of these differences to derive a hypothesis for the function of mtRF1. Results Our model predicts, in agreement with the experimental data, that the 3D structure of mtRF1a allows it to recognize the stop codons UAA and UAG in the A-site of the ribosome. In contrast, we show that mtRF1 likely can only bind the ribosome when the A-site is devoid of mRNA. Furthermore, while mtRF1a will adopt its catalytic conformation, in which it functions as a peptidyl-tRNA hydrolase in the ribosome, only upon binding of a stop codon in the A-site, mtRF1 appears specifically adapted to assume this extended, peptidyl-tRNA hydrolyzing conformation in the absence of mRNA in the A-site. Conclusions We predict that mtRF1 specifically recognizes ribosomes with an empty A-site and is able to function as a peptidyl-tRNA hydrolase in those situations. Stalled ribosomes with empty A-sites that still contain a tRNA bound to a peptide chain can result from the translation of truncated, stop-codon less mRNAs. We hypothesize that mtRF1 recycles such stalled ribosomes, performing a function that is analogous to that of tmRNA in bacteria. Reviewers This article was reviewed by Dr. Eugene Koonin, Prof. Knud H. Nierhaus (nominated by Dr. Sarah Teichmann and Dr. Shamil Sunyaev.

  9. Differential antibiotic sensitivity determined by the large ribosomal subunit in thermophilic archaea.

    OpenAIRE

    Ruggero, D; Londei, P

    1996-01-01

    Hybrid ribosomes obtained by mixing the ribosomal subunits of the extremely thermophilic archaea Sulfolobus solfataricus and Desulfurococcus mobilis were tested for their sensitivity to selected antibiotics. It is shown that structural differences in the large ribosomal subunits determine qualitatively and quantitatively the patterns of response to alpha-sarcin and paromomycin in these species.

  10. Ribosome profiling: a Hi-Def monitor for protein synthesis at the genome-wide scale

    Science.gov (United States)

    Michel, Audrey M; Baranov, Pavel V

    2013-01-01

    Ribosome profiling or ribo-seq is a new technique that provides genome-wide information on protein synthesis (GWIPS) in vivo. It is based on the deep sequencing of ribosome protected mRNA fragments allowing the measurement of ribosome density along all RNA molecules present in the cell. At the same time, the high resolution of this technique allows detailed analysis of ribosome density on individual RNAs. Since its invention, the ribosome profiling technique has been utilized in a range of studies in both prokaryotic and eukaryotic organisms. Several studies have adapted and refined the original ribosome profiling protocol for studying specific aspects of translation. Ribosome profiling of initiating ribosomes has been used to map sites of translation initiation. These studies revealed the surprisingly complex organization of translation initiation sites in eukaryotes. Multiple initiation sites are responsible for the generation of N-terminally extended and truncated isoforms of known proteins as well as for the translation of numerous open reading frames (ORFs), upstream of protein coding ORFs. Ribosome profiling of elongating ribosomes has been used for measuring differential gene expression at the level of translation, the identification of novel protein coding genes and ribosome pausing. It has also provided data for developing quantitative models of translation. Although only a dozen or so ribosome profiling datasets have been published so far, they have already dramatically changed our understanding of translational control and have led to new hypotheses regarding the origin of protein coding genes. © 2013 John Wiley & Sons, Ltd. PMID:23696005

  11. cDNA, genomic sequence cloning and analysis of the ribosomal ...

    African Journals Online (AJOL)

    Ribosomal protein L37A (RPL37A) is a component of 60S large ribosomal subunit encoded by the RPL37A gene, which belongs to the family of ribosomal L37AE proteins, located in the cytoplasm. The complementary deoxyribonucleic acid (cDNA) and the genomic sequence of RPL37A were cloned successfully from giant ...

  12. The Ribosomal RNA Genes on Neurospora crassa Mitochrondrial DNA Are Adjacent

    NARCIS (Netherlands)

    Terpstra, P.; Holtrop, M.

    1977-01-01

    Hybridization of separated 24 S and 17 S ribosomal RNA from Neurospora crassa mitochondrial ribosomes to restriction fragments of mitochondrial DNA leads to the conclusion that the large and small ribosomal RNA are adjacent on the restriction endonuclease cleavage map of the DNA. The distance

  13. Purification and properties of a ribosomal casein kinase from rabbit reticulocytes

    DEFF Research Database (Denmark)

    Issinger, O G

    1977-01-01

    A casein kinase was isolated and purifed from rabbit reticulocytes. About 90% of the enzyme activity co-sedimented with the ribosomal fraction, whereas about 10% of the enzyme activity was found in the ribosome-free supernatant. Both casein kinases (the ribosome-bound enzyme as well as the free...

  14. Affinity of ribosomal protein S8 from mesophilic and (hyper)thermophilic archaea and bacteria for 16S rRNA correlates with the growth temperatures of the organisms.

    Science.gov (United States)

    Gruber, Thomas; Köhrer, Caroline; Lung, Birgit; Shcherbakov, Dmitri; Piendl, Wolfgang

    2003-08-14

    The ribosomal protein S8 plays a pivotal role in the assembly of the 30S ribosomal subunit. Using filter binding assays, S8 proteins from mesophilic, and (hyper)thermophilic species of the archaeal genus Methanococcus and from the bacteria Escherichia coli and Thermus thermophilus were tested for their affinity to their specific 16S rRNA target site. S8 proteins from hyperthermophiles exhibit a 100-fold and S8 from thermophiles exhibit a 10-fold higher affinity than their mesophilic counterparts. Thus, there is a striking correlation of affinity of S8 proteins for their specific RNA binding site and the optimal growth temperatures of the respective organisms. The stability of individual rRNA-protein complexes might modulate the stability of the ribosome, providing a maximum of thermostability and flexibility at the growth temperature of the organism.

  15. ABC-F Proteins Mediate Antibiotic Resistance through Ribosomal Protection.

    Science.gov (United States)

    Sharkey, Liam K R; Edwards, Thomas A; O'Neill, Alex J

    2016-03-22

    Members of the ABC-F subfamily of ATP-binding cassette proteins mediate resistance to a broad array of clinically important antibiotic classes that target the ribosome of Gram-positive pathogens. The mechanism by which these proteins act has been a subject of long-standing controversy, with two competing hypotheses each having gained considerable support: antibiotic efflux versus ribosomal protection. Here, we report on studies employing a combination of bacteriological and biochemical techniques to unravel the mechanism of resistance of these proteins, and provide several lines of evidence that together offer clear support to the ribosomal protection hypothesis. Of particular note, we show that addition of purified ABC-F proteins to anin vitrotranslation assay prompts dose-dependent rescue of translation, and demonstrate that such proteins are capable of displacing antibiotic from the ribosomein vitro To our knowledge, these experiments constitute the first direct evidence that ABC-F proteins mediate antibiotic resistance through ribosomal protection.IMPORTANCEAntimicrobial resistance ranks among the greatest threats currently facing human health. Elucidation of the mechanisms by which microorganisms resist the effect of antibiotics is central to understanding the biology of this phenomenon and has the potential to inform the development of new drugs capable of blocking or circumventing resistance. Members of the ABC-F family, which includelsa(A),msr(A),optr(A), andvga(A), collectively yield resistance to a broader range of clinically significant antibiotic classes than any other family of resistance determinants, although their mechanism of action has been controversial since their discovery 25 years ago. Here we present the first direct evidence that proteins of the ABC-F family act to protect the bacterial ribosome from antibiotic-mediated inhibition. Copyright © 2016 Sharkey et al.

  16. Coupled GTPase and remodelling ATPase activities form a checkpoint for ribosome export.

    Science.gov (United States)

    Matsuo, Yoshitaka; Granneman, Sander; Thoms, Matthias; Manikas, Rizos-Georgios; Tollervey, David; Hurt, Ed

    2014-01-02

    Eukaryotic ribosomes are assembled by a complex pathway that extends from the nucleolus to the cytoplasm and is powered by many energy-consuming enzymes. Nuclear export is a key, irreversible step in pre-ribosome maturation, but mechanisms underlying the timely acquisition of export competence remain poorly understood. Here we show that a conserved Saccharomyces cerevisiae GTPase Nug2 (also known as Nog2, and as NGP-1, GNL2 or nucleostemin 2 in human) has a key role in the timing of export competence. Nug2 binds the inter-subunit face of maturing, nucleoplasmic pre-60S particles, and the location clashes with the position of Nmd3, a key pre-60S export adaptor. Nug2 and Nmd3 are not present on the same pre-60S particles, with Nug2 binding before Nmd3. Depletion of Nug2 causes premature Nmd3 binding to the pre-60S particles, whereas mutations in the G-domain of Nug2 block Nmd3 recruitment, resulting in severe 60S export defects. Two pre-60S remodelling factors, the Rea1 ATPase and its co-substrate Rsa4, are present on Nug2-associated particles, and both show synthetic lethal interactions with nug2 mutants. Release of Nug2 from pre-60S particles requires both its K(+)-dependent GTPase activity and the remodelling ATPase activity of Rea1. We conclude that Nug2 is a regulatory GTPase that monitors pre-60S maturation, with release from its placeholder site linked to recruitment of the nuclear export machinery.

  17. Hydrodynamically driven colloidal assembly in dip coating.

    Science.gov (United States)

    Colosqui, Carlos E; Morris, Jeffrey F; Stone, Howard A

    2013-05-03

    We study the hydrodynamics of dip coating from a suspension and report a mechanism for colloidal assembly and pattern formation on smooth substrates. Below a critical withdrawal speed where the coating film is thinner than the particle diameter, capillary forces induced by deformation of the free surface prevent the convective transport of single particles through the meniscus beneath the film. Capillary-induced forces are balanced by hydrodynamic drag only after a minimum number of particles assemble within the meniscus. The particle assembly can thus enter the thin film where it moves at nearly the withdrawal speed and rapidly separates from the next assembly. The interplay between hydrodynamic and capillary forces produces periodic and regular structures below a critical ratio Ca(2/3)/sqrt[Bo] particles in suspension. The hydrodynamically driven assembly documented here is consistent with stripe pattern formations observed experimentally in dip coating.

  18. Photoaffinity labeling of rat liver ribosomes by N-(2-Nitro-4-azidobenzoyl)puromycin

    International Nuclear Information System (INIS)

    Boehm, H.; Stahl, J.; Bielka, H.

    1979-01-01

    N-(2-nitro-4-azidobenzoyl)-[ 3 H]puromycin (NAB-puromycin) was synthesized as a photoreactive derivative of puromycin in order to detect ribosomal proteins located near the peptidyltransferase centre of rat liver ribosomes. Irradiation of ribosome-NAB-puromycin complexes leads to covalent attachment of the affinity label to proteins of the large ribosomal subunit, in particular to proteins L28/29, and, to a somewhat lower extent, to proteins L4, L6, L10 and L24. The results are discussed in the light of earlier studies performed with other affinity labels that attacked the peptidyltransferase region of rat liver ribosomes. (author)

  19. Structural variation of the ribosomal gene cluster within the class Insecta

    Energy Technology Data Exchange (ETDEWEB)

    Mukha, D.V.; Sidorenko, A.P.; Lazebnaya, I.V. [Vavilov Institute of General Genetics, Moscow (Russian Federation)] [and others

    1995-09-01

    General estimation of ribosomal DNA variation within the class Insecta is presented. It is shown that, using blot-hybridization, one can detect differences in the structure of the ribosomal gene cluster not only between genera within an order, but also between species within a genera, including sibling species. Structure of the ribosomal gene cluster of the Coccinellidae family (ladybirds) is analyzed. It is shown that cloned highly conservative regions of ribosomal DNA of Tetrahymena pyriformis can be used as probes for analyzing ribosomal genes in insects. 24 refs., 4 figs.

  20. Translation activity of chimeric ribosomes composed of Escherichia coli and Bacillus subtilis or Geobacillus stearothermophilus subunits

    Directory of Open Access Journals (Sweden)

    Sayaka Tsuji

    2017-07-01

    Full Text Available Ribosome composition, consisting of rRNA and ribosomal proteins, is highly conserved among a broad range of organisms. However, biochemical studies focusing on ribosomal subunit exchangeability between organisms remain limited. In this study, we show that chimeric ribosomes, composed of Escherichia coli and Bacillus subtilis or E. coli and Geobacillus stearothermophilus subunits, are active for β-galactosidase translation in a highly purified E. coli translation system. Activities of the chimeric ribosomes showed only a modest decrease when using E. coli 30 S subunits, indicating functional conservation of the 50 S subunit between these bacterial species.

  1. Activity of Colicin E3-Treated Ribosomes in Initiation and in Chain Elongation

    Science.gov (United States)

    Tai, Phang-Cheng; Davis, Bernard D.

    1974-01-01

    Colicin E3 (called E3 here) is shown to act on polysomal ribosomes as well as on free ribosomes. This treatment severely inhibits subsequent chain elongation, both on preformed polysomes completing their nascent chains and on ribosomes translating poly(U). Elevated Mg++ concentration relieves this inhibition. However, elevated Mg++ concentration does not relieve the inhibition of ribosomes initiating on natural messenger; hence damage by E3 appears to have an additional, specific effect on some stage in initiation. The effect of damage by E3 resembles that of streptomycin in several respects. This similarity suggests that the two agents may act on closely related sites on the ribosome. PMID:4598289

  2. 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. Copyright © 2011 Elsevier Ltd. All rights reserved.

  3. Fuel assembly cleaning device

    International Nuclear Information System (INIS)

    Kikuchi, Akira.

    1981-01-01

    Purpose: To enable efficient and sufficient cleaning of a fuel assembly even in corners without disassembling the assembly and to effectively remove crud. Constitution: Cleaning water mixed with abrasive is injected into a fuel assembly contained within a cleaning device body to remove crud adhering to the fuel assembly. Since a coolant passage from the opening of the bottom surface is of the fuel assembly to the opening of the top surface is utilized as the cleaning water passage at this, the crud can be removed by the abrasive in the water stream even from narrow gaps of the fuel assembly. (Aizawa, K.)

  4. Using the Ribodeblur pipeline to recover A-sites from yeast ribosome profiling data.

    Science.gov (United States)

    Wang, Hao; Kingsford, Carl; McManus, C Joel

    2018-03-15

    Ribosome profiling has emerged as a powerful technique to study mRNA translation. Ribosome profiling has the potential to determine the relative quantities and locations of ribosomes on mRNA genome wide. Taking full advantage of this approach requires accurate measurement of ribosome locations. However, experimental inconsistencies often obscure the positional information encoded in ribosome profiling data. Here, we describe the Ribodeblur pipeline, a computational analysis tool that uses a maximum likelihood framework to infer ribosome positions from heterogeneous datasets. Ribodeblur is simple to install, and can be run on an average modern Mac or Linux-based laptop. We detail the process of applying the pipeline to high-coverage ribosome profiling data in yeast, and discuss important considerations for potential extension to other organisms. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. Localization of Five Antibiotic Resistances at the Subunit Level in Chloroplast Ribosomes of Chlamydomonas

    Science.gov (United States)

    Schlanger, Gladys; Sager, Ruth

    1974-01-01

    The chloroplast ribosomes from five antibiotic resistant strains of Chlamydomonas, each carrying one mutant gene mapping in chloroplast DNA, have been shown to be resistant to the corresponding antibiotic in a poly(U)-directed amino-acid incorporating assay system. The alteration conferring resistance was localized to the 30S subunit in ribosomes from streptomycin, neamine, and spectinomycin resistant strains, and to the 50S subunit in ribosomes from cleocin and carbomycin resistant strains. Spectinomycin resistant ribosomes showed no cross-resistance to any other drugs, but limited cross-resistance was noted with the other mutant ribosomes. The similarity between these findings and results reported by others with bacterial ribosomes supports our hypothesis that at least some chloroplast ribosomal proteins are coded by genes in chloroplast DNA. Images PMID:4275942

  6. Optical Space Telescope Assembly

    Data.gov (United States)

    National Aeronautics and Space Administration — The Optical Space Telescope Assembly (OSTA) task is to demonstrate the technology readiness of assembling large space telescopes on orbit in 2015. This task is an...

  7. Pactamycin binding site on archaebacterial and eukaryotic ribosomes

    International Nuclear Information System (INIS)

    Tejedor, F.; Amils, R.; Ballesta, J.P.G.

    1987-01-01

    The presence of a photoreactive acetophenone group in the protein synthesis inhibitor pactamycin and the possibility of obtaining active iodinated derivatives that retain full biological activity allow the antibiotic binding site on Saccharomyces cerevisiae and archaebacterium Sulfolobus solfataricus ribosomes to be photoaffinity labeled. Four major labeled proteins have been identified in the yeast ribosomes, i.e., YS10, YS18, YS21/24, and YS30, while proteins AL1a, AS10/L8, AS18/20, and AS21/22 appeared as radioactive spots in S. solfataricus. There seems to be a correlation between some of the proteins labeled in yeast and those previously reported in Escherichia coli indicating that the pactamycin binding sites of both species, which are in the small subunit close to the initiation factors and mRNA binding sites, must have similar characteristics

  8. Ribosome-catalyzed formation of an abnormal peptide analogue

    International Nuclear Information System (INIS)

    Roesser, J.R.; Chorghade, M.S.; Hecht, S.M.

    1986-01-01

    The peptidyl-tRNA analogue N-(chloracetyl) phenylalanyl-tRNA/sup Phe/ was prepared by chemical aminoacylation and prebound to the P site of Escherichia coli ribosomes in response to poly(uridylic acid). Admixture of phenylalanyl-tRNA/sup Phe/ to the A site resulted in the formation of two dipeptides, one of which was found by displacement of chloride ion from the peptidyl-tRNA. This constitutes the first example of ribosome-mediated formation of a peptide of altered connectivity and suggests a need for revision of the current model of peptide bond formation. Also suggested by the present finding is the feasibility of utilizing tRNAs to prepare polypeptides of altered connectivity in an in vitro protein biosynthesizing system. [ 32 P]-oligo(rA), [ 3 H]- and [ 14 C] phenylalanines were used in the assay of the peptidye-tRNA analogue

  9. Evolution of Drosophila ribosomal protein gene core promoters.

    Science.gov (United States)

    Ma, Xiaotu; Zhang, Kangyu; Li, Xiaoman

    2009-03-01

    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, and mammals, demonstrating the evolutionary dynamics of the ribosomal gene regulatory network. We also report a motif present in similar numbers of RPGs among Drosophila species which does not appear to be conserved at the individual RPG gene level. A module-wise stabilizing selection theory is proposed to explain this observation. Overall, our results provide significant insight into the fast-evolving nature of transcriptional regulation in the RPG module.

  10. High frequency of +1 programmed ribosomal frameshifting in Euplotes octocarinatus

    OpenAIRE

    Wang, Ruanlin; Xiong, Jie; Wang, Wei; Miao, Wei; Liang, Aihua

    2016-01-01

    Programmed ?1 ribosomal frameshifting (?1 PRF) has been identified as a mechanism to regulate the expression of many viral genes and some cellular genes. The slippery site of ?1 PRF has been well characterized, whereas the +1 PRF signal and the mechanism involved in +1 PRF remain poorly understood. Previous study confirmed that +1 PRF is required for the synthesis of protein products in several genes of ciliates from the genus Euplotes. To accurately assess the frequency of genes requiring fr...

  11. Ribosomal RNA Kink-turn motif - A flexible molecular hinge

    Czech Academy of Sciences Publication Activity Database

    Rázga, F.; Špačková, Naďa; Réblová, K.; Koča, J.; Leontis, N. B.; Šponer, Jiří

    2004-01-01

    Roč. 22, č. 2 (2004), s. 183-193 ISSN 0739-1102 R&D Projects: GA MŠk LN00A016 Grant - others:Wellcome Trust(GB) GR067507MF; National Institutes of Health(US) 2R15 GM55898 Institutional research plan: CEZ:AV0Z5004920 Keywords : ribosomal RNA * K-turn * molecular dynamics Subject RIV: BO - Biophysics Impact factor: 1.113, year: 2004

  12. Poliovirus tropism and attenuation are determined after internal ribosome entry

    OpenAIRE

    Kauder, Steven E.; Racaniello, Vincent R.

    2004-01-01

    Poliovirus replication is limited to a few organs, including the brain and spinal cord. This restricted tropism may be a consequence of organ-specific differences in translation initiation by the poliovirus internal ribosome entry site (IRES). A C-to-U mutation at base 472 in the IRES of the Sabin type 3 poliovirus vaccine strain, known to attenuate neurovirulence, may further restrict tropism by eliminating viral replication in the CNS. To determine the relationship between IRES-mediated tra...

  13. Membrane module assembly

    Science.gov (United States)

    Kaschemekat, Jurgen

    1994-01-01

    A membrane module assembly adapted to provide a flow path for the incoming feed stream that forces it into prolonged heat-exchanging contact with a heating or cooling mechanism. Membrane separation processes employing the module assembly are also disclosed. The assembly is particularly useful for gas separation or pervaporation.

  14. Photoaffinity labeling of the pactamycin binding site on eubacterial ribosomes

    International Nuclear Information System (INIS)

    Tejedor, F.; Amils, R.; Ballesta, J.P.

    1985-01-01

    Pactamycin, an inhibitor of the initial steps of protein synthesis, has an acetophenone group in its chemical structure that makes the drug a potentially photoreactive molecule. In addition, the presence of a phenolic residue makes it easily susceptible to radioactive labeling. Through iodination, one radioactive derivative of pactamycin has been obtained with biological activities similar to the unmodified drug when tested on in vivo and cell-free systems. With the use of [ 125 I]iodopactamycin, ribosomes of Escherichia coli have been photolabeled under conditions that preserve the activity of the particles and guarantee the specificity of the binding sites. Under these conditions, RNA is preferentially labeled when free, small ribosomal subunits are photolabeled, but proteins are the main target in the whole ribosome. This indicates that an important conformational change takes place in the binding site on association of the two subunits. The major labeled proteins are S2, S4, S18, S21, and L13. These proteins in the pactamycin binding site are probably related to the initiation step of protein synthesis

  15. Ribosome signatures aid bacterial translation initiation site identification.

    Science.gov (United States)

    Giess, Adam; Jonckheere, Veronique; Ndah, Elvis; Chyżyńska, Katarzyna; Van Damme, Petra; Valen, Eivind

    2017-08-30

    While methods for annotation of genes are increasingly reliable, the exact identification of translation initiation sites remains a challenging problem. Since the N-termini of proteins often contain regulatory and targeting information, developing a robust method for start site identification is crucial. Ribosome profiling reads show distinct patterns of read length distributions around translation initiation sites. These patterns are typically lost in standard ribosome profiling analysis pipelines, when reads from footprints are adjusted to determine the specific codon being translated. Utilising these signatures in combination with nucleotide sequence information, we build a model capable of predicting translation initiation sites and demonstrate its high accuracy using N-terminal proteomics. Applying this to prokaryotic translatomes, we re-annotate translation initiation sites and provide evidence of N-terminal truncations and extensions of previously annotated coding sequences. These re-annotations are supported by the presence of structural and sequence-based features next to N-terminal peptide evidence. Finally, our model identifies 61 novel genes previously undiscovered in the Salmonella enterica genome. Signatures within ribosome profiling read length distributions can be used in combination with nucleotide sequence information to provide accurate genome-wide identification of translation initiation sites.

  16. Simulation and analysis of single-ribosome translation

    International Nuclear Information System (INIS)

    Tinoco, Ignacio Jr; Wen, Jin-Der

    2009-01-01

    In the cell, proteins are synthesized by ribosomes in a multi-step process called translation. The ribosome translocates along the messenger RNA to read the codons that encode the amino acid sequence of a protein. Elongation factors, including EF-G and EF-Tu, are used to catalyze the process. Recently, we have shown that translation can be followed at the single-molecule level using optical tweezers; this technique allows us to study the kinetics of translation by measuring the lifetime the ribosome spends at each codon. Here, we analyze the data from single-molecule experiments and fit the data with simple kinetic models. We also simulate the translation kinetics based on a multi-step mechanism from ensemble kinetic measurements. The mean lifetimes from the simulation were consistent with our experimental single-molecule measurements. We found that the calculated lifetime distributions were fit in general by equations with up to five rate-determining steps. Two rate-determining steps were only obtained at low concentrations of elongation factors. These analyses can be used to design new single-molecule experiments to better understand the kinetics and mechanism of translation

  17. Further characterization of ribosome binding to thylakoid membranes. [Pisum sativum

    Energy Technology Data Exchange (ETDEWEB)

    Hurewitz, J.; Jagendorf, A.T.

    1987-05-01

    Previous work indicated more polysomes bound to pea (Pisum sativum cv Progress No. 9) thylakoids in light than in the dark, in vivo. With isolated intact chloroplasts incubated in darkness, addition of MgATP had no effect but 24 to 74% more RNA was thylakoid-bound at pH 8.3 than at pH 7. Thus, the major effect of light on ribosome-binding in vivo may be due to higher stroma pH. In isolated pea chloroplasts, initiation inhibitors (pactamycin and kanamycin) decreased the extent of RNA binding, and elongation inhibitors (lincomycin and streptomycin) increased it. Thus, cycling of ribosomes is controlled by translation, initiation, and termination. Bound RNA accounted for 19 to 24% of the total chloroplast RNA and the incorporation of (/sup 3/H)leucine into thylakoids was proportional to the amount of this bound RNA. These data support the concept that stroma ribosomes are recruited into thylakoid polysomes, which are active in synthesizing thylakoid proteins.

  18. Effect of several ribosomal mutations on speed of elongation

    Energy Technology Data Exchange (ETDEWEB)

    Galas, D.J.; Branscomb, E.W.

    1976-09-28

    Consideration of simple kinetic schemes for the discrimination of charged tRNA at the ribosome has led us to expect changes in the elongation speed to be caused by some ribosomal mutations. We have examined this hypothesis by investigating the effect of several well-studied mutations of E. coli ribosomes on the chain elongation time in the translation of the Z gene of the lactose operon. The lag time (at 37/sup 0/C) in the appearance of the first active, newly-synthesized ..beta..-galactosidase molecule after induction of the operon was measured, and the average elongation time estimated. We found that mutations to resistance to high levels of streptomycin (at the str A locus) fall into two classes; one class exhibits a slow-down in elongation of about 30 percent, the other exhibits little, if any, detectable change. Mutation to paramomycin resistance also causes a significant decrease in speed. On the other hand, mutation to spectinomycin resistance appears not to affect the speed. A common characteristic of streptomycin and paramomycin is that they both are known to cause misreading during translation (and resistance causes a decrease in errors) whereas spectinomycin is known to have no such effect. This evidence, together with kinetic considerations, seems to indicate that mutations which affect the accuracy of translation may also affect elongation speed.

  19. Three tRNAs on the ribosome slow translation elongation.

    Science.gov (United States)

    Choi, Junhong; Puglisi, Joseph D

    2017-12-26

    During protein synthesis, the ribosome simultaneously binds up to three different transfer RNA (tRNA) molecules. Among the three tRNA binding sites, the regulatory role of the exit (E) site, where deacylated tRNA spontaneously dissociates from the translational complex, has remained elusive. Here we use two donor-quencher pairs to observe and correlate both the conformation of ribosomes and tRNAs as well as tRNA occupancy. Our results reveal a partially rotated state of the ribosome wherein all three tRNA sites are occupied during translation elongation. The appearance and lifetime of this state depend on the E-site tRNA dissociation kinetics, which may vary among tRNA species and depends on temperature and ionic strength. The 3-tRNA partially rotated state is not a proper substrate for elongation factor G (EF-G), thus inhibiting translocation until the E-site tRNA dissociates. Our result presents two parallel kinetic pathways during translation elongation, underscoring the ability of E-site codons to modulate the dynamics of protein synthesis.

  20. Structure of Vibrio cholerae ribosome hibernation promoting factor

    International Nuclear Information System (INIS)

    De Bari, Heather; Berry, Edward A.

    2013-01-01

    The X-ray crystal structure of ribosome hibernation promoting factor from V. cholerae has been determined at 2.0 Å resolution. The crystal was phased by two-wavelength MAD using cocrystallized cobalt. The X-ray crystal structure of ribosome hibernation promoting factor (HPF) from Vibrio cholerae is presented at 2.0 Å resolution. The crystal was phased by two-wavelength MAD using cocrystallized cobalt. The asymmetric unit contained two molecules of HPF linked by four Co atoms. The metal-binding sites observed in the crystal are probably not related to biological function. The structure of HPF has a typical β–α–β–β–β–α fold consistent with previous structures of YfiA and HPF from Escherichia coli. Comparison of the new structure with that of HPF from E. coli bound to the Thermus thermophilus ribosome [Polikanov et al. (2012 ▶), Science, 336, 915–918] shows that no significant structural changes are induced in HPF by binding

  1. Dynamic evolution of mitochondrial ribosomal proteins in Holozoa.

    Science.gov (United States)

    Scheel, Bettina M; Hausdorf, Bernhard

    2014-07-01

    We studied the highly dynamic evolution of mitochondrial ribosomal proteins (MRPs) in Holozoa. Most major clades within Holozoa are characterized by gains and/or losses of MRPs. The usefulness of gains of MRPs as rare genomic changes in phylogenetics is undermined by the high frequency of secondary losses. However, phylogenetic analyses of the MRP sequences provide evidence for the Acrosomata hypothesis, a sister group relationship between Ctenophora and Bilateria. An extensive restructuring of the mitochondrial genome and, as a consequence, of the mitochondrial ribosomes occurred in the ancestor of metazoans. The last MRP genes encoded in the mitochondrial genome were either moved to the nuclear genome or were lost. The strong decrease in size of the mitochondrial genome was probably caused by selection for rapid replication of mitochondrial DNA during oogenesis in the metazoan ancestor. A phylogenetic analysis of MRPL56 sequences provided evidence for a horizontal gene transfer of the corresponding MRP gene between metazoans and Dictyostelidae (Amoebozoa). The hypothesis that the requisition of additional MRPs compensated for a loss of rRNA segments in the mitochondrial ribosomes is corroborated by a significant negative correlation between the number of MRPs and length of the rRNA. Newly acquired MRPs evolved faster than bacterial MRPs and positions in eukaryote-specific MRPs were more strongly affected by coevolution than positions in prokaryotic MRPs in accordance with the necessity to fit these proteins into the pre-existing structure of the mitoribosome. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Effect of single base changes and the absence of modified bases in 16S RNA on the reconstitution and function of Escherichia coli 30S ribosomes

    International Nuclear Information System (INIS)

    Denman, R.; Krzyzosiak, W.; Nurse, K.; Ofengand, J.

    1987-01-01

    The gene coding for E. coli 16S rRNA was placed in pUC19 under the control of the strong class III T7 promoter, phi 10, by ligation of the 1490 bp BclI/BstEII fragment of the rrnB operon with appropriate synthetic oligodeoxynucleotides. Such constructs allowed efficient in vitro synthesis of full-length transcripts (up to 900 mol RNA/mol template) free of modified bases. The synthetic RNA could be assembled into 30S subunits upon addition of E. coli 30S ribosomal proteins. The particles co-sedimented with authentic 30S particles and were electron microscopically indistinguishable from them. Upon addition of 50S subunits, codon-dependent P-site binding of tRNA and codon-dependent polypeptide synthesis were >80% of 30S reconstituted from natural 16S RNA and >50% of isolated 30S. UV-induced crosslinking of P-site bound AcVal-tRNA to residue C 1400 was preserved. Changing C 1400 to A had little effect on reconstitution, P-site binding, or polypeptide synthesis. However, the substitution of C 1499 by G markedly inhibited assembly. The effect on P-site binding and polypeptide synthesis is under study. These results show (1) none of the modified bases of 16S RNA are essential for protein synthesis, (2) substitution of A for C 1400 has little functional effect, and (3) position 1400 may be important for ribosome assembly

  3. Sensor mount assemblies and sensor assemblies

    Science.gov (United States)

    Miller, David H [Redondo Beach, CA

    2012-04-10

    Sensor mount assemblies and sensor assemblies are provided. In an embodiment, by way of example only, a sensor mount assembly includes a busbar, a main body, a backing surface, and a first finger. The busbar has a first end and a second end. The main body is overmolded onto the busbar. The backing surface extends radially outwardly relative to the main body. The first finger extends axially from the backing surface, and the first finger has a first end, a second end, and a tooth. The first end of the first finger is disposed on the backing surface, and the tooth is formed on the second end of the first finger.

  4. 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...

  5. Nuclear fuel string assembly

    International Nuclear Information System (INIS)

    Ip, A.K.; Koyanagi, K.; Tarasuk, W.R.

    1976-01-01

    A method of fabricating rodded fuels suitable for use in pressure tube type reactors and in pressure vessel type reactors is described. Fuel rods are secured as an inner and an outer sub-assembly, each rod attached between mounting rings secured to the rod ends. The two sub-assemblies are telescoped together and positioned by spaced thimbles located between them to provide precise positioning while permittng differential axial movement between the sub-assemblies. Such sub-assemblies are particularly suited for mounting as bundle strings. The method provides particular advantages in the assembly of annular-section fuel pins, which includes booster fuel containing enriched fuel material. (LL)

  6. Soldering in electronics assembly

    CERN Document Server

    Judd, Mike

    2013-01-01

    Soldering in Electronics Assembly discusses several concerns in soldering of electronic assemblies. The book is comprised of nine chapters that tackle different areas in electronic assembly soldering. Chapter 1 discusses the soldering process itself, while Chapter 2 covers the electronic assemblies. Chapter 3 talks about solders and Chapter 4 deals with flux. The text also tackles the CS and SC soldering process. The cleaning of soldered assemblies, solder quality, and standards and specifications are also discussed. The book will be of great use to professionals who deal with electronic assem

  7. New arrangements on several species subcomplexes of Triatoma genus based on the chromosomal position of ribosomal genes (Hemiptera - Triatominae).

    Science.gov (United States)

    Pita, Sebastián; Lorite, Pedro; Nattero, Julieta; Galvão, Cleber; Alevi, Kaio C C; Teves, Simone C; Azeredo-Oliveira, Maria T V; Panzera, Francisco

    2016-09-01

    The hemipteran subfamily Triatominae includes 150 blood-sucking species, vectors of Chagas disease. By far the most specious genus is Triatoma, assembled in groups, complexes and subcomplexes based on morphological similarities, geographic distribution and genetic data. However, many molecular studies questioned the species integration of several subcomplexes as monophyletic units. In triatomines, chromosomal position of major ribosomal DNA (rDNA) loci is extremely variable but seems to be species-specific and an evolutionary conserved genetic trait, so that closely related species tend to have ribosomal clusters in the same chromosomal location. Considering that the autosomal position as the ancestral character for all heteropteran species, including triatomines, we suggest that the movement of rDNA loci from autosomes to sex chromosomes rapidly established reproductive barriers between divergent lineages. We proposed that the rDNA translocation from the autosomes to the sex chromosomes restrict reproductive compatibility and eventually promote speciation processes. We analyzed the chromosomal position of 45S rDNA clusters in almost all species of the matogrossensis, rubrovaria, maculata and sordida subcomplexes. The fluorescent in situ hybridization results are discussed considering the available genetic data and we proposed new arrangements in the species that constitute each one of these subcomplexes. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Ribosomes and polyribosomes are present in the squid giant axon: an immunocytochemical study.

    Science.gov (United States)

    Sotelo, J R; Kun, A; Benech, J C; Giuditta, A; Morillas, J; Benech, C R

    1999-05-01

    Ribosomes and polyribosomes were detected by immuno-electron microscopy in the giant axon and small axons of the squid using a polyclonal antibody against rat brain ribosomes. The ribosomal fraction used as antigen was purified by ultracentrifugation on a sucrose density gradient and shown to contain ribosomal RNAs and native ribosomes. The polyclonal antibody raised in rabbits reacted with at least ten proteins on immunoblots of purified rat brain ribosomes as well as with a set of multiple ribosomal proteins prepared from the squid giant fiber lobe. Immunoreactions were performed on cryostat sections of the stellate nerve cut at a distance of more than 3 cm from the stellate ganglion, using pre-embedding techniques. Ribosomes and polyribosomes were identified within the giant axon and small axons using electron microscopic methods, following binding of peroxidase-conjugated anti-rabbit IgG secondary antibody. Polysomes were more frequently localized in peripheral axoplasm, including the cortical layer of the giant axon, and were generally associated with unidentified cytoskeletal filaments or with dense matrix material. The immunochemical demonstration of ribosomes and polyribosomes in the giant axon and small axons of the squid confirms similar observations in the squid and the goldfish obtained with the method of electron spectroscopic imaging, and strongly supports the view that a local system of protein synthesis is present in axons. The immunochemical method here described offers an alternative tool for the selective identification of ribosomes, and is likely to prove of value in the analyses of other axonal systems.

  9. Assembling method for nuclear reactor fuel assembly

    International Nuclear Information System (INIS)

    Kamiwaki, Yoshiharu; Ono, Shunji.

    1996-01-01

    Control rod guide tubes are inserted and assembled in to predetermined openings of a plurality of support lattices arranged in rows at predetermined distance. Sheath heaters are inserted to the guide tubes. The sheath heater comprises a plurality of heater elements therein which are connected to a temperature controller. The temperature of each portion of the sheath heater is controlled by the temperature controller. When the temperature of each portion of the guide tube is made uniform, MOX fuel rods are inserted to vacant openings of the support lattices. This changes the circumferential temperature of the guide tube, but the heat generation amount of each heater element is controlled suitably by the temperature controller. Accordingly, even if MOX fuel rods are inserted as a heat generation member, fuel assembly can be assembled with no assembling errors. (I.N.)

  10. Articulate fuel assembly

    International Nuclear Information System (INIS)

    Noyes, R.C.

    1978-01-01

    An articulated fuel assembly for the core of a fast spectrum reactor comprising an elongated shroud enclosing a cluster of fuel pins, a support foot assembly supporting the fuel assembly in the reactor core and an articulating connector link joining the support foot assembly and the lower end of the elongated shroud is described. The upper end of the elongated shroud and the support foot assembly are adapted to be fixedly restrained against lateral movement when the assembly is placed in the reactor core. The articulating connector link is such as to permit free lateral deflection of the lower end of the shroud relative to the upper end of the shroud and the foot assembly. Such an arrangement icreases the reliability of the fuel assembly and safely accommodates the physical distortions in the fuel assemblies caused by neutron induced swelling of the members and thermally induced expansions thereof by reducing stresses in the structural parts of the assembly and by insuring a negative reactivity for the core as the lower ends of the fuel assemblies are laterally displaced. 4 claims, 4 figures

  11. 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......, of the peptidyl transferase centre. Moreover, each of the protein sites, but particularly those of L2 and L23, lies at the centre of RNA domains where they can maximally influence both the assembly of secondary binding proteins and the function of the RNA region....

  12. Simple peptides derived from the ribosomal core potentiate RNA polymerase ribozyme function

    Science.gov (United States)

    Tagami, Shunsuke; Attwater, James; Holliger, Philipp

    2017-04-01

    The emergence of functional interactions between nucleic acids and polypeptides was a key transition in the origin of life and remains at the heart of all biology. However, how and why simple non-coded peptides could have become critical for RNA function is unclear. Here, we show that putative ancient peptide segments from the cores of both ribosomal subunits enhance RNA polymerase ribozyme (RPR) function, as do derived homopolymeric peptides comprising lysine or the non-proteinogenic lysine analogues ornithine or, to a lesser extent, diaminobutyric acid, irrespective of chirality or chiral purity. Lysine decapeptides enhance RPR function by promoting holoenzyme assembly through primer-template docking, accelerate RPR evolution, and allow RPR-catalysed RNA synthesis at near physiological (≥1 mM) Mg2+ concentrations, enabling templated RNA synthesis within membranous protocells. Our results outline how compositionally simple, mixed-chirality peptides may have augmented the functional potential of early RNAs and promoted the emergence of the first protocells.

  13. Evolutionary conservation of the ribosomal biogenesis factor Rbm19/Mrd1: implications for function.

    Directory of Open Access Journals (Sweden)

    Yvonne Kallberg

    Full Text Available Ribosome biogenesis in eukaryotes requires coordinated folding and assembly of a pre-rRNA into sequential pre-rRNA-protein complexes in which chemical modifications and RNA cleavages occur. These processes require many small nucleolar RNAs (snoRNAs and proteins. Rbm19/Mrd1 is one such protein that is built from multiple RNA-binding domains (RBDs. We find that Rbm19/Mrd1 with five RBDs is present in all branches of the eukaryotic phylogenetic tree, except in animals and Choanoflagellates, that instead have a version with six RBDs and Microsporidia which have a minimal Rbm19/Mrd1 protein with four RBDs. Rbm19/Mrd1 therefore evolved as a multi-RBD protein very early in eukaryotes. The linkers between the RBDs have conserved properties; they are disordered, except for linker 3, and position the RBDs at conserved relative distances from each other. All but one of the RBDs have conserved properties for RNA-binding and each RBD has a specific consensus sequence and a conserved position in the protein, suggesting a functionally important modular design. The patterns of evolutionary conservation provide information for experimental analyses of the function of Rbm19/Mrd1. In vivo mutational analysis confirmed that a highly conserved loop 5-β4-strand in RBD6 is essential for function.

  14. Evolutionary conservation of the ribosomal biogenesis factor Rbm19/Mrd1: implications for function.

    Science.gov (United States)

    Kallberg, Yvonne; Segerstolpe, Åsa; Lackmann, Fredrik; Persson, Bengt; Wieslander, Lars

    2012-01-01

    Ribosome biogenesis in eukaryotes requires coordinated folding and assembly of a pre-rRNA into sequential pre-rRNA-protein complexes in which chemical modifications and RNA cleavages occur. These processes require many small nucleolar RNAs (snoRNAs) and proteins. Rbm19/Mrd1 is one such protein that is built from multiple RNA-binding domains (RBDs). We find that Rbm19/Mrd1 with five RBDs is present in all branches of the eukaryotic phylogenetic tree, except in animals and Choanoflagellates, that instead have a version with six RBDs and Microsporidia which have a minimal Rbm19/Mrd1 protein with four RBDs. Rbm19/Mrd1 therefore evolved as a multi-RBD protein very early in eukaryotes. The linkers between the RBDs have conserved properties; they are disordered, except for linker 3, and position the RBDs at conserved relative distances from each other. All but one of the RBDs have conserved properties for RNA-binding and each RBD has a specific consensus sequence and a conserved position in the protein, suggesting a functionally important modular design. The patterns of evolutionary conservation provide information for experimental analyses of the function of Rbm19/Mrd1. In vivo mutational analysis confirmed that a highly conserved loop 5-β4-strand in RBD6 is essential for function.

  15. Structure–function insights reveal the human ribosome as a cancer target for antibiotics

    Science.gov (United States)

    Myasnikov, Alexander G.; Kundhavai Natchiar, S.; Nebout, Marielle; Hazemann, Isabelle; Imbert, Véronique; Khatter, Heena; Peyron, Jean-François; Klaholz, Bruno P.

    2016-01-01

    Many antibiotics in clinical use target the bacterial ribosome by interfering with the protein synthesis machinery. However, targeting the human ribosome in the case of protein synthesis deregulations such as in highly proliferating cancer cells has not been investigated at the molecular level up to now. Here we report the structure of the human 80S ribosome with a eukaryote-specific antibiotic and show its anti-proliferative effect on several cancer cell lines. The structure provides insights into the detailed interactions in a ligand-binding pocket of the human ribosome that are required for structure-assisted drug design. Furthermore, anti-proliferative dose response in leukaemic cells and interference with synthesis of c-myc and mcl-1 short-lived protein markers reveals specificity of a series of eukaryote-specific antibiotics towards cytosolic rather than mitochondrial ribosomes, uncovering the human ribosome as a promising cancer target. PMID:27665925

  16. Control of ribosome traffic by position-dependent choice of synonymous codons

    DEFF Research Database (Denmark)

    Mitarai, Namiko; Pedersen, Steen

    2013-01-01

    Messenger RNA (mRNA) encodes a sequence of amino acids by using codons. For most amino acids, there are multiple synonymous codons that can encode the amino acid. The translation speed can vary from one codon to another, thus there is room for changing the ribosome speed while keeping the amino...... acid sequence and hence the resulting protein. Recently, it has been noticed that the choice of the synonymous codon, via the resulting distribution of slow- and fast-translated codons, affects not only on the average speed of one ribosome translating the mRNA but also might have an effect on nearby...... ribosomes by affecting the appearance of 'traffic jams' where multiple ribosomes collide and form queues. To test this 'context effect' further, we here investigate the effect of the sequence of synonymous codons on the ribosome traffic by using a ribosome traffic model with codon-dependent rates, estimated...

  17. Computational resources for ribosome profiling: from database to Web server and software.

    Science.gov (United States)

    Wang, Hongwei; Wang, Yan; Xie, Zhi

    2017-08-14

    Ribosome profiling is emerging as a powerful technique that enables genome-wide investigation of in vivo translation at sub-codon resolution. The increasing application of ribosome profiling in recent years has achieved remarkable progress toward understanding the composition, regulation and mechanism of translation. This benefits from not only the awesome power of ribosome profiling but also an extensive range of computational resources available for ribosome profiling. At present, however, a comprehensive review on these resources is still lacking. Here, we survey the recent computational advances guided by ribosome profiling, with a focus on databases, Web servers and software tools for storing, visualizing and analyzing ribosome profiling data. This review is intended to provide experimental and computational biologists with a reference to make appropriate choices among existing resources for the question at hand. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. Mutations in 23 S ribosomal RNA perturb transfer RNA selection and can lead to streptomycin dependence.

    Science.gov (United States)

    Bilgin, N; Ehrenberg, M

    1994-01-21

    Escherichia coli ribosomes with a G to C transversion at position 2661 in 23 S ribosomal RNA are more accurate in tRNA selection than wild-type ribosomes. This enhanced accuracy is due to improved initial selection of ternary complexes rather than proofreading of aminoacyl tRNAs. The 2661C mutation reduces the binding rate of cognate ternary complexes to the A-site. This binding rate deficiency becomes dramatic when ribosomes also harbour an S12 mutation with a streptomycin-resistant, hyperaccurate phenotype. In this case, severe loss of kinetic efficiency in EF-Tu function leads to cell death. Streptomycin restores viability by increasing the association rate of ternary complex to these doubly altered ribosomes. The binding rate of EF-G to 2661C ribosomes is also reduced while the translocation rate is unaffected.

  19. Senescent changes in the ribosomes of animal cells in vivo and in vitro

    Science.gov (United States)

    Miquel, J.; Johnson, J. E., Jr.

    1979-01-01

    The paper examines RNA-ribosomal changes observed in protozoa and fixed postmitotic cells, as well as the characteristics of intermitotic cells. Attention is given to a discussion of the implications of the reported ribosomal changes as to the senescent deterioration of protein synthesis and physiological functions. A survey of the literature suggests that, while the data on ribosomal change in dividing cells both in vivo and in vitro are inconclusive, there is strong histological and biochemical evidence in favor of some degree of quantitative ribosomal loss in fixed postmitotic cells. Since these decreases in ribosomes are demonstrated in differential cells from nematodes, insects and mammals, they may represent a universal manifestation of cytoplasmic senescence in certain types of fixed postmitotic animal cells. The observed variability in ribosomal loss for cells belonging to the same type suggests that this involution phenomenon is rather related to the wear and tear suffered by a particular cell.

  20. [The isolation of ribosomes from Acholeplasma and the study of their physicochemical characteristics].

    Science.gov (United States)

    Korobkova, E S; Panchenko, L P; Skripal', I G

    1992-01-01

    Ribosomes are produced from Acholeplasma laidlawii PG-8 and A. laidlawii var. granulum str. 118. It is proved as possible to use the standard method of differential centrifugation for isolation of mollicute ribosomes. The physico-chemical properties of acholesplasma ribosomes are studied. The protein content for A. laidlawii PG-8 amounted to 39.6%, rRNA content--60.4% and for A. laidlawii var. granulum--39.1 and 60.8%, respectively. The RNA: protein ratio equals 1.5:1, the ratio of optic density indicates at 260 and 280 nm--2, that is peculiar to treated preparations of ribosomes. Sedimentation coefficient of acholeplasma ribosomes is 70S. The produced preparations of acholeplasma ribosomes can be used subsequently for creating the system of translation in vitro to study the biosynthesis processes of mollicute protein.

  1. Structural features of the binding site for ribosomal protein S8 in Escherichia coli 16S rRNA defined using NMR spectroscopy.

    Science.gov (United States)

    Kalurachchi, K; Uma, K; Zimmermann, R A; Nikonowicz, E P

    1997-03-18

    Ribosomal protein S8 of Escherichia coli plays a key role in 30S ribosomal subunit assembly through its interaction with 16S rRNA. S8 also participates in the translational regulation of ribosomal protein expression through its interaction with spc operon mRNA. The binding site for protein S8 within the 16S rRNA encompasses nucleotides G588 to G604 and C634 to C651 and is composed of two base paired helical regions that flank a phylogenetically conserved core element containing nine residues. We have investigated the structure of the rRNA binding site for S8 both in the free state and in the presence of protein using NMR spectroscopy. The integrity of the two helical segments has been verified, and the presence of G597 x C643 and A596 x U644 base pairs within the conserved core, predicted from comparative analysis, have been confirmed. In addition, we have identified a base triple within the core that is composed of residues A595 x (A596 x U644). The NMR data suggest that S8-RNA interaction is accomplished without significant changes in the RNA. Nonetheless, S8 binding promotes formation of the U598 x A640 base pair and appears to stabilize the G597 x C643 and A596 x U644 base pairs.

  2. Structural features of the binding site for ribosomal protein S8 in Escherichia coli 16S rRNA defined using NMR spectroscopy

    Science.gov (United States)

    Kalurachchi, K.; Uma, K.; Zimmermann, R. A.; Nikonowicz, E. P.

    1997-01-01

    Ribosomal protein S8 of Escherichia coli plays a key role in 30S ribosomal subunit assembly through its interaction with 16S rRNA. S8 also participates in the translational regulation of ribosomal protein expression through its interaction with spc operon mRNA. The binding site for protein S8 within the 16S rRNA encompasses nucleotides G588 to G604 and C634 to C651 and is composed of two base paired helical regions that flank a phylogenetically conserved core element containing nine residues. We have investigated the structure of the rRNA binding site for S8 both in the free state and in the presence of protein using NMR spectroscopy. The integrity of the two helical segments has been verified, and the presence of G597·C643 and A596·U644 base pairs within the conserved core, predicted from comparative analysis, have been confirmed. In addition, we have identified a base triple within the core that is composed of residues A595·(A596· U644). The NMR data suggest that S8–RNA interaction is accomplished without significant changes in the RNA. Nonetheless, S8 binding promotes formation of the U598·A640 base pair and appears to stabilize the G597·C643 and A596·U644 base pairs. PMID:9122161

  3. Improved Assembly for Gas Shielding During Welding or Brazing

    Science.gov (United States)

    Gradl, Paul; Baker, Kevin; Weeks, Jack

    2009-01-01

    An improved assembly for inert-gas shielding of a metallic joint is designed to be useable during any of a variety of both laser-based and traditional welding and brazing processes. The basic purpose of this assembly or of a typical prior related assembly is to channel the flow of a chemically inert gas to a joint to prevent environmental contamination of the joint during the welding or brazing process and, if required, to accelerate cooling upon completion of the process.

  4. An accuracy center in the ribosome conserved over 2 billion years.

    OpenAIRE

    Alksne, L E; Anthony, R A; Liebman, S W; Warner, J R

    1993-01-01

    The accuracy of translation in Escherichia coli is profoundly influenced by three interacting ribosomal proteins, S12, S4, and S5. Mutations at lysine-42 of S12, originally isolated as causing resistance to streptomycin, increase accuracy. Countervailing "ribosomal ambiguity mutations" (ram) in S4 or S5 decrease accuracy. In the eukaryotic ribosome of Saccharomyces cerevisiae, mutations in SUP46 and SUP44, encoding the proteins equivalent to S4 and S5, lead to omnipotent suppression--i.e., to...

  5. Is efficiency of suppressor tRNAs controlled at the level of ribosomal proofreading in vivo?

    OpenAIRE

    Faxén, M; Kirsebom, L A; Isaksson, L A

    1988-01-01

    Ribosomal rpsD mutations did not stimulate nonsense suppressor tRNAs in a general manner according to their increased ribosomal ambiguity and decreased proofreading efficiency. Streptomycin, which stimulates error production by blocking proofreading in vitro, did not increase efficiency of suppressor tRNAs in strains with normal or streptomycin-resistant (rpsL) ribosomes. It did so only in combination with one rpsL mutation which is associated with streptomycin pseudodependence.

  6. A protein interaction map of the kalimantacin biosynthesis assembly line

    Directory of Open Access Journals (Sweden)

    Birgit Uytterhoeven

    2016-11-01

    Full Text Available The antimicrobial secondary metabolite kalimantacin is produced by a hybrid polyketide/ non-ribosomal peptide system in Pseudomonas fluorescens BCCM_ID9359. In this study, the kalimantacin biosynthesis gene cluster is analyzed by yeast two-hybrid analysis, creating a protein-protein interaction map of the entire assembly line. In total, 28 potential interactions were identified, of which 13 could be confirmed further. These interactions include the dimerization of ketosynthase domains, a link between assembly line modules 9 and 10, and a specific interaction between the trans-acting enoyl reductase BatK and the carrier proteins of modules 8 and 10. These interactions reveal fundamental insight into the biosynthesis of secondary metabolites.This study is the first to reveal interactions in a complete biosynthetic pathway. Similar future studies could build a strong basis for engineering strategies in such clusters.

  7. Assembly tool design

    International Nuclear Information System (INIS)

    Kanamori, Naokazu; Nakahira, Masataka; Ohkawa, Yoshinao; Tada, Eisuke; Seki, Masahiro

    1996-06-01

    The reactor core of the International Thermonuclear Experimental Reactor (ITER) is assembled with a number of large and asymmetric components within a tight tolerance in order to assure the structural integrity for various loads and to provide the tritium confinement. In addition, the assembly procedure should be compatible with remote operation since the core structures will be activated by 14-MeV neutrons once it starts operation and thus personal access will be prohibited. Accordingly, the assembly procedure and tool design are quite essential and should be designed from the beginning to facilitate remote operation. According to the ITER Design Task Agreement, the Japan Atomic Energy Research Institute (JAERI) has performed design study to develop the assembly procedures and associated tool design for the ITER tokamak assembly. This report describes outlines of the assembly tools and the remaining issues obtained in this design study. (author)

  8. 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.

  9. Fuel assembly storage pool

    International Nuclear Information System (INIS)

    Hiranuma, Hiroshi.

    1976-01-01

    Object: To remove limitation of the number of storage of fuel assemblies to increase the number of storage thereof so as to relatively reduce the water depth required for shielding radioactive rays. Structure: Fuel assembly storage rack containers for receiving a plurality of spent fuel assembly racks are stacked in multi-layer fashion within a storage pool filled with water for shielding radioactive rays and removing heat. (Furukawa, Y.)

  10. Ribosome-inactivating proteins and other lectins from Adenia (Passifloraceae).

    Science.gov (United States)

    Pelosi, Emanuele; Lubelli, Chiara; Polito, Letizia; Barbieri, Luigi; Bolognesi, Andrea; Stirpe, Fiorenzo

    2005-11-01

    The caudices of 10 Adenia species contain galactose-binding lectins that were purified by affinity chromatography. All lectins but three agglutinate human erythrocytes. Six lectins consist of two unequal chains, which can be separated by reduction, and inhibit protein synthesis both by a rabbit reticulocyte lysate and by HeLa and Raji cells. The lectins from A. goetzii, A. lanceolata and A. stenodactyla had the highest cytotoxicity, inhibiting cell protein synthesis with IC50s (concentration inhibiting by 50%) below 0.1 ng/ml, and deadenylate DNA, thus being type 2 ribosome-inactivating proteins.

  11. Unusual ribosomal RNA of the intestinal parasite Giardia lamblia.

    OpenAIRE

    Edlind, T D; Chakraborty, P R

    1987-01-01

    The anaerobic protozoan Giardia lamblia is a common intestinal parasite in humans, but is poorly defined at molecular and phylogenetic levels. We report here a structural characterization of the ribosomal RNA (rRNA) and rRNA genes of G. lamblia. Gel electrophoresis under native or non-denaturing conditions identified two high molecular weight rRNA species corresponding to the 16-18S and 23-28S rRNAs. Surprisingly, both species (1300 and 2300 nucleotides long, respectively) were considerably s...

  12. Potential roles for ubiquitin and the proteasome during ribosome biogenesis

    Czech Academy of Sciences Publication Activity Database

    Stavreva, D. A.; Kawasaki, M.; Dundr, M.; Koberna, Karel; Müller, W. G.; Tsujimura-Takahashi, T.; Komatsu, W.; Hayano, T.; Isobe, T.; Raška, Ivan; Misteli, T.; Takahashi, N.; McNally, J. G.

    2006-01-01

    Roč. 26, č. 13 (2006), s. 5131-5145 ISSN 0270-7306 R&D Projects: GA MŠk(CZ) LC535; GA ČR(CZ) GA304/05/0374; GA ČR(CZ) GA304/04/0692 Grant - others:NIH(US) Intramural Research Program; Ministry of Education(JP) Pioneer Research grant Institutional research plan: CEZ:AV0Z50110509 Keywords : the role of the ubikvitin * proteasome system in ribosome biogenesis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.773, year: 2006

  13. Ribosomal RNA: nature's own polymerase-amplified target for diagnosis.

    Science.gov (United States)

    Waters, A P; McCuthan, T F

    1990-02-01

    The technological criteria for diagnostic tests for tropical diseases are strict and limited. One method of considerable potential for field use is the exploitation of regions of species-specific sequences of ribosomal RNA (rRNA). Oligodeoxynucleotide probes can be used to detect the specific RNa regions in question. Andy Waters and Tom McCutchan describe how simple rRNA methodologies for Plasmodium can detect and identify parasites as well as follow different stages in their development. The potential of this methodology extends to monitoring both epidemiological parameters and the efficacy of novel drug regimes.

  14. ex vivo DNA assembly

    Directory of Open Access Journals (Sweden)

    Adam B Fisher

    2013-10-01

    Full Text Available Even with decreasing DNA synthesis costs there remains a need for inexpensive, rapid and reliable methods for assembling synthetic DNA into larger constructs or combinatorial libraries. Advances in cloning techniques have resulted in powerful in vitro and in vivo assembly of DNA. However, monetary and time costs have limited these approaches. Here, we report an ex vivo DNA assembly method that uses cellular lysates derived from a commonly used laboratory strain of Escherichia coli for joining double-stranded DNA with short end homologies embedded within inexpensive primers. This method concurrently shortens the time and decreases costs associated with current DNA assembly methods.

  15. Target Assembly Facility

    Data.gov (United States)

    Federal Laboratory Consortium — The Target Assembly Facility integrates new armor concepts into actual armored vehicles. Featuring the capability ofmachining and cutting radioactive materials, it...

  16. Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit

    Science.gov (United States)

    Hashem, Yaser; Des Georges, Amedee; Dhote, Vidya; Langlois, Robert; Liao, Hstau Y.; Grassucci, Robert A.; Pestova, Tatyana V.; Hellen, Christopher U. T.; Frank, Joachim

    2013-11-01

    Hepatitis C virus (HCV) and classical swine fever virus (CSFV) messenger RNAs contain related (HCV-like) internal ribosome entry sites (IRESs) that promote 5'-end independent initiation of translation, requiring only a subset of the eukaryotic initiation factors (eIFs) needed for canonical initiation on cellular mRNAs. Initiation on HCV-like IRESs relies on their specific interaction with the 40S subunit, which places the initiation codon into the P site, where it directly base-pairs with eIF2-bound initiator methionyl transfer RNA to form a 48S initiation complex. However, all HCV-like IRESs also specifically interact with eIF3 (refs 2, 5, 6, 7, 9, 10, 11, 12), but the role of this interaction in IRES-mediated initiation has remained unknown. During canonical initiation, eIF3 binds to the 40S subunit as a component of the 43S pre-initiation complex, and comparison of the ribosomal positions of eIF3 and the HCV IRES revealed that they overlap, so that their rearrangement would be required for formation of ribosomal complexes containing both components. Here we present a cryo-electron microscopy reconstruction of a 40S ribosomal complex containing eIF3 and the CSFV IRES. Remarkably, although the position and interactions of the CSFV IRES with the 40S subunit in this complex are similar to those of the HCV IRES in the 40S-IRES binary complex, eIF3 is completely displaced from its ribosomal position in the 43S complex, and instead interacts through its ribosome-binding surface exclusively with the apical region of domain III of the IRES. Our results suggest a role for the specific interaction of HCV-like IRESs with eIF3 in preventing ribosomal association of eIF3, which could serve two purposes: relieving the competition between the IRES and eIF3 for a common binding site on the 40S subunit, and reducing formation of 43S complexes, thereby favouring translation of viral mRNAs.

  17. Combined Effect of the Cfr Methyltransferase and Ribosomal Protein L3 Mutations on Resistance to Ribosome-Targeting Antibiotics.

    Science.gov (United States)

    Pakula, Kevin K; Hansen, Lykke H; Vester, Birte

    2017-09-01

    Several groups of antibiotics inhibit bacterial growth by binding to bacterial ribosomes. Mutations in ribosomal protein L3 have been associated with resistance to linezolid and tiamulin, which both bind at the peptidyl transferase center in the ribosome. Resistance to these and other antibiotics also occurs through methylation of 23S rRNA at position A2503 by the methyltransferase Cfr. The mutations in L3 and the cfr gene have been found together in clinical isolates, raising the question of whether they have a combined effect on antibiotic resistance or growth. We transformed a plasmid-borne cfr gene into a uL3-depleted Escherichia coli strain containing either wild-type L3 or L3 with one of seven mutations, G147R, Q148F, N149S, N149D, N149R, Q150L, or T151P, expressed from plasmid-carried rplC genes. The L3 mutations are well tolerated, with small to moderate growth rate decreases. The presence of Cfr has a very minor influence on the growth rate. The resistance of the transformants to linezolid, tiamulin, florfenicol, and Synercid (a combination of quinupristin and dalfopristin [Q-D]) was measured by MIC assays. The resistance from Cfr was, in all cases, stronger than the effects of the L3 mutations, but various effects were obtained with the combinations of Cfr and L3 mutations ranging from a synergistic to an antagonistic effect. Linezolid and tiamulin susceptibility varied greatly among the L3 mutations, while no significant effects on florfenicol and Q-D susceptibility were seen. This study underscores the complex interplay between various resistance mechanisms and cross-resistance, even from antibiotics with overlapping binding sites. Copyright © 2017 American Society for Microbiology.

  18. Fluorescence studies on a streptomycin-induced conformational change in ribosomes which correlates with misreading.

    Science.gov (United States)

    Hanas, J S; Simpson, M V

    1986-05-25

    The fluorescent reagent N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (I-AEDANS) was employed to detect and study the previously reported conformational change in the Escherichia coli ribosome induced by streptomycin. Labeling of ribosomes with this probe, which results in the derivatization of proteins S18 and L31', described earlier, inhibits neither their ribosomal protein synthesizing nor misreading ability. To calculate the amount of streptomycin bound to the ribosome, we determined the K'D for streptomycin, which is 0.24 micron, indicating that under our conditions, bound streptomycin/ribosome molar ratios are low, not in excess of 1. Under these conditions, streptomycin addition induces fluorescence quenching by 15% but does not affect streptomycin-resistant ribosomes. Maximal misreading occurs at these same ratios. Removal of AEDANS-L31' from the ribosomes drastically reduces streptomycin-induced quenching indicating the involvement of the environment of this protein in streptomycin action. The finding that streptomycin decreases AEDANS-L31' affinity for the ribosome supports this view. Streptomycin has been shown to bind to the 30 S subunit protein S4 while the 50 S protein L31' has been shown to be localized at the subunit interface. Thus, the observation that streptomycin influences this 50 S subunit protein L31', combined with the tight correlation between the effects of streptomycin on quenching and on misreading, strongly suggests that this antibiotic induces a conformational change at the subunit interface of the ribosome, and that this results in misreading. Polyuridylic acid also induces a conformational change in the ribosome but the polynucleotide and streptomycin seem to act independently. Streptomycin-resistant ribosomes, which undergo neither streptomycin-induced fluorescence nor streptomycin-induced misreading, are resistant to misreading induced by high Mg2+ as well.

  19. 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.

  20. Genetic diversity of Entamoeba: Novel ribosomal lineages from cockroaches.

    Directory of Open Access Journals (Sweden)

    Tetsuro Kawano

    Full Text Available Our current taxonomic perspective on Entamoeba is largely based on small-subunit ribosomal RNA genes (SSU rDNA from Entamoeba species identified in vertebrate hosts with minor exceptions such as E. moshkovskii from sewage water and E. marina from marine sediment. Other Entamoeba species have also been morphologically identified and described from non-vertebrate species such as insects; however, their genetic diversity remains unknown. In order to further disclose the diversity of the genus, we investigated Entamoeba spp. in the intestines of three cockroach species: Periplaneta americana, Blaptica dubia, and Gromphadorhina oblongonota. We obtained 134 Entamoeba SSU rDNA sequences from 186 cockroaches by direct nested PCR using the DNA extracts of intestines from cockroaches, followed by scrutinized BLASTn screening and phylogenetic analyses. All the sequences identified in this study were distinct from those reported from known Entamoeba species, and considered as novel Entamoeba ribosomal lineages. Furthermore, they were positioned at the base of the clade of known Entamoeba species and displayed remarkable degree of genetic diversity comprising nine major groups in the three cockroach species. This is the first report of the diversity of SSU rDNA sequences from Entamoeba in non-vertebrate host species, and should help to understand the genetic diversity of the genus Entamoeba.

  1. Translational autocontrol of the Escherichia coli ribosomal protein S15.

    Science.gov (United States)

    Portier, C; Dondon, L; Grunberg-Manago, M

    1990-01-20

    When rpsO, the gene encoding the ribosomal protein S15 in Escherichia coli, is carried by a multicopy plasmid, the mRNA synthesis rate of S15 increases with the gene dosage but the rate of synthesis of S15 does not rise. A translational fusion between S15 and beta-galactosidase was introduced on the chromosome in a delta lac strain and the expression of beta-galactosidase studied under different conditions. The presence of S15 in trans represses the beta-galactosidase level five- to sixfold, while the synthesis rate of the S15-beta-galactosidase mRNA decreases by only 30 to 50%. These data indicate that S15 is subject to autogenous translational control. Derepressed mutants were isolated and sequenced. All the point mutations map in the second codon of S15, suggesting a location for the operator site that is very near to the translation initiation codon. However, the creation of deletion mutations shows that the operator extends into the 5' non-coding part of the message, thus overlapping the ribosome loading site.

  2. The Expanding Toolkit of Translating Ribosome Affinity Purification.

    Science.gov (United States)

    Dougherty, Joseph D

    2017-12-13

    Translating ribosome affinity purification is a method initially developed for profiling mRNA from genetically defined cell types in complex tissues. It has been applied both to identify target molecules in cell types that are important for controlling a variety of behaviors in the brain, and to understand the molecular consequences on those cells due to experimental manipulations, ranging from drugs of abuse to disease-causing mutations. Since its inception, a variety of methodological advances are opening new avenues of investigation. These advances include a variety of new methods for targeting cells for translating ribosome affinity purification by features such as their projections or activity, additional tags and mouse reagents increasing the flexibility of the system, and new modifications of the method specifically focused on studying the regulation of translation. The latter includes methods to assess cell type-specific regulation of translation in specific subcellular compartments. Here, I provide a summary of these recent advances and resources, highlighting both new experimental opportunities and areas for future technical development. Copyright © 2017 the authors 0270-6474/17/3712079-09$15.00/0.

  3. Genetic diversity of Entamoeba: Novel ribosomal lineages from cockroaches

    Science.gov (United States)

    Kawano, Tetsuro; Imada, Mihoko; Chamavit, Pennapa; Kobayashi, Seiki; Hashimoto, Tetsuo

    2017-01-01

    Our current taxonomic perspective on Entamoeba is largely based on small-subunit ribosomal RNA genes (SSU rDNA) from Entamoeba species identified in vertebrate hosts with minor exceptions such as E. moshkovskii from sewage water and E. marina from marine sediment. Other Entamoeba species have also been morphologically identified and described from non-vertebrate species such as insects; however, their genetic diversity remains unknown. In order to further disclose the diversity of the genus, we investigated Entamoeba spp. in the intestines of three cockroach species: Periplaneta americana, Blaptica dubia, and Gromphadorhina oblongonota. We obtained 134 Entamoeba SSU rDNA sequences from 186 cockroaches by direct nested PCR using the DNA extracts of intestines from cockroaches, followed by scrutinized BLASTn screening and phylogenetic analyses. All the sequences identified in this study were distinct from those reported from known Entamoeba species, and considered as novel Entamoeba ribosomal lineages. Furthermore, they were positioned at the base of the clade of known Entamoeba species and displayed remarkable degree of genetic diversity comprising nine major groups in the three cockroach species. This is the first report of the diversity of SSU rDNA sequences from Entamoeba in non-vertebrate host species, and should help to understand the genetic diversity of the genus Entamoeba. PMID:28934335

  4. Identification of Acinetobacter baumannii via amplified ribosomal DNA restriction analysis

    Directory of Open Access Journals (Sweden)

    Delsuz Rezaei

    2014-02-01

    Full Text Available Background: Acinetobacter baumannii is a multi-resistant opportunistic nosocomial pathogen responsible for hospital outbreaks worldwide. In addition to common microscopic and biochemical methods, the Amplified Ribosomal DNA Restriction Analysis (ARDRA was tested to identify Acinetobacter genomic species (DNA groups. Methods: In this study, standard biochemical tests were used for identification of isolates. The genomic species of Acinetobacter spp. was confirmed by Amplified Ribosomal DNA Restriction Analysis (ARDRA. PCR products of 16S rRNA were digested with AluI, MboI and HhaI restriction enzymes. Results: ARDRA proved to be a rapid and reliable method for identification of Acinetobacter baumannii (genome species 2 of the Acinetobacter genomic species, including the closely related genomic species (genomic species 1 (A. calcoaceticus, 2 (A. baumannii, 3, and TU13. Conclusion: The results of this study suggested that ARDRA with AluI, MboI and HhaI restriction enzymes can be used for identificationof A. baumannii which may help to elucidate the ecology and clinical significance of different species of this genus. Since ARDRA is performed by universal primers of 16S rDNA gene, it is expected to be applicable to identifying most bacterial species.

  5. Vertical pump assembly

    International Nuclear Information System (INIS)

    Dohnal, M.; Rosel, J.; Skarka, V.

    1988-01-01

    The mounting is described of the drive assembly of a vertical pump for nuclear power plants in areas with seismic risk. The assembly is attached to the building floor using flexible and damping elements. The design allows producing seismically resistant pumps without major design changes in the existing types of vertical pumps. (E.S.). 1 fig

  6. Assembling Transgender Moments

    Science.gov (United States)

    Greteman, Adam J.

    2017-01-01

    In this article, the author seeks to assemble moments--scholarly, popular, and aesthetic--in order to explore the possibilities that emerge as moments collect in education's encounters with the needs, struggles, and possibilities of transgender lives and practices. Assembling moments, the author argues, illustrates the value of "moments"…

  7. Extending reference assembly models

    DEFF Research Database (Denmark)

    Church, Deanna M.; Schneider, Valerie A.; Steinberg, Karyn Meltz

    2015-01-01

    The human genome reference assembly is crucial for aligning and analyzing sequence data, and for genome annotation, among other roles. However, the models and analysis assumptions that underlie the current assembly need revising to fully represent human sequence diversity. Improved analysis tools...... and updated data reporting formats are also required....

  8. Perspective: Geometrically frustrated assemblies

    Science.gov (United States)

    Grason, Gregory M.

    2016-09-01

    This perspective will overview an emerging paradigm for self-organized soft materials, geometrically frustrated assemblies, where interactions between self-assembling elements (e.g., particles, macromolecules, proteins) favor local packing motifs that are incompatible with uniform global order in the assembly. This classification applies to a broad range of material assemblies including self-twisting protein filament bundles, amyloid fibers, chiral smectics and membranes, particle-coated droplets, curved protein shells, and phase-separated lipid vesicles. In assemblies, geometric frustration leads to a host of anomalous structural and thermodynamic properties, including heterogeneous and internally stressed equilibrium structures, self-limiting assembly, and topological defects in the equilibrium assembly structures. The purpose of this perspective is to (1) highlight the unifying principles and consequences of geometric frustration in soft matter assemblies; (2) classify the known distinct modes of frustration and review corresponding experimental examples; and (3) describe outstanding questions not yet addressed about the unique properties and behaviors of this broad class of systems.

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

    Science.gov (United States)

    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 approximately 26,000 fold tighter than the equivalent interaction from Escherichia coli. Deletion analysis demonstrated that binding to the minimal 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 rRNA. PMID:22079365

  10. Self-assembled nanostructures

    CERN Document Server

    Zhang, Jin Z; Liu, Jun; Chen, Shaowei; Liu, Gang-yu

    2003-01-01

    Nanostructures refer to materials that have relevant dimensions on the nanometer length scales and reside in the mesoscopic regime between isolated atoms and molecules in bulk matter. These materials have unique physical properties that are distinctly different from bulk materials. Self-Assembled Nanostructures provides systematic coverage of basic nanomaterials science including materials assembly and synthesis, characterization, and application. Suitable for both beginners and experts, it balances the chemistry aspects of nanomaterials with physical principles. It also highlights nanomaterial-based architectures including assembled or self-assembled systems. Filled with in-depth discussion of important applications of nano-architectures as well as potential applications ranging from physical to chemical and biological systems, Self-Assembled Nanostructures is the essential reference or text for scientists involved with nanostructures.

  11. High utilization fuel assembly

    International Nuclear Information System (INIS)

    Camden, T.M. Jr.

    1986-01-01

    A nuclear fuel assembly is described comprising an array of parallel arranged guide tubes, an inlet nozzle attached to one end of the guide tubes, an outlet nozzle attached to the other end of the guide tubes, grids having the openings therethrough attached to and spaced along the length of the guide tubes, and of parallel arranged fuel rod assemblies each having an upper end and a lower end. The fuel rod assemblies are fitted within the openings in the grids, the fuel rod assemblies being arranged axially offset relative to each adjacent fuel rod assembly and comprising an upper fuel rod and a lower axially aligned fuel rod with a gap therebetween. The gap between the fuel rods each is axially offset relative to each adjacent gap so as to eliminate an axial gap across the core

  12. Novel mRNA-specific effects of ribosome drop-off on translation rate and polysome profile.

    Science.gov (United States)

    Bonnin, Pierre; Kern, Norbert; Young, Neil T; Stansfield, Ian; Romano, M Carmen

    2017-05-01

    The well established phenomenon of ribosome drop-off plays crucial roles in translational accuracy and nutrient starvation responses during protein translation. When cells are under stress conditions, such as amino acid starvation or aminoacyl-tRNA depletion due to a high level of recombinant protein expression, ribosome drop-off can substantially affect the efficiency of protein expression. Here we introduce a mathematical model that describes the effects of ribosome drop-off on the ribosome density along the mRNA and on the concomitant protein synthesis rate. Our results show that ribosome premature termination may lead to non-intuitive ribosome density profiles, such as a ribosome density which increases from the 5' to the 3' end. Importantly, the model predicts that the effects of ribosome drop-off on the translation rate are mRNA-specific, and we quantify their resilience to drop-off, showing that the mRNAs which present ribosome queues are much less affected by ribosome drop-off than those which do not. Moreover, among those mRNAs that do not present ribosome queues, resilience to drop-off correlates positively with the elongation rate, so that sequences using fast codons are expected to be less affected by ribosome drop-off. This result is consistent with a genome-wide analysis of S. cerevisiae, which reveals that under favourable growth conditions mRNAs coding for proteins involved in the translation machinery, known to be highly codon biased and using preferentially fast codons, are highly resilient to ribosome drop-off. Moreover, in physiological conditions, the translation rate of mRNAs coding for regulatory, stress-related proteins, is less resilient to ribosome drop-off. This model therefore allows analysis of variations in the translational efficiency of individual mRNAs by accounting for the full range of known ribosome behaviours, as well as explaining mRNA-specific variations in ribosome density emerging from ribosome profiling studies.

  13. The location of protein S8 and surrounding elements of 16S rRNA in the 70S ribosome from combined use of directed hydroxyl radical probing and X-ray crystallography.

    Science.gov (United States)

    Lancaster, L; Culver, G M; Yusupova, G Z; Cate, J H; Yusupov, M M; Noller, H F

    2000-05-01

    Ribosomal protein S8, which is essential for the assembly of the central domain of 16S rRNA, is one of the most thoroughly studied RNA-binding proteins. To map its surrounding RNA in the ribosome, we carried out directed hydroxyl radical probing of 16S rRNA using Fe(II) tethered to nine different positions on the surface of protein S8 in 70S ribosomes. Hydroxyl radical-induced cleavage was observed near the classical S8-binding site in the 620 stem, and flanking the other S8-footprinted regions of the central domain at the three-helix junction near position 650 and the 825 and 860 stems. In addition, cleavage near the 5' terminus of 16S rRNA, in the 300 region of its 5' domain, and in the 1070 region of its 3'-major domain provide information about the proximity to S8 of RNA elements not directly involved in its binding. These data, along with previous footprinting and crosslinking results, allowed positioning of protein S8 and its surrounding RNA elements in a 7.8-A map of the Thermus thermophilus 70S ribosome. The resulting model is in close agreement with the extensive body of data from previous studies using protein-protein and protein-RNA crosslinking, chemical and enzymatic footprinting, and genetics.

  14. Patterns of major divergence between the internal transcribed spacers of ribosomal DNA in Xenopus borealis and Xenopus laevis, and of minimal divergence within ribosomal coding regions.

    OpenAIRE

    Furlong, J C; Maden, B E

    1983-01-01

    We have determined the nucleotide sequences of the two internal transcribed spacers, the adjacent ribosomal coding sequences and the boundary between the external transcribed spacer and the 18S coding sequence in a cloned ribosomal transcription unit from Xenopus borealis. The transcribed spacers differ very extensively from those of X. laevis. Nevertheless, embedded in the internal transcribed spacers are several short sequence elements which are identical between the two species. These cons...

  15. TESS Lens-Bezel Assembly Modal Testing

    Science.gov (United States)

    Dilworth, Brandon J.; Karlicek, Alexandra

    2017-01-01

    The Transiting Exoplanet Survey Satellite (TESS) program, led by the Kavli Institute for Astrophysics and Space Research at the Massachusetts Institute of Technology (MIT) will be the first-ever spaceborne all-sky transit survey. MIT Lincoln Laboratory is responsible for the cameras, including the lens assemblies, detector assemblies, lens hoods, and camera mounts. TESS is scheduled to be launched in August of 2017 with the primary goal to detect small planets with bright host starts in the solar neighborhood, so that detailed characterizations of the planets and their atmospheres can be performed. The TESS payload consists of four identical cameras and a data handling unit. Each camera consists of a lens assembly with seven optical elements and a detector assembly with four charge-coupled devices (CCDs) including their associated electronics. The optical prescription requires that several of the lenses are in close proximity to a neighboring element. A finite element model (FEM) was developed to estimate the relative deflections between each lens-bezel assembly under launch loads to predict that there are adequate clearances preventing the lenses from making contact. Modal tests using non-contact response measurements were conducted to experimentally estimate the modal parameters of the lens-bezel assembly, and used to validate the initial FEM assumptions. Key Words Non-contact measurements, modal analysis, model validation

  16. Hypoxic stress-induced changes in ribosomes of maize seedling roots

    International Nuclear Information System (INIS)

    Bailey-Serres, J.; Freeling, M.

    1990-01-01

    The hypoxic stress response of Zea mays L. seedling roots involves regulation of gene expression at transcriptional and posttranscriptional levels. We investigated the effect of hypoxia on the translational machinery of seedling roots. The levels of monoribosomes and ribosomal subunits increased dramatically within 1 hour of stress. Prolonged hypoxia resulted in continued accumulation of nontranslating ribosomes, as well as increased levels of small polyribosomes. The return of seedlings to normal aerobic conditions resulted in recovery of normal polyribosome levels. Comparison of ribosomal proteins from control and hypoxic roots revealed differences in quantity and electrophoretic mobility. In vivo labeling of roots with [ 35 S]methionine revealed variations in newly synthesized ribosomal proteins. In vivo labeling of roots with [ 32 P]orthophosphate revealed a major reduction in the phosphorylation of a 31 kilodalton ribosomal protein in hypoxic stressed roots. In vitro phosphorylation of ribosomal proteins by endogenous kinases was used to probe for differences in ribosome structure and composition. The patterns of in vitro kinased phosphoproteins of ribosomes from control and hypoxic roots were not identical. Variation in phosphoproteins of polyribosomes from control and hypoxic roots, as well as among polyribosomes from hypoxic roots were observed. These results indicate that modification of the translational machinery occurs in response to hypoxic stress

  17. cDNA, genomic cloning and sequence analysis of ribosomal protein ...

    African Journals Online (AJOL)

    Ribosomal protein S4X (RPS4X) is one of the 40S ribosomal proteins encoded by the RPS4X gene. The cDNA and the genomic sequence of RPS4X were cloned successfully from giant panda (Ailuropoda melanoleuca) using reverse transcriptase-polymerase chain reaction (RT-PCR) and touchdown-PCR technology ...

  18. Large facilities and the evolving ribosome, the cellular machine for genetic-code translation.

    Science.gov (United States)

    Yonath, Ada

    2009-10-06

    Well-focused X-ray beams, generated by advanced synchrotron radiation facilities, yielded high-resolution diffraction data from crystals of ribosomes, the cellular nano-machines that translate the genetic code into proteins. These structures revealed the decoding mechanism, localized the mRNA path and the positions of the tRNA molecules in the ribosome and illuminated the interactions of the ribosome with initiation, release and recycling factors. They also showed that the ribosome is a ribozyme whose active site is situated within a universal symmetrical region that is embedded in the otherwise asymmetric ribosome structure. As this highly conserved region provides the machinery required for peptide bond formation and for ribosome polymerase activity, it may be the remnant of the proto-ribosome, a dimeric pre-biotic machine that formed peptide bonds and non-coded polypeptide chains. Synchrotron radiation also enabled the determination of structures of complexes of ribosomes with antibiotics targeting them, which revealed the principles allowing for their clinical use, revealed resistance mechanisms and showed the bases for discriminating pathogens from hosts, hence providing valuable structural information for antibiotics improvement.

  19. Control of ribosome traffic by position-dependent choice of synonymous codons

    International Nuclear Information System (INIS)

    Mitarai, Namiko; Pedersen, Steen

    2013-01-01

    Messenger RNA (mRNA) encodes a sequence of amino acids by using codons. For most amino acids, there are multiple synonymous codons that can encode the amino acid. The translation speed can vary from one codon to another, thus there is room for changing the ribosome speed while keeping the amino acid sequence and hence the resulting protein. Recently, it has been noticed that the choice of the synonymous codon, via the resulting distribution of slow- and fast-translated codons, affects not only on the average speed of one ribosome translating the mRNA but also might have an effect on nearby ribosomes by affecting the appearance of ‘traffic jams’ where multiple ribosomes collide and form queues. To test this ‘context effect’ further, we here investigate the effect of the sequence of synonymous codons on the ribosome traffic by using a ribosome traffic model with codon-dependent rates, estimated from experiments. We compare the ribosome traffic on wild-type (WT) sequences and sequences where the synonymous codons were swapped randomly. By simulating translation of 87 genes, we demonstrate that the WT sequences, especially those with a high bias in codon usage, tend to have the ability to reduce ribosome collisions, hence optimizing the cellular investment in the translation apparatus. The magnitude of such reduction of the translation time might have a significant impact on the cellular growth rate and thereby have importance for the survival of the species. (paper)

  20. Organization of ribosomes and nucleoids in Escherichia coli cells during growth and in quiescence.

    Science.gov (United States)

    Chai, Qian; Singh, Bhupender; Peisker, Kristin; Metzendorf, Nicole; Ge, Xueliang; Dasgupta, Santanu; Sanyal, Suparna

    2014-04-18

    We have examined the distribution of ribosomes and nucleoids in live Escherichia coli cells under conditions of growth, division, and in quiescence. In exponentially growing cells translating ribosomes are interspersed among and around the nucleoid lobes, appearing as alternative bands under a fluorescence microscope. In contrast, inactive ribosomes either in stationary phase or after treatment with translation inhibitors such as chloramphenicol, tetracycline, and streptomycin gather predominantly at the cell poles and boundaries with concomitant compaction of the nucleoid. However, under all conditions, spatial segregation of the ribosomes and the nucleoids is well maintained. In dividing cells, ribosomes accumulate on both sides of the FtsZ ring at the mid cell. However, the distribution of the ribosomes among the new daughter cells is often unequal. Both the shape of the nucleoid and the pattern of ribosome distribution are also modified when the cells are exposed to rifampicin (transcription inhibitor), nalidixic acid (gyrase inhibitor), or A22 (MreB-cytoskeleton disruptor). Thus we conclude that the intracellular organization of the ribosomes and the nucleoids in bacteria are dynamic and critically dependent on cellular growth processes (replication, transcription, and translation) as well as on the integrity of the MreB cytoskeleton.

  1. Organization of Ribosomes and Nucleoids in Escherichia coli Cells during Growth and in Quiescence*

    Science.gov (United States)

    Chai, Qian; Singh, Bhupender; Peisker, Kristin; Metzendorf, Nicole; Ge, Xueliang; Dasgupta, Santanu; Sanyal, Suparna

    2014-01-01

    We have examined the distribution of ribosomes and nucleoids in live Escherichia coli cells under conditions of growth, division, and in quiescence. In exponentially growing cells translating ribosomes are interspersed among and around the nucleoid lobes, appearing as alternative bands under a fluorescence microscope. In contrast, inactive ribosomes either in stationary phase or after treatment with translation inhibitors such as chloramphenicol, tetracycline, and streptomycin gather predominantly at the cell poles and boundaries with concomitant compaction of the nucleoid. However, under all conditions, spatial segregation of the ribosomes and the nucleoids is well maintained. In dividing cells, ribosomes accumulate on both sides of the FtsZ ring at the mid cell. However, the distribution of the ribosomes among the new daughter cells is often unequal. Both the shape of the nucleoid and the pattern of ribosome distribution are also modified when the cells are exposed to rifampicin (transcription inhibitor), nalidixic acid (gyrase inhibitor), or A22 (MreB-cytoskeleton disruptor). Thus we conclude that the intracellular organization of the ribosomes and the nucleoids in bacteria are dynamic and critically dependent on cellular growth processes (replication, transcription, and translation) as well as on the integrity of the MreB cytoskeleton. PMID:24599955

  2. The Ribosomal RNA is a Useful Marker to Visualize Rhizobia Interacting with Legume Plants

    Science.gov (United States)

    Rinaudi, Luciana; Isola, Maria C.; Giordano, Walter

    2004-01-01

    Symbiosis between rhizobia and leguminous plants leads to the formation of nitrogen-fixing root nodules. In the present article, we recommend the use of the ribosomal RNA (rRNA) isolated from legume nodules in an experimental class with the purpose of introducing students to the structure of eukaryotic and prokaryotic ribosomes and of…

  3. cDNA, genomic cloning and sequence analysis of ribosomal protein ...

    African Journals Online (AJOL)

    enoh

    2012-03-13

    Mar 13, 2012 ... Ribosomal protein S4X (RPS4X) is one of the 40S ribosomal proteins encoded by the RPS4X gene. The. cDNA and the genomic sequence of RPS4X were cloned successfully from giant panda (Ailuropoda melanoleuca) using reverse transcriptase-polymerase chain reaction (RT-PCR) and touchdown- ...

  4. The antibiotic micrococcin acts on protein L11 at the ribosomal GTPase centre

    DEFF Research Database (Denmark)

    Porse, B T; Cundliffe, E; Garrett, Roger Antony

    1999-01-01

    -type strain, whereas other mutants carrying single-site substitutions within an 11 amino acid residue segment of the N-terminal domain of L11 grow normally. Protein L11 binds to 23 S rRNA within the ribosomal GTPase centre which regulates GTP hydrolysis on ribosomal factors. Micrococcin binding within the r...

  5. rRNA:mRNA pairing alters the length and the symmetry of mRNA-protected fragments in ribosome profiling experiments

    OpenAIRE

    O?Connor, Patrick B. F.; Li, Gene-Wei; Weissman, Jonathan S.; Atkins, John F.; Baranov, Pavel V.

    2013-01-01

    Motivation: Ribosome profiling is a new technique that allows monitoring locations of translating ribosomes on mRNA at a whole transcriptome level. A recent ribosome profiling study demonstrated that internal Shine?Dalgarno (SD) sequences have a major global effect on translation rates in bacteria: ribosomes pause at SD sites in mRNA. Therefore, it is important to understand how SD sites effect mRNA movement through the ribosome and generation of ribosome footprints. Results: Here, we provide...

  6. Phosphorylation of ribosomal proteins induced by auxins in maize embryonic tissues

    International Nuclear Information System (INIS)

    Perez, L.; Aguilar, R.; Mendez, A.P.; de Jimenez, E.S.

    1990-01-01

    The effect of auxin on ribosomal protein phosphorylation of germinating maize (Zea mays) tissues was investigated. Two-dimensional gel electrophoresis and autoradiography of [ 32 P] ribosomal protein patterns for natural and synthetic auxin-treated tissues were performed. Both the rate of 32 P incorporation and the electrophoretic patterns were dependent on 32 P pulse length, suggesting that active protein phosphorylation-dephosphorylation occurred in small and large subunit proteins, in control as well as in auxin-treated tissues. The effect of ribosomal protein phosphorylation on in vitro translation was tested. Measurements of poly(U) translation rates as a function of ribosome concentration provided apparent K m values significantly different for auxin-treated and nontreated tissues. These findings suggest that auxin might exert some kind of translational control by regulating the phosphorylated status of ribosomal proteins

  7. Streptomycin sensitivity of ribosomes isolated from a streptomycin-producing Streptomyces griseus.

    Science.gov (United States)

    Valu, G; Szabó, G

    1979-01-01

    The streptomycin sensitivity of ribosomes derived from a streptomycin-producing Streptomyces griseus was examined in a polyuridylic acid directed 14C-phenylalanine incorporating system. In order to get reproducible results it is essential to use cell-free extracts which do not inactivate streptomycin. This condition can be fulfilled by the combination of washed ribosomes of the streptomycin-producing strain and the 110 000 g supernatant of the streptomycin-nonproducing variant of S. griseus, because the streptomycin-phosphorylating activity can be washed out from ribosomes of younger streptomycin-producing cultures, and the streptomycin-nonproducing S. griseus does not have any streptomycin-inactivating capacity. In this amino acid polymerizing system the ribosomes of the streptomycin-producing strain were as sensitive to streptomycin as the ribosomes of the nonproducing variant or of Escherichia coli.

  8. Nuclear fuel assembly repair

    International Nuclear Information System (INIS)

    Bassler, E.A.; Stavsky, R.

    1986-01-01

    In response to utility needs to recover investment in nuclear fuel assemblies, Westinghouse Electric Corporation has developed tools and equipment to repair damaged fuel assemblies in an economical and safe manner, to enable utilities to reinsert these assemblies in the core. There are two possible repair techniques - bottom nozzle reconstitution and top nozzle reconstitution. Both techniques have been approved through formal design review; prototype tools have been built and successfully tested. The tools are modular in nature, easily transportable, and designed to fit the spent fuel pool at a reactor site. (author)

  9. TPX assembly plan

    International Nuclear Information System (INIS)

    Knutson, D.

    1993-01-01

    The TPX machine will be assembled in the TFTR Test Cell at the Plasma Physics Laboratory, utilizing the existing TFTR machine foundation. Preparation of the area for assembly will begin after completion of the decontamination and decommissioning phase on TFTR and certification that the radiation levels remaining, if any, are consistent with the types of operations planned. Assembly operations begin with the arrival of the first components, and conclude, approximately 24 months later, with the successful completion of the integrated systems tests and the achievement of a first plasma

  10. DC source assemblies

    Science.gov (United States)

    Campbell, Jeremy B; Newson, Steve

    2013-02-26

    Embodiments of DC source assemblies of power inverter systems of the type suitable for deployment in a vehicle having an electrically grounded chassis are provided. An embodiment of a DC source assembly comprises a housing, a DC source disposed within the housing, a first terminal, and a second terminal. The DC source also comprises a first capacitor having a first electrode electrically coupled to the housing, and a second electrode electrically coupled to the first terminal. The DC source assembly further comprises a second capacitor having a first electrode electrically coupled to the housing, and a second electrode electrically coupled to the second terminal.

  11. 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.

  12. A mutation in the 530 loop of Escherichia coli 16S ribosomal RNA causes resistance to streptomycin.

    Science.gov (United States)

    Melançon, P; Lemieux, C; Brakier-Gingras, L

    1988-01-01

    Oligonucleotide-directed mutagenesis was used to introduce an A to C transversion at position 523 in the 16S ribosomal RNA gene of Escherichia coli rrnB operon cloned in plasmid pKK3535. E. coli cells transformed with the mutated plasmid were resistant to streptomycin. The mutated ribosomes isolated from these cells were not stimulated by streptomycin to misread the message in a poly(U)-directed assay. They were also restrictive to the stimulation of misreading by other error-promoting related aminoglycoside antibiotics such as neomycin, kanamycin or gentamicin, which do not compete for the streptomycin binding site. The 530 loop where the mutation in the 16S rRNA is located has been mapped at the external surface of the 30S subunit, and is therefore distal from the streptomycin binding site at the subunit interface. Our results support the conclusion that the mutation at position 523 in the 16S rRNA does not interfere with the binding of streptomycin, but prevents the drug from inducing conformational changes in the 530 loop which account for its miscoding effect. Since this effect primarily results from a perturbation of the translational proofreading control, our results also provide evidence that the 530 loop of the 16S rRNA is involved in this accuracy control. Images PMID:3054810

  13. Fasciola hepatica - where is 28S ribosomal RNA?

    Science.gov (United States)

    Haçarız, Orçun; Sayers, Gearóid

    2013-10-01

    Advanced molecular biology techniques are currently used to develop new effective strategies against fasciolosis. Assessment of the quality of extracted total RNA is an important step prior to commencing many molecular biology methods such as transcriptomics. However, RNA quality assessment is complicated for some organisms, including Fasciola hepatica, by the absence of a 28S rRNA peak/band, when assessed with modern protocols. In this study, electrophoretic profiles of F. hepatica ribosomal RNAs were evaluated using microfluidics capillary based and conventional non-denaturing gel electrophoresis methods. An important modification to recommended protocols, the exclusion of heat-denaturation step, in the microfluidics capillary based electrophoresis is critical to visualise the expected 28S rRNA and obtain an RNA integrity number (RIN). The intensity of the 28S rRNA band is reduced by the effect of non-denaturing gel electrophoresis. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Globular conformation of some ribosomal proteins in solution

    International Nuclear Information System (INIS)

    Serdyuk, I.N.; Spirin, A.S.

    1978-01-01

    The possibility that such RNA-binding proteins of the 30 S subparticle as S4, S7, S8 and S16 exist in the form of compact globules in solution has been explored experimentally. These proteins have been studied in D 2 O solution by neutron scattering to measure their radii of gyration. This type of radiation using D 2 O as a solvent provides the maximum 'contrast', that is the maximum difference between the scattering of the protein and the solvent. It allowed measurements to be made using protein at <= 1.5 mg/ml. The radii of gyration for the ribosomal proteins S4, S7, S8 and S16 were found to be relatively small corresponding to the radii of gyration of compact globular proteins of the same molecular weights. (Auth.)

  15. Expanding the Entamoeba Universe: New Hosts Yield Novel Ribosomal Lineages.

    Science.gov (United States)

    Jacob, Alison S; Busby, Eloise J; Levy, Abigail D; Komm, Natasha; Clark, C Graham

    2016-01-01

    Removing the requirement for cell culture has led to a substantial increase in the number of lineages of Entamoeba recognized as distinct. Surveying the range of potential host species for this parasite genus has barely been started and it is clear that additional sampling of the same host in different locations often identifies additional diversity. In this study, using small subunit ribosomal RNA gene sequencing, we identify four new lineages of Entamoeba, including the first report of Entamoeba from an elephant, and extend the host range of some previously described lineages. In addition, examination of microbiome data from a number of host animals suggests that substantial Entamoeba diversity remains to be uncovered. © 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists.

  16. Ubiquitination of newly synthesized proteins at the ribosome.

    Science.gov (United States)

    Wang, Feng; Canadeo, Larissa A; Huibregtse, Jon M

    2015-07-01

    Newly synthesized proteins can be misfolded or damaged because of errors during synthesis or environmental insults (e.g., heat shock), placing a significant burden on protein quality control systems. In addition, numerous human diseases are associated with a deficiency in eliminating aberrant proteins or accumulation of aggregated proteins. Understanding the mechanisms of protein quality control and disposal pathways for misfolded proteins is therefore crucial for therapeutic intervention in these diseases. Quality control processes function at many points in the life cycle of proteins, and a subset act at the actual site of protein synthesis, the ribosome. Here we summarize recent advances in the role of the ubiquitin proteasome system in protein quality control during the process of translation. Copyright © 2015 Elsevier B.V. and Société française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  17. Mechanisms for ribotoxin-induced ribosomal RNA cleavage.

    Science.gov (United States)

    He, Kaiyu; Zhou, Hui-Ren; Pestka, James J

    2012-11-15

    The Type B trichothecene deoxynivalenol (DON), a ribotoxic mycotoxin known to contaminate cereal-based foods, induces ribosomal RNA (rRNA) cleavage in the macrophage via p38-directed activation of caspases. Here we employed the RAW 264.7 murine macrophage model to test the hypothesis that this rRNA cleavage pathway is similarly induced by other ribotoxins. Capillary electrophoresis confirmed that the antibiotic anisomycin (≥25ng/ml), the macrocylic trichothecene satratoxin G (SG) (≥10ng/ml) and ribosome-inactivating protein ricin (≥300ng/ml) induced 18s and 28s rRNA fragmentation patterns identical to that observed for DON. Also, as found for DON, inhibition of p38, double-stranded RNA-activated kinase (PKR) and hematopoietic cell kinase (Hck) suppressed MAPK anisomycin-induced rRNA cleavage, while, in contrast, their inhibition did not affect SG- and ricin-induced rRNA fragmentation. The p53 inhibitor pifithrin-μ and pan caspase inhibitor Z-VAD-FMK suppressed rRNA cleavage induced by anisomycin, SG and ricin, indicating that these ribotoxins shared with DON a conserved downstream pathway. Activation of caspases 8, 9 and 3 concurrently with apoptosis further suggested that rRNA cleavage occurred in parallel with both extrinsic and intrinsic pathways of programmed cell death. When specific inhibitors of cathepsins L and B (lysosomal cysteine cathepsins active at cytosolic neutral pH) were tested, only the former impaired anisomycin-, SG-, ricin- and DON-induced rRNA cleavage. Taken together, the data suggest that (1) all four ribotoxins induced p53-dependent rRNA cleavage via activation of cathepsin L and caspase 3, and (2) activation of p53 by DON and anisomycin involved p38 whereas SG and ricin activated p53 by an alternative mechanism. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. Ribosomal protein gene knockdown causes developmental defects in zebrafish.

    Directory of Open Access Journals (Sweden)

    Tamayo Uechi

    Full Text Available The ribosomal proteins (RPs form the majority of cellular proteins and are mandatory for cellular growth. RP genes have been linked, either directly or indirectly, to various diseases in humans. Mutations in RP genes are also associated with tissue-specific phenotypes, suggesting a possible role in organ development during early embryogenesis. However, it is not yet known how mutations in a particular RP gene result in specific cellular changes, or how RP genes might contribute to human diseases. The development of animal models with defects in RP genes will be essential for studying these questions. In this study, we knocked down 21 RP genes in zebrafish by using morpholino antisense oligos to inhibit their translation. Of these 21, knockdown of 19 RPs resulted in the development of morphants with obvious deformities. Although mutations in RP genes, like other housekeeping genes, would be expected to result in nonspecific developmental defects with widespread phenotypes, we found that knockdown of some RP genes resulted in phenotypes specific to each gene, with varying degrees of abnormality in the brain, body trunk, eyes, and ears at about 25 hours post fertilization. We focused further on the organogenesis of the brain. Each knocked-down gene that affected the morphogenesis of the brain produced a different pattern of abnormality. Among the 7 RP genes whose knockdown produced severe brain phenotypes, 3 human orthologs are located within chromosomal regions that have been linked to brain-associated diseases, suggesting a possible involvement of RP genes in brain or neurological diseases. The RP gene knockdown system developed in this study could be a powerful tool for studying the roles of ribosomes in human diseases.

  19. The fragmented mitochondrial ribosomal RNAs of Plasmodium falciparum.

    Science.gov (United States)

    Feagin, Jean E; Harrell, Maria Isabel; Lee, Jung C; Coe, Kevin J; Sands, Bryan H; Cannone, Jamie J; Tami, Germaine; Schnare, Murray N; Gutell, Robin R

    2012-01-01

    The mitochondrial genome in the human malaria parasite Plasmodium falciparum is most unusual. Over half the genome is composed of the genes for three classic mitochondrial proteins: cytochrome oxidase subunits I and III and apocytochrome b. The remainder encodes numerous small RNAs, ranging in size from 23 to 190 nt. Previous analysis revealed that some of these transcripts have significant sequence identity with highly conserved regions of large and small subunit rRNAs, and can form the expected secondary structures. However, these rRNA fragments are not encoded in linear order; instead, they are intermixed with one another and the protein coding genes, and are coded on both strands of the genome. This unorthodox arrangement hindered the identification of transcripts corresponding to other regions of rRNA that are highly conserved and/or are known to participate directly in protein synthesis. The identification of 14 additional small mitochondrial transcripts from P. falciparum and the assignment of 27 small RNAs (12 SSU RNAs totaling 804 nt, 15 LSU RNAs totaling 1233 nt) to specific regions of rRNA are supported by multiple lines of evidence. The regions now represented are highly similar to those of the small but contiguous mitochondrial rRNAs of Caenorhabditis elegans. The P. falciparum rRNA fragments cluster on the interfaces of the two ribosomal subunits in the three-dimensional structure of the ribosome. All of the rRNA fragments are now presumed to have been identified with experimental methods, and nearly all of these have been mapped onto the SSU and LSU rRNAs. Conversely, all regions of the rRNAs that are known to be directly associated with protein synthesis have been identified in the P. falciparum mitochondrial genome and RNA transcripts. The fragmentation of the rRNA in the P. falciparum mitochondrion is the most extreme example of any rRNA fragmentation discovered.

  20. Nuclear reactor spacer assembly

    International Nuclear Information System (INIS)

    Anthony, A.J.; Groves, M.D.

    1979-01-01

    A fuel assembly for a nuclear reactor is disclosed wherein the fuel element receiving and supporting grid is comprised of a first metal, the guide tubes which pass through the grid assembly are comprised of a second metal and the grid is supported on the guide tubes by means of expanded sleeves located intermediate the grid and guide tubes. The fuel assembly is fabricated by inserting the sleeves, of initial outer diameter commensurate with the guide tube outer diameters, through the holes in the grid assembly provided for the guide tubes and thereafter expanding the sleeves radially outwardly along their entire length such that the guide tubes can subsequently be passed through the sleeves. The step of radial expansion, as a result of windows provided in the sleeves having dimensions commensurate with the geometry of the grid, mechanically captures the grid and simultaneously preloads the sleeve against the grid whereby relative motion between the grid and guide tube will be precluded

  1. Automated Assembly Center (AAC)

    Science.gov (United States)

    Stauffer, Robert J.

    1993-01-01

    The objectives of this project are as follows: to integrate advanced assembly and assembly support technology under a comprehensive architecture; to implement automated assembly technologies in the production of high-visibility DOD weapon systems; and to document the improved cost, quality, and lead time. This will enhance the production of DOD weapon systems by utilizing the latest commercially available technologies combined into a flexible system that will be able to readily incorporate new technologies as they emerge. Automated assembly encompasses the following areas: product data, process planning, information management policies and framework, three schema architecture, open systems communications, intelligent robots, flexible multi-ability end effectors, knowledge-based/expert systems, intelligent workstations, intelligent sensor systems, and PDES/PDDI data standards.

  2. Marine Acoustic Sensor Assembly

    National Research Council Canada - National Science Library

    Ruffa, Anthony A

    2007-01-01

    A marine acoustic sensor assembly includes an acoustic panel having a forward surface and an after surface, a laser scanner oriented so as to project a laser beam onto the acoustic panel after surface...

  3. Mesoscale Polymer Assemblies

    Science.gov (United States)

    Choudhary, Satyan; Pham, Jonathan; Crosby, Alfred

    2015-03-01

    Materials encompassing structural hierarchy and multi-functionality allow for remarkable physical properties across different length scales. Mesoscale Polymer (MSP) assemblies provide a critical link, from nanometer to centimeter scales, in the definition of such hierarchical structures. Recent focus has been on exploiting these MSP assemblies for optical, electronic, photonics and biological applications. We demonstrate a novel fabrication method for MSP assemblies. Current fabrication methods restrict the length scale and volume of such assemblies. A new method developed uses a simple piezo-actuated motion for de-pinning of a polymer solution trapped by capillary forces between a flexible blade and a rigid substrate. The advantages of new method include ability to make MSP of monodisperse length and to fabricate sufficient volumes of MSP to study their physical properties and functionality in liquid dispersions. We demonstrate the application of MSP as filler for soft materials, providing rheological studies of the MSP with surrounding matrices.

  4. Federal Assembly, Prague

    Czech Academy of Sciences Publication Activity Database

    Hnídková, Vendula

    -, č. 37 (2011), s. 70 ISSN 1573-3815 Institutional research plan: CEZ:AV0Z80330511 Keywords : Czech architecture of the 20th century * Karel Prager * federal assembly Subject RIV: AL - Art, Architecture , Cultural Heritage

  5. Nuclear fuel assembly

    International Nuclear Information System (INIS)

    Betten, P.R.

    1976-01-01

    Under the invention the fuel assembly is particularly suitable for liquid metal cooled fast neutron breeder reactors. Hence, according to the invention a fuel assembly cladding includes inward corrugations with respect to the remainder of the cladding according to a recurring pattern determined by the pitch of the metal wire helically wound round the fuel rods of the assembly. The parts of the cladding pressed inwards correspond to the areas in which the wire encircling the peripheral fuel rods is generally located apart from the cladding, thereby reducing the play between the cladding and the peripheral fuel rods situated in these areas. The reduction in the play in turn improves the coolant flow in the internal secondary channels of the fuel assembly to the detriment of the flow in the peripheral secondary channels and thereby establishes a better coolant fluid temperature profile [fr

  6. Assembling Sustainable Territories

    DEFF Research Database (Denmark)

    Vandergeest, Peter; Ponte, Stefano; Bush, Simon

    2015-01-01

    The authors show how certification assembles ‘sustainable’ territories through a complex layering of regulatory authority in which both government and nongovernment entities claim rule-making authority, sometimes working together, sometimes in parallel, sometimes competitively. It is argued...

  7. VIRUS instrument collimator assembly

    Science.gov (United States)

    Marshall, Jennifer L.; DePoy, Darren L.; Prochaska, Travis; Allen, Richard D.; Williams, Patrick; Rheault, Jean-Philippe; Li, Ting; Nagasawa, Daniel Q.; Akers, Christopher; Baker, David; Boster, Emily; Campbell, Caitlin; Cook, Erika; Elder, Alison; Gary, Alex; Glover, Joseph; James, Michael; Martin, Emily; Meador, Will; Mondrik, Nicholas; Rodriguez-Patino, Marisela; Villanueva, Steven; Hill, Gary J.; Tuttle, Sarah; Vattiat, Brian; Lee, Hanshin; Chonis, Taylor S.; Dalton, Gavin B.; Tacon, Mike

    2014-07-01

    The Visual Integral-Field Replicable Unit Spectrograph (VIRUS) instrument is a baseline array 150 identical fiber fed optical spectrographs designed to support observations for the Hobby-Eberly Telescope Dark Energy Experiment (HETDEX). The collimator subassemblies of the instrument have been assembled in a production line and are now complete. Here we review the design choices and assembly practices used to produce a suite of identical low-cost spectrographs in a timely fashion using primarily unskilled labor.

  8. Nuclear fuel assembly

    International Nuclear Information System (INIS)

    Helmersson, S.

    1982-05-01

    The fuel assembly has a square-shaped cross section and it is put together of four quadratic assemblies each having seventeen positions for fuel rods, which are situated in a lattice formed by a pattern of triangles and squares. Nine of the positions correspond to the junction of a square lattice which has four squares, whereas eight rods are outside the quadratic past. (G.B.)

  9. Polymer Directed Protein Assemblies

    Directory of Open Access Journals (Sweden)

    Patrick van Rijn

    2013-05-01

    Full Text Available Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e.g., virus particles. Viruses are a multi-protein assembly of which the morphology is dictated by poly-nucleotides namely RNA or DNA. This “biopolymer” directs the proteins and imposes limitations on the structure like the length or diameter of the particle. Not only do these bionanoparticles use polymer-directed self-assembly, also processes like amyloid formation are in a way a result of directed protein assembly by partial unfolded/misfolded biopolymers namely, polypeptides. The combination of proteins and synthetic polymers, inspired by the natural processes, are therefore regarded as a highly promising area of research. Directed protein assembly is versatile with respect to the possible interactions which brings together the protein and polymer, e.g., electrostatic, v.d. Waals forces or covalent conjugation, and possible combinations are numerous due to the large amounts of different polymers and proteins available. The protein-polymer interacting behavior and overall morphology is envisioned to aid in clarifying protein-protein interactions and are thought to entail some interesting new functions and properties which will ultimately lead to novel bio-hybrid materials.

  10. NMR structure determination of the binding site for ribosomal protein S8 from Escherichia coli 16 S rRNA.

    Science.gov (United States)

    Kalurachchi, K; Nikonowicz, E P

    1998-07-24

    Many cellular processes involve the preferential interaction of an RNA molecule with a specific protein. A detailed analysis of the individual protein and RNA components of these interactions can provide unique insights into the structural features important for protein-RNA recognition. Ribosomal protein S8 of Escherichia coli plays a key role in 30 S ribosomal subunit assembly through its interaction with 16 S rRNA. The binding site for protein S8 comprises a portion of helix 21, nucleotides G588 to G604 and C634 to C651. This region forms a base-paired helix that is interrupted by a non-Watson-Crick segment composed of nine phylogenetically conserved nucleotides. We have investigated the detailed structure of the conserved segment and the interaction of this region with metal ions using NMR spectroscopy. Twenty-four of the 40 calculated structures converged to similar conformations and were grouped into two families. The main difference between the families is the orientation of the base of U641. The rms deviation between the heavy-atoms of the ten lowest-energy structures is 1.24 A. The orientations of the G597.C643 base-pair and A595.(A596.U644) base-triple within the conserved core have been defined and appear to extend the proximal segment of helix 21 into the phylogenetically conserved core. The base of A642 terminates this helix by stacking against C643 and the base of U641 forms hydrogen bonds with core nucleotides. The conserved core also contains a Mg2+-binding site that promotes stabilization of the secondary and tertiary structure elements of the core. A model for the interaction of S8 with its RNA-binding site is proposed. Copyright 1998 Academic Press.

  11. A Portrait of Ribosomal DNA Contacts with Hi-C Reveals 5S and 45S rDNA Anchoring Points in the Folded Human Genome.

    Science.gov (United States)

    Yu, Shoukai; Lemos, Bernardo

    2016-12-31

    Ribosomal RNAs (rRNAs) account for >60% of all RNAs in eukaryotic cells and are encoded in the ribosomal DNA (rDNA) arrays. The rRNAs are produced from two sets of loci: the 5S rDNA array resides exclusively on human chromosome 1, whereas the 45S rDNA array resides on the short arm of five human acrocentric chromosomes. The 45S rDNA gives origin to the nucleolus, the nuclear organelle that is the site of ribosome biogenesis. Intriguingly, 5S and 45S rDNA arrays exhibit correlated copy number variation in lymphoblastoid cells (LCLs). Here we examined the genomic architecture and repeat content of the 5S and 45S rDNA arrays in multiple human genome assemblies (including PacBio MHAP assembly) and ascertained contacts between the rDNA arrays and the rest of the genome using Hi-C datasets from two human cell lines (erythroleukemia K562 and lymphoblastoid cells). Our analyses revealed that 5S and 45S arrays each have thousands of contacts in the folded genome, with rDNA-associated regions and genes dispersed across all chromosomes. The rDNA contact map displayed conserved and disparate features between two cell lines, and pointed to specific chromosomes, genomic regions, and genes with evidence of spatial proximity to the rDNA arrays; the data also showed a lack of direct physical interaction between the 5S and 45S rDNA arrays. Finally, the analysis identified an intriguing organization in the 5S array with Alu and 5S elements adjacent to one another and organized in opposite orientation along the array. Portraits of genome folding centered on the ribosomal DNA array could help understand the emergence of concerted variation, the control of 5S and 45S expression, as well as provide insights into an organelle that contributes to the spatial localization of human chromosomes during interphase. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  12. Ribosomal studies on the 70S ribosome of E.coli by means of neutron scattering; Strukturuntersuchungen am 70S-Ribosom von E.coli unter Anwendung von Neutronenstreuung

    Energy Technology Data Exchange (ETDEWEB)

    Burkhardt, N. [GKSS-Forschungszentrum Geesthacht GmbH (Germany). Inst. fuer Werkstofforschung

    1997-12-31

    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 ({sup 1}H) for deuterium ({sup 2}H), 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.) [Deutsch] Ribosomen sind Ribonukleinsaeure-Protein Komplexe, die in allen lebenden Organismen die Proteinbiosynthese katalysieren. Strukturmodelle fuer das prokaryontische 70S-Ribosom beruhen derzeit vorwiegend auf elektronenmikroskopischen Untersuchungen und beschreiben im wesentlichen die aeussere Oberflaeche des Partikels. Informationen ueber die innere Struktur des Ribosoms koennen Messungen mit

  13. RNA self-assembly and RNA nanotechnology.

    Science.gov (United States)

    Grabow, Wade W; Jaeger, Luc

    2014-06-17

    as the ribosome, large ribozymes, and riboswitches. Thus, the next step in synthetic RNA design will involve new ways to implement these same types of dynamic and responsive architectures into nanostructures functioning as real nanomachines in and outside the cell. RNA nanotechnology will likely garner broader utility and influence with a greater focus on the interplay between thermodynamic and kinetic influences on RNA self-assembly and using natural RNAs as guiding principles.

  14. Understanding Biases in Ribosome Profiling Experiments Reveals Signatures of Translation Dynamics in Yeast.

    Directory of Open Access Journals (Sweden)

    Jeffrey A Hussmann

    2015-12-01

    Full Text Available Ribosome profiling produces snapshots of the locations of actively translating ribosomes on messenger RNAs. These snapshots can be used to make inferences about translation dynamics. Recent ribosome profiling studies in yeast, however, have reached contradictory conclusions regarding the average translation rate of each codon. Some experiments have used cycloheximide (CHX to stabilize ribosomes before measuring their positions, and these studies all counterintuitively report a weak negative correlation between the translation rate of a codon and the abundance of its cognate tRNA. In contrast, some experiments performed without CHX report strong positive correlations. To explain this contradiction, we identify unexpected patterns in ribosome density downstream of each type of codon in experiments that use CHX. These patterns are evidence that elongation continues to occur in the presence of CHX but with dramatically altered codon-specific elongation rates. The measured positions of ribosomes in these experiments therefore do not reflect the amounts of time ribosomes spend at each position in vivo. These results suggest that conclusions from experiments in yeast using CHX may need reexamination. In particular, we show that in all such experiments, codons decoded by less abundant tRNAs were in fact being translated more slowly before the addition of CHX disrupted these dynamics.

  15. Ribosomal proteins as biomarkers for bacterial identification by mass spectrometry in the clinical microbiology laboratory.

    Science.gov (United States)

    Suarez, Stéphanie; Ferroni, Agnès; Lotz, Aurélie; Jolley, Keith A; Guérin, Philippe; Leto, Julie; Dauphin, Brunhilde; Jamet, Anne; Maiden, Martin C J; Nassif, Xavier; Armengaud, Jean

    2013-09-01

    Whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for identification of microorganisms that is increasingly used in microbiology laboratories. This identification is based on the comparison of the tested isolate mass spectrum with reference databases. Using Neisseria meningitidis as a model organism, we showed that in one of the available databases, the Andromas database, 10 of the 13 species-specific biomarkers correspond to ribosomal proteins. Remarkably, one biomarker, ribosomal protein L32, was subject to inter-strain variability. The analysis of the ribosomal protein patterns of 100 isolates for which whole genome sequences were available, confirmed the presence of inter-strain variability in the molecular weight of 29 ribosomal proteins, thus establishing a correlation between the sequence type (ST) and/or clonal complex (CC) of each strain and its ribosomal protein pattern. Since the molecular weight of three of the variable ribosomal proteins (L30, L31 and L32) was included in the spectral window observed by MALDI-TOF MS in clinical microbiology, i.e., 3640-12000 m/z, we were able by analyzing the molecular weight of these three ribosomal proteins to classify each strain in one of six subgroups, each of these subgroups corresponding to specific STs and/or CCs. Their detection by MALDI-TOF allows therefore a quick typing of N. meningitidis isolates. © 2013 Elsevier B.V. All rights reserved.

  16. Ribosomal stress induces L11- and p53-dependent apoptosis in mouse pluripotent stem cells.

    Science.gov (United States)

    Morgado-Palacin, Lucia; Llanos, Susana; Serrano, Manuel

    2012-02-01

    Ribosome biogenesis is the most demanding energetic process in proliferating cells and it is emerging as a critical sensor of cellular homeostasis. Upon disturbance of ribosome biogenesis, specific free ribosomal proteins, most notably L11, bind and inhibit Mdm2, resulting in activation of the tumor suppressor p53. This pathway has been characterized in somatic and cancer cells, but its function in embryonic pluripotent cells has remained unexplored. Here, we show that treatment with low doses of Actinomycin D or depletion of ribosomal protein L37, two well-established inducers of ribosomal stress, activate p53 in an L11-dependent manner in mouse embryonic stem cells (ESCs) and in induced pluripotent stem cells (iPSCs). Activation of p53 results in transcriptional induction of p53 targets, including p21, Mdm2, Pidd, Puma, Noxa and Bax. Finally, ribosomal stress elicits L11- and p53-dependent apoptosis in ESCs/iPSCs. These results extend to pluripotent cells the functionality of the ribosomal stress pathway and we speculate that this could be a relevant cellular checkpoint during early embryogenesis.

  17. Human Assisted Assembly Processes

    Energy Technology Data Exchange (ETDEWEB)

    CALTON,TERRI L.; PETERS,RALPH R.

    2000-01-01

    Automatic assembly sequencing and visualization tools are valuable in determining the best assembly sequences, but without Human Factors and Figure Models (HFFMs) it is difficult to evaluate or visualize human interaction. In industry, accelerating technological advances and shorter market windows have forced companies to turn to an agile manufacturing paradigm. This trend has promoted computerized automation of product design and manufacturing processes, such as automated assembly planning. However, all automated assembly planning software tools assume that the individual components fly into their assembled configuration and generate what appear to be a perfectly valid operations, but in reality the operations cannot physically be carried out by a human. Similarly, human figure modeling algorithms may indicate that assembly operations are not feasible and consequently force design modifications; however, if they had the capability to quickly generate alternative assembly sequences, they might have identified a feasible solution. To solve this problem HFFMs must be integrated with automated assembly planning to allow engineers to verify that assembly operations are possible and to see ways to make the designs even better. Factories will very likely put humans and robots together in cooperative environments to meet the demands for customized products, for purposes including robotic and automated assembly. For robots to work harmoniously within an integrated environment with humans the robots must have cooperative operational skills. For example, in a human only environment, humans may tolerate collisions with one another if they did not cause much pain. This level of tolerance may or may not apply to robot-human environments. Humans expect that robots will be able to operate and navigate in their environments without collisions or interference. The ability to accomplish this is linked to the sensing capabilities available. Current work in the field of cooperative

  18. High-Resolution Melting Curve Analysis of the 16S Ribosomal Gene to Detect and Identify Pathogenic and Saprophytic Leptospira Species in Colombian Isolates.

    Science.gov (United States)

    Peláez Sánchez, Ronald G; Quintero, Juan Álvaro López; Pereira, Martha María; Agudelo-Flórez, Piedad

    2017-05-01

    AbstractIt is important to identify the circulating Leptospira agent to enhance the performance of serodiagnostic tests by incorporating specific antigens of native species, develop vaccines that take into account the species/serovars circulating in different regions, and optimize prevention and control strategies. The objectives of this study were to develop a polymerase chain reaction (PCR)-high-resolution melting (HRM) assay for differentiating between species of the genus Leptospira and to verify its usefulness in identifying unknown samples to species level. A set of primers from the initial region of the 16S ribosomal gene was designed to detect and differentiate the 22 species of Leptospira . Eleven reference strains were used as controls to establish the reference species and differential melting curves. Twenty-five Colombian Leptospira isolates were studied to evaluate the usefulness of the PCR-HRM assay in identifying unknown samples to species level. This identification was confirmed by sequencing and phylogenetic analysis of the 16S ribosomal gene. Eleven Leptospira species were successfully identified, except for Leptospira meyeri / Leptospira yanagawae because the sequences were 100% identical. The 25 isolates from humans, animals, and environmental water sources were identified as Leptospira santarosai (twelve), Leptospira interrogans (nine), and L. meyeri / L. yanagawae (four). The species verification was 100% concordant between PCR-HRM and phylogenetic analysis of the 16S ribosomal gene. The PCR-HRM assay designed in this study is a useful tool for identifying Leptospira species from isolates.

  19. Purification and characterization of ribosomal proteins L27 and L30 having antimicrobial activity produced by the Lactobacillus salivarius SGL 03.

    Science.gov (United States)

    Pidutti, P; Federici, F; Brandi, J; Manna, L; Rizzi, E; Marini, U; Cecconi, D

    2018-02-01

    The aim of this study was to investigate the antimicrobial potential of proteins secreted by a new strain of Lactobacillus salivarius. The secretome of L. salivarius SGL 03 strain was analysed by gel-assisted fractionation and MS/MS to identify low-molecular-mass proteins. This strategy allowed us to identify 10 secreted proteins. Then, a combination of heterologous expression and agar well diffusion was used to characterize them as to their antimicrobial activity, mechanisms of action and stability. Our findings indicate that L27 and L30 proteins of the 50S ribosomal subunit have antimicrobial activity against Streptococcus pyogenes, Streptococcus uberis and Enterococcus faecium. In addition, both proteins are bactericidal against S. pyogenes and maintain their antimicrobial activity after different protease treatments, at acidic pH, after heat treatment, and if stored in a refrigerated ambient at least at 4°C. The overall results demonstrated that the L27 and L30 ribosomal proteins are of interest as new antimicrobial molecules to prevent the growth of S. pyogenes, S. uberis and E. faecium. Our results provide the first insight into the extra-ribosomal activity of L27 and L30 secreted proteins of L. salivarius. This study demonstrated the capacity of L. salivarius SGL 03 to produce antimicrobial molecules and suggested this strain as a promising probiotic candidate. © 2017 The Society for Applied Microbiology.

  20. Ribosomal trafficking is reduced in Schwann cells following induction of myelination.

    Science.gov (United States)

    Love, James M; Shah, Sameer B

    2015-01-01

    Local synthesis of proteins within the Schwann cell periphery is extremely important for efficient process extension and myelination, when cells undergo dramatic changes in polarity and geometry. Still, it is unclear how ribosomal distributions are developed and maintained within Schwann cell projections to sustain local translation. In this multi-disciplinary study, we expressed a plasmid encoding a fluorescently labeled ribosomal subunit (L4-GFP) in cultured primary rat Schwann cells. This enabled the generation of high-resolution, quantitative data on ribosomal distributions and trafficking dynamics within Schwann cells during early stages of myelination, induced by ascorbic acid treatment. Ribosomes were distributed throughout Schwann cell projections, with ~2-3 bright clusters along each projection. Clusters emerged within 1 day of culture and were maintained throughout early stages of myelination. Three days after induction of myelination, net ribosomal movement remained anterograde (directed away from the Schwann cell body), but ribosomal velocity decreased to about half the levels of the untreated group. Statistical and modeling analysis provided additional insight into key factors underlying ribosomal trafficking. Multiple regression analysis indicated that net transport at early time points was dependent on anterograde velocity, but shifted to dependence on anterograde duration at later time points. A simple, data-driven rate kinetics model suggested that the observed decrease in net ribosomal movement was primarily dictated by an increased conversion of anterograde particles to stationary particles, rather than changes in other directional parameters. These results reveal the strength of a combined experimental and theoretical approach in examining protein localization and transport, and provide evidence of an early establishment of ribosomal populations within Schwann cell projections with a reduction in trafficking following initiation of myelination.

  1. Revisiting the Structures of Several Antibiotics Bound to the Bacterial Ribosome

    Energy Technology Data Exchange (ETDEWEB)

    D Bulkley; C Innis; G Blaha; T Steitz

    2011-12-31

    The increasing prevalence of antibiotic-resistant pathogens reinforces the need for structures of antibiotic-ribosome complexes that are accurate enough to enable the rational design of novel ribosome-targeting therapeutics. Structures of many antibiotics in complex with both archaeal and eubacterial ribosomes have been determined, yet discrepancies between several of these models have raised the question of whether these differences arise from species-specific variations or from experimental problems. Our structure of chloramphenicol in complex with the 70S ribosome from Thermus thermophilus suggests a model for chloramphenicol bound to the large subunit of the bacterial ribosome that is radically different from the prevailing model. Further, our structures of the macrolide antibiotics erythromycin and azithromycin in complex with a bacterial ribosome are indistinguishable from those determined of complexes with the 50S subunit of Haloarcula marismortui, but differ significantly from the models that have been published for 50S subunit complexes of the eubacterium Deinococcus radiodurans. Our structure of the antibiotic telithromycin bound to the T. thermophilus ribosome reveals a lactone ring with a conformation similar to that observed in the H. marismortui and D. radiodurans complexes. However, the alkyl-aryl moiety is oriented differently in all three organisms, and the contacts observed with the T. thermophilus ribosome are consistent with biochemical studies performed on the Escherichia coli ribosome. Thus, our results support a mode of macrolide binding that is largely conserved across species, suggesting that the quality and interpretation of electron density, rather than species specificity, may be responsible for many of the discrepancies between the models.

  2. Revisiting the structures of several antibiotics bound to the bacterial ribosome

    Energy Technology Data Exchange (ETDEWEB)

    Bulkley, David; Innis, C. Axel; Blaha, Gregor; Steitz, Thomas A. (Yale)

    2010-10-08

    The increasing prevalence of antibiotic-resistant pathogens reinforces the need for structures of antibiotic-ribosome complexes that are accurate enough to enable the rational design of novel ribosome-targeting therapeutics. Structures of many antibiotics in complex with both archaeal and eubacterial ribosomes have been determined, yet discrepancies between several of these models have raised the question of whether these differences arise from species-specific variations or from experimental problems. Our structure of chloramphenicol in complex with the 70S ribosome from Thermus thermophilus suggests a model for chloramphenicol bound to the large subunit of the bacterial ribosome that is radically different from the prevailing model. Further, our structures of the macrolide antibiotics erythromycin and azithromycin in complex with a bacterial ribosome are indistinguishable from those determined of complexes with the 50S subunit of Haloarcula marismortui, but differ significantly from the models that have been published for 50S subunit complexes of the eubacterium Deinococcus radiodurans. Our structure of the antibiotic telithromycin bound to the T. thermophilus ribosome reveals a lactone ring with a conformation similar to that observed in the H. marismortui and D. radiodurans complexes. However, the alkyl-aryl moiety is oriented differently in all three organisms, and the contacts observed with the T. thermophilus ribosome are consistent with biochemical studies performed on the Escherichia coli ribosome. Thus, our results support a mode of macrolide binding that is largely conserved across species, suggesting that the quality and interpretation of electron density, rather than species specificity, may be responsible for many of the discrepancies between the models.

  3. A Numbers Game: Ribosome Densities, Bacterial Growth, and Antibiotic-Mediated Stasis and Death

    Directory of Open Access Journals (Sweden)

    Bruce R. Levin

    2017-02-01

    Full Text Available We postulate that the inhibition of growth and low rates of mortality of bacteria exposed to ribosome-binding antibiotics deemed bacteriostatic can be attributed almost uniquely to these drugs reducing the number of ribosomes contributing to protein synthesis, i.e., the number of effective ribosomes. We tested this hypothesis with Escherichia coli K-12 MG1655 and constructs that had been deleted for 1 to 6 of the 7 rRNA (rrn operons. In the absence of antibiotics, constructs with fewer rrn operons have lower maximum growth rates and longer lag phases than those with more ribosomal operons. In the presence of the ribosome-binding “bacteriostatic” antibiotics tetracycline, chloramphenicol, and azithromycin, E. coli strains with 1 and 2 rrn operons are killed at a substantially higher rate than those with more rrn operons. This increase in the susceptibility of E. coli with fewer rrn operons to killing by ribosome-targeting bacteriostatic antibiotics is not reflected in their greater sensitivity to killing by the bactericidal antibiotic ciprofloxacin, which does not target ribosomes, but also to killing by gentamicin, which does. Finally, when such strains are exposed to these ribosome-targeting bacteriostatic antibiotics, the time before these bacteria start to grow again when the drugs are removed, referred to as the post-antibiotic effect (PAE, is markedly greater for constructs with fewer rrn operons than for those with more rrn operons. We interpret the results of these other experiments reported here as support for the hypothesis that the reduction in the effective number of ribosomes due to binding to these structures provides a sufficient explanation for the action of bacteriostatic antibiotics that target these structures.

  4. Crystal Structure of Hypusine-Containing Translation Factor eIF5A Bound to a Rotated Eukaryotic Ribosome

    Science.gov (United States)

    Shin, Byung-Sik; Rigger, Lukas; Yusupova, Gulnara; Micura, Ronald; Dever, Thomas E.; Yusupov, Marat

    2017-01-01

    Eukaryotic translation initiation factor eIF5A promotes protein synthesis by resolving polyproline-induced ribosomal stalling. Here, we report a 3.25-Å resolution crystal structure of eIF5A bound to the yeast 80S ribosome. The structure reveals a previously unseen conformation of an eIF5A–ribosome complex and highlights a possible functional link between conformational changes of the ribosome during protein synthesis and the eIF5A–ribosome association. PMID:27196944

  5. Defining the structural requirements for a helix in 23 S ribosomal RNA that confers erythromycin resistance

    DEFF Research Database (Denmark)

    Douthwaite, S; Powers, T; Lee, J Y

    1989-01-01

    The helix spanning nucleotides 1198 to 1247 (helix 1200-1250) in Escherichia coli 23 S ribosomal RNA (rRNA) is functionally important in protein synthesis, and deletions in this region confer erythromycin resistance. In order to define the structural requirements for resistance, we have dissected....... However, removal of either these or non-conserved nucleotides from helix 1200-1250 measurably reduces the efficiency of 23 S RNA in forming functional ribosomes. We have used chemical probing and a modified primer extension method to investigate erythromycin binding to wild-type and resistant ribosomes...

  6. The Arabidopsis TOR Kinase Specifically Regulates the Expression of Nuclear Genes Coding for Plastidic Ribosomal Proteins and the Phosphorylation of the Cytosolic Ribosomal Protein S6.

    Science.gov (United States)

    Dobrenel, Thomas; Mancera-Martínez, Eder; Forzani, Céline; Azzopardi, Marianne; Davanture, Marlène; Moreau, Manon; Schepetilnikov, Mikhail; Chicher, Johana; Langella, Olivier; Zivy, Michel; Robaglia, Christophe; Ryabova, Lyubov A; Hanson, Johannes; Meyer, Christian

    2016-01-01

    Protein translation is an energy consuming process that has to be fine-tuned at both the cell and organism levels to match the availability of resources. The target of rapamycin kinase (TOR) is a key regulator of a large range of biological processes in response to environmental cues. In this study, we have investigated the effects of TOR inactivation on the expression and regulation of Arabidopsis ribosomal proteins at different levels of analysis, namely from transcriptomic to phosphoproteomic. TOR inactivation resulted in a coordinated down-regulation of the transcription and translation of nuclear-encoded mRNAs coding for plastidic ribosomal proteins, which could explain the chlorotic phenotype of the TOR silenced plants. We have identified in the 5' untranslated regions (UTRs) of this set of genes a conserved sequence related to the 5' terminal oligopyrimidine motif, which is known to confer translational regulation by the TOR kinase in other eukaryotes. Furthermore, the phosphoproteomic analysis of the ribosomal fraction following TOR inactivation revealed a lower phosphorylation of the conserved Ser240 residue in the C-terminal region of the 40S ribosomal protein S6 (RPS6). These results were confirmed by Western blot analysis using an antibody that specifically recognizes phosphorylated Ser240 in RPS6. Finally, this antibody was used to follow TOR activity in plants. Our results thus uncover a multi-level regulation of plant ribosomal genes and proteins by the TOR kinase.

  7. Prevention of nuclear war

    International Nuclear Information System (INIS)

    Anon.

    1990-01-01

    Removing the threat of a nuclear war-as the General Assembly formally stated in the Final Document of its first special session devoted to disarmament, in 1978-is considered to be the task of the present day. In that Document, the General Assembly sought to establish principles, guidelines and procedures for preventing nuclear war. It declared that to that end, it was imperative to remove the threat of nuclear weapons, to halt and reverse the nuclear-arms race until the total elimination of nuclear weapons and their delivery systems had been achieved (see chapter iv), and to prevent the proliferation of nuclear weapons (see chapter VII). At the same time, it called for other measures designed to prevent the outbreak of nuclear war and to lessen the danger of the treat or use of nuclear weapons. The Assembly's clear call for action was dictated by the awareness that there was no insuperable barrier dividing peace from war and that, unless nations brought the spiralling nuclear-arms race to an end, the day might come when nuclear weapons would actually be used, with catastrophic consequences. In adopting the Final Document, the international community achieved, for the first time, a consensus on an international disarmament strategy having as its immediate goal the elimination of the danger of a nuclear war and the implementation of measures to halt and reverse the arms race. The General Assembly, at its second special session on disarmament, in 1982, reaffirmed the validity of the 1978 Final Document. This paper reports that nuclear issues and in particular the prevention of nuclear war remain, however, major concerns of all States. Undoubtedly, all nations have a vital interest in the negotiation of effective measures for her prevention of nuclear war, since nuclear weapons pose a unique threat to human survival. If nuclear war were to occur, its consequences would be global, not simple regional

  8. Dietary ascorbic acid normalizes ribosomal efficiency for collagen production in skin of streptozotocin-induced diabetic rats

    International Nuclear Information System (INIS)

    Schneir, M.; Imberman, M.; Ramamurthy, N.; Golub, L.

    1987-01-01

    The objective of this study was to quantify the contribution of both ribosome amount and ribosomal efficiency to decreased collagen production in skin of diabetic rats and diabetic rats treated with dietary ascorbic acid. Male Sprague-Dawley rats were distributed equally into the following categories: non-diabetic controls; diabetics; ascorbic acid-treated diabetics. On day-20, all rats were injected with ( 3 H)proline and killed after 2 h. Absolute rate of collagen production, ribosome content, and ribosomal efficiency of collagen production were quantified. Also ribosomal efficiency was quantified for ribosomes in sucrose-gradient fractionated post-mitochondrial supernatants. The results indicate that decreased ribosomal efficiency was responsible for 70% of the decreased collagen production with 30% caused by decreased ribosome content, when measured for total skin or sucrose gradient-isolated ribosomes. At both levels of analysis, ascorbic acid treatment normalized ribosomal efficiency, indicating diabetes-mediated decreased ribosomal efficiency for collagen production is related to a co-translational event, such as procollagen underhydroxylation

  9. Photovoltaic self-assembly.

    Energy Technology Data Exchange (ETDEWEB)

    Lavin, Judith; Kemp, Richard Alan; Stewart, Constantine A.

    2010-10-01

    This late-start LDRD was focused on the application of chemical principles of self-assembly on the ordering and placement of photovoltaic cells in a module. The drive for this chemical-based self-assembly stems from the escalating prices in the 'pick-and-place' technology currently used in the MEMS industries as the size of chips decreases. The chemical self-assembly principles are well-known on a molecular scale in other material science systems but to date had not been applied to the assembly of cells in a photovoltaic array or module. We explored several types of chemical-based self-assembly techniques, including gold-thiol interactions, liquid polymer binding, and hydrophobic-hydrophilic interactions designed to array both Si and GaAs PV chips onto a substrate. Additional research was focused on the modification of PV cells in an effort to gain control over the facial directionality of the cells in a solvent-based environment. Despite being a small footprint research project worked on for only a short time, the technical results and scientific accomplishments were significant and could prove to be enabling technology in the disruptive advancement of the microelectronic photovoltaics industry.

  10. Ribosomal biosynthesis of α-amanitin in Galerina marginata

    Science.gov (United States)

    Luo, Hong; Hallen-Adams, Heather E.; Scott-Craig, John S.; Walton, Jonathan D.

    2014-01-01

    Amatoxins, including α-amanitin, are bicyclic octapeptides found in mushrooms (Agaricomycetes, Agaricales) of certain species in the genera Amanita, Galerina, Lepiota, and Conocybe. Amatoxins and the chemically similar phallotoxins are synthesized on ribosomes in Amanita bisporigera, Amanita phalloides, and Amanita ocreata. In order to determine if amatoxins are synthesized by a similar mechanism in another, distantly related mushroom, we obtained genome survey sequence data from a monokaryotic isolate of Galerina marginata, which produces α-amanitin. The genome of G. marginata contains two copies of the α-amanitin gene (GmAMA1-1 and GmAMA1-2). The α-amanitin proprotein sequences of G. marginata (35 amino acids) are highly divergent from AMA1 of A. bisporigera except for the toxin region itself (IWGIGCNP in single-letter amino acid code) and the amino acids immediately upstream (N[A/S]TRLP). G. marginata does not contain any related toxin-encoding sequences besides GmAMA1-1 and GmAMA1-2. DNA from two other α-amanitin-producing isolates of Galerina (G. badipes and G. venenata) hybridized to GmAMA1, whereas DNA from the toxin non-producing species Galerina hybrida did not. Expression of the GmAMA1 genes was induced by growth on low carbon. RNASeq evidence indicates that both copies of GmAMA1 are expressed approximately equally. A prolyl oligopeptidase (POP) is strongly implicated in processing of the cyclic peptide toxins of A. bisporigera and Conocybe apala. G. marginata has two predicted POP genes; one, like AbPOPB of A. bisporigera, is present only in the toxin-producing isolates of Galerina and the other, like AbPOPA of A. bisporigera, is present in all species. Our results indicate that G. marginata biosynthesizes amatoxins on ribosomes by a pathway similar to Amanita species, involving a genetically encoded proprotein of 35 amino acids that is post-translationally processed by a POP. However, due to the high degree of divergence, the evolutionary

  11. Mechanisms for ribotoxin-induced ribosomal RNA cleavage

    Energy Technology Data Exchange (ETDEWEB)

    He, Kaiyu [Department of Microbiology and Molecular Genetics (United States); Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Zhou, Hui-Ren [Food Science and Human Nutrition (United States); Pestka, James J., E-mail: pestka@msu.edu [Department of Microbiology and Molecular Genetics (United States); Food Science and Human Nutrition (United States); Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States)

    2012-11-15

    The Type B trichothecene deoxynivalenol (DON), a ribotoxic mycotoxin known to contaminate cereal-based foods, induces ribosomal RNA (rRNA) cleavage in the macrophage via p38-directed activation of caspases. Here we employed the RAW 264.7 murine macrophage model to test the hypothesis that this rRNA cleavage pathway is similarly induced by other ribotoxins. Capillary electrophoresis confirmed that the antibiotic anisomycin (≥ 25 ng/ml), the macrocylic trichothecene satratoxin G (SG) (≥ 10 ng/ml) and ribosome-inactivating protein ricin (≥ 300 ng/ml) induced 18s and 28s rRNA fragmentation patterns identical to that observed for DON. Also, as found for DON, inhibition of p38, double-stranded RNA-activated kinase (PKR) and hematopoietic cell kinase (Hck) suppressed MAPK anisomycin-induced rRNA cleavage, while, in contrast, their inhibition did not affect SG- and ricin-induced rRNA fragmentation. The p53 inhibitor pifithrin-μ and pan caspase inhibitor Z-VAD-FMK suppressed rRNA cleavage induced by anisomycin, SG and ricin, indicating that these ribotoxins shared with DON a conserved downstream pathway. Activation of caspases 8, 9 and 3 concurrently with apoptosis further suggested that rRNA cleavage occurred in parallel with both extrinsic and intrinsic pathways of programmed cell death. When specific inhibitors of cathepsins L and B (lysosomal cysteine cathepsins active at cytosolic neutral pH) were tested, only the former impaired anisomycin-, SG-, ricin- and DON-induced rRNA cleavage. Taken together, the data suggest that (1) all four ribotoxins induced p53-dependent rRNA cleavage via activation of cathepsin L and caspase 3, and (2) activation of p53 by DON and anisomycin involved p38 whereas SG and ricin activated p53 by an alternative mechanism. Highlights: ► Deoxynivalenol (DON) anisomycin, satratoxin G (SG) and ricin are ribotoxins. ► Ribotoxins induce 18s and 28s rRNA cleavage in the RAW 264.7 macrophage model. ► Ribotoxins induce rRNA cleavage via

  12. Magnetic nanoparticle assemblies

    CERN Document Server

    Trohidou, Kalliopi N

    2014-01-01

    Magnetic nanoparticles with diameters in the range of a few nanometers are today at the cutting edge of modern technology and innovation because of their use in numerous applications ranging from engineering to biomedicine. A great deal of scientific interest has been focused on the functionalization of magnetic nanoparticle assemblies. The understanding of interparticle interactions is necessary to clarify the physics of these assemblies and their use in the development of high-performance magnetic materials. This book reviews prominent research studies on the static and dynamic magnetic properties of nanoparticle assemblies, gathering together experimental and computational techniques in an effort to reveal their optimized magnetic properties for biomedical use and as ultra-high magnetic recording media.

  13. Integrated magnetic transformer assembly

    DEFF Research Database (Denmark)

    2014-01-01

    The present invention relates to an integrated magnetics transformer assembly comprising a first magnetically permeable core forming a first substantially closed magnetic flux path and a second magnetically permeable core forming a second substantially closed magnetic flux path. A first input...... inductor winding is wound around a first predetermined segment of the first magnetically permeable core and a second input inductor winding is wound around a first predetermined segment of the second magnetically permeable core. The integrated magnetics transformer assembly further comprises a first output......-winding of the first output inductor winding and the first half-winding of the second output inductor winding are configured to produce aligned, i.e. in the same direction, magnetic fluxes through the first substantially closed magnetic flux path. The integrated magnetics transformer assembly is well- suited for use...

  14. Reactor fuel assembly

    International Nuclear Information System (INIS)

    Anthony, A.J.; Groves, M.D.

    1980-01-01

    A nuclear reactor fuel assembly having a lower end fitting and actuating means interacting therewith for holding the assembly down on the core support stand against the upward flow of coolant. Locking means for interacting with projections on the support stand are carried by the lower end fitting and are actuated by the movement of an actuating rod operated from above the top of the assembly. In one embodiment of the invention the downward movement of the actuating rod forces a latched spring to move outward into locking engagement with a shoulder on the support stand projections. In another embodiment, the actuating rod is rotated to effect the locking between the end fitting and the projection. (author)

  15. Power module assembly

    Science.gov (United States)

    Campbell, Jeremy B [Torrance, CA; Newson, Steve [Redondo Beach, CA

    2011-11-15

    A power module assembly of the type suitable for deployment in a vehicular power inverter, wherein the power inverter has a grounded chassis, is provided. The power module assembly comprises a conductive base layer electrically coupled to the chassis, an insulating layer disposed on the conductive base layer, a first conductive node disposed on the insulating layer, a second conductive node disposed on the insulating layer, wherein the first and second conductive nodes are electrically isolated from each other. The power module assembly also comprises a first capacitor having a first electrode electrically connected to the conductive base layer, and a second electrode electrically connected to the first conductive node, and further comprises a second capacitor having a first electrode electrically connected to the conductive base layer, and a second electrode electrically connected to the second conductive node.

  16. Liaison based assembly design

    Energy Technology Data Exchange (ETDEWEB)

    Ames, A.; Kholwadwala, D.; Wilson, R.H.

    1996-12-01

    Liaison Based Assembly Design extends the current information infrastructure to support design in terms of kinematic relationships between parts, or liaisons. These liaisons capture information regarding contact, degrees-of-freedom constraints and containment relationships between parts in an assembly. The project involved defining a useful collection of liaison representations, investigating their properties, and providing for maximum use of the data in downstream applications. We tested our ideas by implementing a prototype system involving extensions to Pro/Engineer and the Archimedes assembly planner. With an expanded product model, the design system is more able to capture design intent. When a product update is attempted, increased knowledge availability improves our ability to understand the effect of design changes. Manufacturing and analysis disciplines benefit from having liaison information available, so less time is wasted arguing over incomplete design specifications and our enterprise can be more completely integrated.

  17. Transfer of fuel assemblies

    International Nuclear Information System (INIS)

    Vuckovich, M.; Burkett, J. P.; Sallustio, J.

    1984-01-01

    Fuel assemblies of a nuclear reactor are transferred during fueling or refueling or the like by a crane. The work-engaging fixture of the crane picks up an assembly, removes it from this slot, transfers it to the deposit site and deposits it in its slot at the deposit site. The control for the crane includes a strain gauge connected to the crane line which raises and lowers the load. The strain gauge senses the load on the crane. The signal from the strain gauge is compared with setpoints; a high-level setpoint, a low-level setpoint and a slack-line setpoint. If the strain gauge signal exceeds the high-level setpoint, the line drive is disabled. This event may occur during raising of a fuel assembly which encounters resistance. The high-level setpoint may be overridden under proper precautions. The line drive is also disabled if the strain gauge signal is less than the low-level setpoint. This event occurs when a fuel assembly being deposited contacts the bottom of its slot or an obstruction in, or at the entry to the slot. To preclude lateral movement and possible damage to a fuel assembly suspended from the crane line, the traverse drive of the crane is disabled once the strain-gauge exceets the lov-level setpoint. The traverse drive can only be enabled after the strain-gauge signal is less than the slack-line set-point. This occurs when the lines has been set in slack-line setting. When the line is tensioned after slack-li ne setting, the traverse drive remains enabled only if the line has been disconnected from the fuel assembly

  18. Hand Controller Assembly

    Science.gov (United States)

    Bandera, Pablo (Inventor); Buchele, Paul (Inventor)

    2015-01-01

    A user input device for a vehicular electrical system is provided. The user input device includes a handle sized and shaped to be gripped by a human hand and a gimbal assembly within the handle. The gimbal assembly includes a first gimbal component, a second gimbal component coupled to the first gimbal component such that the second gimbal component is rotatable relative to the first gimbal component about a first axis, and a third gimbal component coupled to the second gimbal component such that the third gimbal component is rotatable relative to the second gimbal component about a second axis.

  19. Assembling an aesthetic.

    Science.gov (United States)

    Candela, Emily

    2012-12-01

    Recent research informing and related to the study of three-dimensional scientific models is assembled here in a way that explores an aesthetic, specifically, of touch. I concentrate on the materiality of models, drawing on insights from the history and philosophy of science, design and metaphysics. This article chronicles the ways in which touch, or material interactions, operate in the world of 3D models, and its role in what models mean and do. I end with a call for greater attention to scientific process, described as assembly of and within science, which is revealed by this focus on touch. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Nuclear fuel assembly

    International Nuclear Information System (INIS)

    1975-01-01

    The nuclear fuel assembly described includes a cluster of fuel elements supported at a distance from each other so that their axes are parallel in order to establish secondary channels between them reserved for the coolant. Several ducts for an auxiliary cooling fluid are arranged in the cluster. The wall of each duct is pierced with coolant ejection holes which are placed circumferentially to a pre-determined pattern established according to the position of the duct in the cluster and by the axial distance of the ejection hole along the duct. This assembly is intended for reactors cooled by light or heavy water [fr

  1. Assembling RNA Nanoparticles.

    Science.gov (United States)

    Xiao, Shou-Jun

    2017-01-01

    RNA nanoparticles are designed and self-assembled according to noncanonical interactions of naturally conserved RNA motifs and/or canonical Watson-Crick base-pairing interactions, which have potential applications in gene therapy and nanomedicine. These artificially engineered nanoparticles are mainly synthesized from in vitro transcribed RNAs, purified by denaturing and native polyacrylamide gel electrophoresis (PAGE), and characterized with native PAGE, AFM, and TEM technologies. The protocols of in vitro transcription, denaturing and native PAGE, and RNA nanoparticle self-assembly are described in detail.

  2. Structure of a two-domain N-terminal fragment of ribosomal protein L10 from Methanococcus jannaschii reveals a specific piece of the archaeal ribosomal stalk.

    Science.gov (United States)

    Kravchenko, Olesya; Mitroshin, Ivan; Nikonov, Stanislav; Piendl, Wolfgang; Garber, Maria

    2010-06-04

    Ribosomal stalk is involved in the formation of the so-called "GTPase-associated site" and plays a key role in the interaction of ribosome with translation factors and in the control of translation accuracy. The stalk is formed by two or three copies of the L7/L12 dimer bound to the C-terminal tail of protein L10. The N-terminal domain of L10 binds to a segment of domain II of 23S rRNA near the binding site for ribosomal protein L11. The structure of bacterial L10 in complex with three L7/L12 N-terminal dimers has been determined in the isolated state, and the structure of the first third of archaeal L10 bound to domain II of 23S rRNA has been solved within the Haloarcula marismortui 50S ribosomal subunit. A close structural similarity between the RNA-binding domain of archaeal L10 and the RNA-binding domain of bacterial L10 has been demonstrated. In this work, a long RNA-binding N-terminal fragment of L10 from Methanococcus jannaschii has been isolated and crystallized. The crystal structure of this fragment (which encompasses two-thirds of the protein) has been solved at 1.6 A resolution. The model presented shows the structure of the RNA-binding domain and the structure of the adjacent domain that exist in archaeal L10 and eukaryotic P0 proteins only. Furthermore, our model incorporated into the structure of the H. marismortui 50S ribosomal subunit allows clarification of the structure of the archaeal ribosomal stalk base. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  3. Dynamic enzyme docking to the ribosome coordinates N-terminal processing with polypeptide folding.

    Science.gov (United States)

    Sandikci, Arzu; Gloge, Felix; Martinez, Michael; Mayer, Matthias P; Wade, Rebecca; Bukau, Bernd; Kramer, Günter

    2013-07-01

    Newly synthesized polypeptides undergo various cotranslational maturation steps, including N-terminal enzymatic processing, chaperone-assisted folding and membrane targeting, but the spatial and temporal coordination of these steps is unclear. We show that Escherichia coli methionine aminopeptidase (MAP) associates with ribosomes through a charged loop that is crucial for nascent-chain processing and cell viability. MAP competes with peptide deformylase (PDF), the first enzyme to act on nascent chains, for binding sites at the ribosomal tunnel exit. PDF has extremely fast association and dissociation kinetics, which allows it to frequently sample ribosomes and ensure the processing of nascent chains after their emergence. Premature recruitment of the chaperone trigger factor, or polypeptide folding, negatively affect processing efficiency. Thus, the fast ribosome association kinetics of PDF and MAP are crucial for the temporal separation of nascent-chain processing from later maturation events, including chaperone recruitment and folding.

  4. 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

    -His is among the fusion proteins used in our previous study for ribosomal coupling of C-terminally His-tagged green fluorescent protein. To assess the efficiency of separation of target protein from ribosomes, by site-specific proteolysis, we required monoclonal antibodies directed against rpL23 and GFP. We......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......) followed by expression in an rpL23 deficient strain of E. coli. This allowed for the isolation of ribsomes with covalently coupled target proteins which could be efficiently purified by centrifugation after in vitro proteolysis at a specific site incorporated between rpL23 and the target protein. rpL23-GFP...

  5. Ribosomal RNA analysis in the diagnosis of Diamond-Blackfan Anaemia.

    Science.gov (United States)

    Quarello, Paola; Garelli, Emanuela; Carando, Adriana; Mancini, Cecilia; Foglia, Luiselda; Botto, Carlotta; Farruggia, Piero; De Keersmaecker, Kim; Aspesi, Anna; Ellis, Steve R; Dianzani, Irma; Ramenghi, Ugo

    2016-03-01

    Diamond-Blackfan anaemia (DBA) is an inherited disease characterized by pure erythroid aplasia that has been tagged as a 'ribosomopathy'. We report a multi-centre study focused on the analysis of rRNA processing of 53 Italian DBA patients using capillary electrophoresis analysis of rRNA maturation of the 40S and 60S ribosomal subunits. The ratio of 28S/18S rRNA was higher in patients with mutated ribosomal proteins (RPs) of the small ribosomal subunit. In contrast, patients with mutated RPs of the large ribosomal subunit (RPLs) had a lower 28S/18S ratio. The assay reported here would be amenable for development as a diagnostic tool. © 2016 John Wiley & Sons Ltd.

  6. Structure of the quaternary complex between SRP, SR, and translocon bound to the translating ribosome.

    Science.gov (United States)

    Jomaa, Ahmad; Fu, Yu-Hsien Hwang; Boehringer, Daniel; Leibundgut, Marc; Shan, Shu-Ou; Ban, Nenad

    2017-05-19

    During co-translational protein targeting, the signal recognition particle (SRP) binds to the translating ribosome displaying the signal sequence to deliver it to the SRP receptor (SR) on the membrane, where the signal peptide is transferred to the translocon. Using electron cryo-microscopy, we have determined the structure of a quaternary complex of the translating Escherichia coli ribosome, the SRP-SR in the 'activated' state and the translocon. Our structure, supported by biochemical experiments, reveals that the SRP RNA adopts a kinked and untwisted conformation to allow repositioning of the 'activated' SRP-SR complex on the ribosome. In addition, we observe the translocon positioned through interactions with the SR in the vicinity of the ribosome exit tunnel where the signal sequence is extending beyond its hydrophobic binding groove of the SRP M domain towards the translocon. Our study provides new insights into the mechanism of signal sequence transfer from the SRP to the translocon.

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

    Directory of Open Access Journals (Sweden)

    Stanisław Weidner

    2014-01-01

    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.

  8. Tsukamurella tyrosinosolvens intravascular catheter infection identified using 16S ribosomal DNA sequencing.

    Science.gov (United States)

    Sheridan, Elizabeth A S; Warwick, Simon; Chan, Anthony; Dall'Antonia, Martino; Koliou, Maria; Sefton, Armine

    2003-03-01

    Cultures of blood from a hemodialysis line repeatedly yielded a gram-positive rod. The organism was identified as Tsukamurella tyrosinosolvens by 16S ribosomal DNA sequencing, and the patient was treated successfully by removal of the line.

  9. Maturation of the 90S pre-ribosome requires Mrd1 dependent U3 snoRNA and 35S pre-rRNA structural rearrangements.

    Science.gov (United States)

    Lackmann, Fredrik; Belikov, Sergey; Burlacu, Elena; Granneman, Sander; Wieslander, Lars

    2018-01-24

    In eukaryotes, ribosome biogenesis requires folding and assembly of the precursor rRNA (pre-rRNA) with a large number of proteins and snoRNPs into huge RNA-protein complexes. In spite of intense genetic, biochemical and high-resolution cryo-EM studies in Saccharomyces cerevisiae, information about the structure of the 35S pre-rRNA is limited. To overcome this, we performed high-throughput SHAPE chemical probing on the 35S pre-rRNA within 90S pre-ribosomes. We focused our analyses on external (5'ETS) and internal (ITS1) transcribed spacers as well as the 18S rRNA region. We show that in the 35S pre-rRNA, the central pseudoknot is not formed and the central core of the 18S rRNA is in an open configuration but becomes more constrained in 20S pre-rRNA. The essential ribosome biogenesis protein Mrd1 influences the structure of the 18S rRNA region locally and is involved in organizing the central pseudoknot and surrounding structures. We demonstrate that U3 snoRNA dynamically interacts with the 35S pre-rRNA and that Mrd1 is required for disrupting U3 snoRNA base pairing interactions in the 5'ETS. We propose that the dynamic U3 snoRNA interactions and Mrd1 are essential for establishing the structure of the central core of 18S rRNA that is required for processing and 40S subunit function. © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Ribosomal DNA Nanoprobes studied by Fourier Transform Infrared spectroscopy

    Science.gov (United States)

    Fagundes, Jaciara; Castilho, Maiara L.; Téllez Soto, Claudio A.; Vieira, Laís de Souza; Canevari, Renata A.; Fávero, Priscila P.; Martin, Airton A.; Raniero, Leandro

    2014-01-01

    Paracoccidioides brasiliensis (P. brasiliensis) is a thermo-dimorphic fungus that causes paracoccidioidomycosis. Brazil epidemiological data shows that endemic areas are the subtropical regions, especially where agricultural activities predominate such as the Southeast, South, and Midwest. There are several tests to diagnose paracoccidioidomycosis, but they have many limitations such as low sensitivity, high cost, and a cross-reacting problem. In this work, gold nanoprobes were used to identify P. brasiliensis as an alternative diagnostic technique, which is easier to apply, costs less, and has great potential for application. The specific Ribosomal sequence of P. brasiliensis DNA was amplified and used to design the nanoprobes using a thiol-modified oligonucleotide. The results of positive and negative tests were done by UV-visible and Fourier Transform Infrared (FT-IR) measurements. The deconvolution of FT-IR sample spectra showed differences in the vibrational modes from the hydrogen bridge NHN and NHO bands that form the double helix DNA for samples matching the DNA sequence of nanoprobes that could be used to classify the samples.

  11. Divergent paralogues of ribosomal DNA in eucalypts (Myrtaceae).

    Science.gov (United States)

    Bayly, Michael J; Ladiges, Pauline Y

    2007-07-01

    The presence of divergent paralogues of nuclear ribosomal DNA, from the 18S-5.8S-26S cistron, is reported in members of Eucalyptus subg. Eucalyptus. These paralogues, which include non-functional pseudogenes, probably diverged prior to the differentiation of species groups in subg. Eucalyptus. When compared with presumably functional sequences, the pseudogenes show greater sequence variation between species, particularly in the 5.8S gene. They are also characterised by reduced GC content, associated with a reduced number of CpG and CpNpG methylation sites, and an increase in the inferred number of methylation-induced substitutions. Some pseudogenes also lack motifs that are usually conserved in plants, both in ITS1 and the 5.8S gene. Two main lineages of pseudogenes are identified, one isolated from a group of western Australian species, one from a group of eastern Australian species. It is not clear whether these two lineages of pseudogenes are orthologous, or represent independent divergences from functional sequence types. The presence of divergent rDNA paralogues highlights the need for caution when interpreting eucalypt phylogenies based on ITS sequences.

  12. Universal PCR primers for ribosomal protein gene introns of fish

    Directory of Open Access Journals (Sweden)

    Seinen Chow

    2016-01-01

    Full Text Available Abstract Human ribosomal protein (RP gene sequences with respect to intron/exon structures and corresponding cDNA or genomic data of fish species were obtained from the GenBank database. Based on conserved exon sequences, 128 primer pairs for 41 genes were designed for exon-primed intron-crossing (EPIC polymerase chain reaction (PCR. In reference to the draft genome sequences of the Pacific bluefin tuna (Thunnus orientalis, 12 primer pairs expected to amplify introns of the bluefin tuna with lengths of 500–1000 bp were selected and applied to six distantly related fish species belonging to the Orders Clupeiformes, Tetraodontiformes, Pleuronectiformes, Perciformes, Scorpaeniformes, and Anguilliformes. PCR amplification was observed for at least four species in each primer pair, and all fragments were larger than those expected for intronless amplification. Single fragment amplification was observed for at least seven primer pairs per species. Fragment sizes of the bluefin tuna for nine primer pairs corresponded to those expected from the genomic data. Thus, our primer pairs are potentially applicable to a wide variety of fish species and serve as an initial step for isolating single-copy nuclear DNA sequences.

  13. Inferring gene expression from ribosomal promoter sequences, a crowdsourcing approach.

    Science.gov (United States)

    Meyer, Pablo; Siwo, Geoffrey; Zeevi, Danny; Sharon, Eilon; Norel, Raquel; Segal, Eran; Stolovitzky, Gustavo

    2013-11-01

    The Gene Promoter Expression Prediction challenge consisted of predicting gene expression from promoter sequences in a previously unknown experimentally generated data set. The challenge was presented to the community in the framework of the sixth Dialogue for Reverse Engineering Assessments and Methods (DREAM6), a community effort to evaluate the status of systems biology modeling methodologies. Nucleotide-specific promoter activity was obtained by measuring fluorescence from promoter sequences fused upstream of a gene for yellow fluorescence protein and inserted in the same genomic site of yeast Saccharomyces cerevisiae. Twenty-one teams submitted results predicting the expression levels of 53 different promoters from yeast ribosomal protein genes. Analysis of participant predictions shows that accurate values for low-expressed and mutated promoters were difficult to obtain, although in the latter case, only when the mutation induced a large change in promoter activity compared to the wild-type sequence. As in previous DREAM challenges, we found that aggregation of participant predictions provided robust results, but did not fare better than the three best algorithms. Finally, this study not only provides a benchmark for the assessment of methods predicting activity of a specific set of promoters from their sequence, but it also shows that the top performing algorithm, which used machine-learning approaches, can be improved by the addition of biological features such as transcription factor binding sites.

  14. Recombinant tagging system using ribosomal frameshifting to monitor protein expression.

    Science.gov (United States)

    Han, Se Jong; Cho, Sayeon; Lowehhaupt, Ky; Park, So-Young; Sim, Sang Jun; Kim, Yang-Gyun

    2013-03-01

    For rapid and accurate quantitation of recombinant proteins during expression and after purification, we introduce a new tagging strategy that expresses both target proteins and limitedly tagged target proteins together in a single cell at a constant ratio by utilizing cis-elements of programmed -1 ribosomal frameshifting (-1RFS) as an embedded device. -1RFS is an alternative reading mechanism that effectively controls protein expression by many viruses. When a target gene is fused to the enhanced green fluorescent protein (EGFP) gene with a -1RFS element implanted between them, the unfused target and the target-GFP fusion proteins are expressed at a fixed ratio. The expression ratio between these two protein products is adjustable simply by changing -1RFS signals. This limited-tagging system would be valuable for the real-time monitoring of protein expression when optimizing expression condition for a new protein, and in monitoring large-scale bioprocesses without a large metabolic burden on host cells. Furthermore, this strategy allows for the direct measurement of the quantity of a protein on a chip surface and easy application to proteomewide study of gene products. Copyright © 2012 Wiley Periodicals, Inc.

  15. 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

    2016-04-16

    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.

  16. Visualizing the 16-Membered Ring Macrolides Tildipirosin and Tilmicosin Bound to Their Ribosomal Site

    DEFF Research Database (Denmark)

    Poehlsgaard, Jacob; Møller Andersen, Niels; Warrass, Ralf

    2012-01-01

    The veterinary antibiotic tildipirosin (20,23-dipiperidinyl-mycaminosyl-tylonolide, Zuprevo) was developed recently to treat bovine and swine respiratory tract infections caused by bacterial pathogens such as Pasteurella multocida. Tildipirosin is a derivative of the naturally occurring compound...... of P. multocida and Escherichia coli ribosomes. The drugs nevertheless differ in how they occupy this site. Interactions of the two piperidine components, which are unique to tildipirosin, distinguish this drug from tylosin and tilmicosin. The 23-piperidine of tildipirosin contacts ribosomal residues...

  17. FSscan: a mechanism-based program to identify +1 ribosomal frameshift hotspots

    OpenAIRE

    Liao, Pei-Yu; Choi, Yong Seok; Lee, Kelvin H.

    2009-01-01

    In +1 programmed ribosomal frameshifting (PRF), ribosomes skip one nucleotide toward the 3?-end during translation. Most of the genes known to demonstrate +1 PRF have been discovered by chance or by searching homologous genes. Here, a bioinformatic framework called FSscan is developed to perform a systematic search for potential +1 frameshift sites in the Escherichia coli genome. Based on a current state of the art understanding of the mechanism of +1 PRF, FSscan calculates scores for a 16-nt...

  18. Production of RNA-protein cross links in γ irradiated E. Coli ribosomes

    International Nuclear Information System (INIS)

    Ekert, Bernard; Giocanti, Nicole

    1976-01-01

    γ irradiation in de-aerated conditions of E. coli MRE 600 ribosomes, labelled with 14 C uracil, leads to a decrease of extractibility of 14 C RNA by lithium chloride 4 M-urea 8 M. On the other hand, the radioactivity of the protein fraction increases with irradiation. These results strongly suggest that RNA-protein cross links are formed in irradiated ribosomes [fr

  19. Structure of the E. coli ribosome-EF-Tu complex at cryo-EM.

    Science.gov (United States)

    Fischer, Niels; Neumann, Piotr; Konevega, Andrey L; Bock, Lars V; Ficner, Ralf; Rodnina, Marina V; Stark, Holger

    2015-04-23

    Single particle electron cryomicroscopy (cryo-EM) has recently made significant progress in high-resolution structure determination of macromolecular complexes due to improvements in electron microscopic instrumentation and computational image analysis. However, cryo-EM structures can be highly non-uniform in local resolution and all structures available to date have been limited to resolutions above 3 Å. Here we present the cryo-EM structure of the 70S ribosome from Escherichia coli in complex with elongation factor Tu, aminoacyl-tRNA and the antibiotic kirromycin at 2.65-2.9 Å resolution using spherical aberration (Cs)-corrected cryo-EM. Overall, the cryo-EM reconstruction at 2.9 Å resolution is comparable to the best-resolved X-ray structure of the E. coli 70S ribosome (2.8 Å), but provides more detailed information (2.65 Å) at the functionally important ribosomal core. The cryo-EM map elucidates for the first time the structure of all 35 rRNA modifications in the bacterial ribosome, explaining their roles in fine-tuning ribosome structure and function and modulating the action of antibiotics. We also obtained atomic models for flexible parts of the ribosome such as ribosomal proteins L9 and L31. The refined cryo-EM-based model presents the currently most complete high-resolution structure of the E. coli ribosome, which demonstrates the power of cryo-EM in structure determination of large and dynamic macromolecular complexes.

  20. A conserved chloramphenicol binding site at the entrance to the ribosomal peptide exit tunnel

    DEFF Research Database (Denmark)

    Long, Katherine S; Porse, Bo T

    2003-01-01

    , of E.coli 23S rRNA and G2084 (2058 in E.coli numbering) in domain V of H.halobium 23S rRNA. The modification sites overlap with a portion of the macrolide binding site and cluster at the entrance to the peptide exit tunnel. The data correlate with the recently reported chloramphenicol binding site...... on an archaeal ribosome and suggest that a similar binding site is present on the E.coli ribosome....

  1. Choking Prevention

    Science.gov (United States)

    ... Healthy Living Healthy Living Healthy Living Nutrition Fitness Sports Oral Health Emotional Wellness Growing Healthy Sleep Safety & Prevention Safety & Prevention Safety and Prevention Immunizations At Home ...

  2. Fire resistant PV shingle assembly

    Science.gov (United States)

    Lenox, Carl J.

    2012-10-02

    A fire resistant PV shingle assembly includes a PV assembly, including PV body, a fire shield and a connection member connecting the fire shield below the PV body, and a support and inter-engagement assembly. The support and inter-engagement assembly is mounted to the PV assembly and comprises a vertical support element, supporting the PV assembly above a support surface, an upper interlock element, positioned towards the upper PV edge, and a lower interlock element, positioned towards the lower PV edge. The upper interlock element of one PV shingle assembly is inter-engageable with the lower interlock element of an adjacent PV shingle assembly. In some embodiments the PV shingle assembly may comprise a ventilation path below the PV body. The PV body may be slidably mounted to the connection member to facilitate removal of the PV body.

  3. 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

    ) 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...

  4. 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.

  5. Mutational analysis of S12 protein and implications for the accuracy of decoding by the ribosome.

    Science.gov (United States)

    Sharma, Divya; Cukras, Anthony R; Rogers, Elizabeth J; Southworth, Daniel R; Green, Rachel

    2007-12-07

    The fidelity of aminoacyl-tRNA selection by the ribosome depends on a conformational switch in the decoding center of the small ribosomal subunit induced by cognate but not by near-cognate aminoacyl-tRNA. The aminoglycosides paromomycin and streptomycin bind to the decoding center and induce related structural rearrangements that explain their observed effects on miscoding. Structural and biochemical studies have identified ribosomal protein S12 (as well as specific nucleotides in 16S ribosomal RNA) as a critical molecular contributor in distinguishing between cognate and near-cognate tRNA species as well as in promoting more global rearrangements in the small subunit, referred to as "closure." Here we use a mutational approach to define contributions made by two highly conserved loops in S12 to the process of tRNA selection. Most S12 variant ribosomes tested display increased levels of fidelity (a "restrictive" phenotype). Interestingly, several variants, K42A and R53A, were substantially resistant to the miscoding effects of paromomycin. Further characterization of the compromised paromomycin response identified a probable second, fidelity-modulating binding site for paromomycin in the 16S ribosomal RNA that facilitates closure of the small subunit and compensates for defects associated with the S12 mutations.

  6. Ribosomal accretion, apriorism and the phylogenetic method: A response to Petrov and Williams

    Directory of Open Access Journals (Sweden)

    Derek eCaetano-Anollés

    2015-06-01

    Full Text Available Historical (ideographic and non-historical (nomothetic studies of ribosomal accretion appear to arrive at diametrically opposite conclusions. Phylogenetic analysis of thousands of RNA molecules and protein structures in hundreds of genomes supports the structural origin of the ribosome in RNA decoding and ribosomal mechanics. Predictions from extant features in a handful of rRNA structural models of the large ribosomal subunit support its origin in protein biosynthesis. In recent correspondence, one of us reported that correcting dismissals of conflicting data and avoiding unwarranted assumptions of the nomethetic method reconciled conclusions. In response, Petrov and Williams dismissed our arguments claiming we did not understand their ‘BS’ algorithmic model of ribosomal apical growth. Instead, they controverted the historical approach. Here we show that their objections to the phylogenetic method are unjustified, that the BS algorithm subjectively guarantees back-in-time molecular deconstructions towards the protein biosynthetic core, and that processes of ribosomal growth are much more complex. We prompt abandoning apriorism, decreasing ad hoc hypotheses and integrating historical and non-historical scientific methods.

  7. Real-time tRNA transit on single translating ribosomes at codon resolution

    Science.gov (United States)

    Uemura, Sotaro; Aitken, Colin Echeverría; Korlach, Jonas; Flusberg, Benjamin A.; Turner, Stephen W.; Puglisi, Joseph D.

    2015-01-01

    Translation by the ribosome occurs by a complex mechanism involving the coordinated interaction of multiple nucleic acid and protein ligands. Here we have used zero-mode waveguides (ZMWs) and sophisticated detection instrumentation to allow real-time observation of translation at physiologically-relevant (μM) ligand concentrations. Translation at each codon is monitored by stable binding of tRNAs – labeled with distinct fluorophores – to translating ribosomes, allowing direct detection of the identity of tRNA molecules bound to the ribosome, and therefore, the underlying mRNA sequence. We observe the transit of tRNAs on single translating ribosomes and have determined the number of tRNA molecules simultaneously bound to the ribosome, at each codon of an mRNA. Our results show that ribosomes are only briefly occupied by two tRNAs and that release of deacylated tRNA from the E site is uncoupled from binding of A-site tRNA and occurs rapidly after translocation. The methods outlined here have broad application to the study of mRNA sequences, and the mechanism and regulation of translation. PMID:20393556

  8. The complete structure of the large subunit of the mammalian mitochondrial ribosome.

    Science.gov (United States)

    Greber, Basil J; Boehringer, Daniel; Leibundgut, Marc; Bieri, Philipp; Leitner, Alexander; Schmitz, Nikolaus; Aebersold, Ruedi; Ban, Nenad

    2014-11-13

    Mitochondrial ribosomes (mitoribosomes) are extensively modified ribosomes of bacterial descent specialized for the synthesis and insertion of membrane proteins that are critical for energy conversion and ATP production inside mitochondria. Mammalian mitoribosomes, which comprise 39S and 28S subunits, have diverged markedly from the bacterial ribosomes from which they are derived, rendering them unique compared to bacterial, eukaryotic cytosolic and fungal mitochondrial ribosomes. We have previously determined at 4.9 Å resolution the architecture of the porcine (Sus scrofa) 39S subunit, which is highly homologous to the human mitoribosomal large subunit. Here we present the complete atomic structure of the porcine 39S large mitoribosomal subunit determined in the context of a stalled translating mitoribosome at 3.4 Å resolution by cryo-electron microscopy and chemical crosslinking/mass spectrometry. The structure reveals the locations and the detailed folds of 50 mitoribosomal proteins, shows the highly conserved mitoribosomal peptidyl transferase active site in complex with its substrate transfer RNAs, and defines the path of the nascent chain in mammalian mitoribosomes along their idiosyncratic exit tunnel. Furthermore, we present evidence that a mitochondrial tRNA has become an integral component of the central protuberance of the 39S subunit where it architecturally substitutes for the absence of the 5S ribosomal RNA, a ubiquitous component of all cytoplasmic ribosomes.

  9. Diverse Regulators of Human Ribosome Biogenesis Discovered by Changes in Nucleolar Number

    Directory of Open Access Journals (Sweden)

    Katherine I. Farley-Barnes

    2018-02-01

    Full Text Available Ribosome biogenesis is a highly regulated, essential cellular process. Although studies in yeast have established some of the biological principles of ribosome biogenesis, many of the intricacies of its regulation in higher eukaryotes remain unknown. To understand how ribosome biogenesis is globally integrated in human cells, we conducted a genome-wide siRNA screen for regulators of nucleolar number. We found 139 proteins whose depletion changed the number of nucleoli per nucleus from 2–3 to only 1 in human MCF10A cells. Follow-up analyses on 20 hits found many (90% to be essential for the nucleolar functions of rDNA transcription (7, pre-ribosomal RNA (pre-rRNA processing (16, and/or global protein synthesis (14. This genome-wide analysis exploits the relationship between nucleolar number and function to discover diverse cellular pathways that regulate the making of ribosomes and paves the way for further exploration of the links between ribosome biogenesis and human disease.

  10. Ribosome Collision Is Critical for Quality Control during No-Go Decay.

    Science.gov (United States)

    Simms, Carrie L; Yan, Liewei L; Zaher, Hani S

    2017-10-19

    No-go decay (NGD) is a eukaryotic quality control mechanism that evolved to cope with translational arrests. The process is characterized by an endonucleolytic cleavage near the stall sequence, but the mechanistic details are unclear. Our analysis of cleavage sites indicates that cleavage requires multiple ribosomes on the mRNA. We also show that reporters harboring stall sequences near the initiation codon, which cannot accommodate multiple ribosomes, are not subject to NGD. Consistent with our model, we uncover an inverse correlation between ribosome density per mRNA and cleavage efficiency. Furthermore, promoting global ribosome collision in vivo resulted in ubiquitination of ribosomal proteins, suggesting that collision is sensed by the cell to initiate downstream quality control processes. Collectively, our data suggest that NGD and subsequent quality control are triggered by ribosome collision. This model provides insight into the regulation of quality control processes and the manner by which they reduce off-target effects. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Ribosome excursions during mRNA translocation mediate broad branching of frameshift pathways.

    Science.gov (United States)

    Yan, Shannon; Wen, Jin-Der; Bustamante, Carlos; Tinoco, Ignacio

    2015-02-26

    Programmed ribosomal frameshifting produces alternative proteins from a single transcript. -1 frameshifting occurs on Escherichia coli's dnaX mRNA containing a slippery sequence AAAAAAG and peripheral mRNA structural barriers. Here, we reveal hidden aspects of the frameshifting process, including its exact location on the mRNA and its timing within the translation cycle. Mass spectrometry of translated products shows that ribosomes enter the -1 frame from not one specific codon but various codons along the slippery sequence and slip by not just -1 but also -4 or +2 nucleotides. Single-ribosome translation trajectories detect distinctive codon-scale fluctuations in ribosome-mRNA displacement across the slippery sequence, representing multiple ribosomal translocation attempts during frameshifting. Flanking mRNA structural barriers mechanically stimulate the ribosome to undergo back-and-forth translocation excursions, broadly exploring reading frames. Both experiments reveal aborted translation around mutant slippery sequences, indicating that subsequent fidelity checks on newly adopted codon position base pairings lead to either resumed translation or early termination. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Nanotechnology: A molecular assembler

    Science.gov (United States)

    Kelly, T. Ross; Snapper, Marc L.

    2017-09-01

    The idea of nanometre-scale machines that can assemble molecules has long been thought of as the stuff of science fiction. Such a machine has now been built -- and might herald a new model for organic synthesis. See Letter p.374

  13. Ordinary General Assembly

    CERN Multimedia

    Staff Association

    2011-01-01

    Tuesday 12 April at 14.00 Council Chamber, Bldg 503 In conformity with the Statutes of the Staff Association, an ordinary General Assembly is organized once a year (article IV.2.1). Agenda   Adoption of the Agenda Approval of the Draft Minutes of the Ordinary General Assembly of 20 April 2010 Presentation and approval of the Activity Report 2010 Presentation and approval of the Financial Report 2010 Presentation and approval of the Auditors Report 2010 Programme for 2011 Presentation et and approval of the draft budget and subscription rate 2012 Election of the Election Committee Election of the Board of Auditors Miscellaneous We remind members of article IV.3.4 in the Statutes of the Association which reads: “After having dealt with all the items on the agenda, the members may, with the consent of the Assembly, have other matters discussed, but decisions may be taken only on the items listed on the agenda. Nevertheless, the Assembly ma...

  14. Ordinary General Assembly

    CERN Multimedia

    Staff Association

    2011-01-01

    Tuesday 12 April at 14.00 Council Chamber, Bldg 503 In conformity with the Statutes of the Staff Association, an ordinary General Assembly is organized once a year (article IV.2.1). Agenda   Adoption of the Agenda Approval of the Draft Minutes of the Ordinary General Assembly of 20 April 2010 Presentation and approval of the Activity Report 2010 Presentation and approval of the Financial Report 2010 Presentation and approval of the Auditors Report 2010 Programme for 2011 Presentation and approval of the draft budget and subscription rate 2012 Election of the Election Committee Election of the Board of Auditors Miscellaneous We remind members of article IV.3.4 in the Statutes of the Association which reads: “After having dealt with all the items on the agenda, the members may, with the consent of the Assembly, have other matters discussed, but decisions may be taken only on the items listed on the agenda. Nevertheless, the Assembly may r...

  15. Ordinary General Assembly

    CERN Multimedia

    Staff Association

    2010-01-01

    Tuesday 20 April at 10.00 Council Chamber, Bldg 503 In conformity with the Statutes of the Staff Association, an ordinary General Assembly is organized once a year (article IV.2.1). Agenda   Adoption of the Agenda Approval of the Draft Minutes of the Ordinary General Assembly of 12 May 2009 Presentation and approval of the Activity Report 2009 Presentation and approval of the Financial Report 2009 Presentation and approval of the Auditors Report 2009 Programme for 2010 Presentation et and approval of the draft budget and subscription rate 2010 Election of the Election Committee Election of the Board of Auditors Miscellaneous We remind members of article IV.3.4 in the Statutes of the Association which reads: “After having dealt with all the items on the agenda, the members may, with the consent of the Assembly, have other matters discussed, but decisions may be taken only on the items listed on the agenda. Nevertheless, the Assembly may require t...

  16. America's Assembly Line

    DEFF Research Database (Denmark)

    Nye, David Edwin

    A social history of the assembly line, invented in 1913. Both praised as a boon to consumers and as a curse for workers, it has been satirized, imitated, and celebrated for 100 years. It has inspired fiction, comedy, cafeteria layouts, and suburban housing. It transformed industrial labor...

  17. Industrial Assembly Cases

    DEFF Research Database (Denmark)

    Ellekilde, Lars-Peter; Buch, Jacob Pørksen; Iversen, Thorbjørn Mosekjær

    This technical report presents 13 different industrial assembly tasks, which are composed of 70 different operations. The report is written to provide an overview and do as such not contain product specific information such as object weights, dimensions etc. The operations are classified into a set...

  18. Assembly of primary cilia

    DEFF Research Database (Denmark)

    Pedersen, Lotte B; Veland, Iben R; Schrøder, Jacob M

    2008-01-01

    knowledge about IFT is based on studies performed in Chlamydomonas and Caenorhabditis elegans. Therefore, our review of the IFT literature includes studies performed in these two model organisms. The role of several non-IFT proteins (e.g., centrosomal proteins) in the ciliary assembly process is also...... discussed. Developmental Dynamics, 2008. (c) 2008 Wiley-Liss, Inc....

  19. Spool assembly support analysis

    International Nuclear Information System (INIS)

    Norman, B.F.

    1994-01-01

    This document provides the wind/seismic analysis and evaluation for the pump pit spool assemblies. Hand calculations were used for the analysis. UBC, AISC, and load factors were used in this evaluation. The results show that the actual loads are under the allowable loads and all requirements are met

  20. Nanoparticle assemblies and superstructures

    National Research Council Canada - National Science Library

    Kotov, Nicholas A

    2006-01-01

    ... building blocks of larger and more complex systems. Therefore, the present challenge of nanoscale science is to shift from making certain building blocks to organizing them in one-, two-, and three-dimensional structures. Such assemblies and superstructures are the next logical step in the development of nanoscience and nanotechnology. In this re...

  1. Macroscopic magnetic Self assembly

    NARCIS (Netherlands)

    Löthman, Per Arvid

    2018-01-01

    Exploring the macroscopic scale's similarities to the microscale is part and parcel of this thesis as reflected in the research question: what can we learn about the microscopic scale by studying the macroscale? Investigations of the environment in which the self-assembly takes place, and the

  2. Assembling sustainable territories

    NARCIS (Netherlands)

    Vandergeest, Peter; Ponte, Stefano; Bush, Simon

    2015-01-01

    The authors show how certification assembles ‘sustainable’ territories through a complex layering of regulatory authority in which both government and nongovernment entities claim rule-making authority, sometimes working together, sometimes in parallel, sometimes competitively. It is argued that

  3. The putative E. histolytica orthologue of the human ribosomal RNA ...

    Indian Academy of Sciences (India)

    Initiation of rDNA transcription requires the assembly of a specific multi-protein complex at the rDNA promoter containing the RNA Pol I with auxiliary factors. One of these factors is known as Rrn3P in yeast and Transcription Initiation Factor IA (TIF-IA) in mammals. Rrn3p/TIF-IA serves as a bridge between RNA Pol I and the ...

  4. Defining the specificity of cotranslationally acting chaperones by systematic analysis of mRNAs associated with ribosome-nascent chain complexes.

    Directory of Open Access Journals (Sweden)

    Marta del Alamo

    2011-07-01

    Full Text Available Polypeptides exiting the ribosome must fold and assemble in the crowded environment of the cell. Chaperones and other protein homeostasis factors interact with newly translated polypeptides to facilitate their folding and correct localization. Despite the extensive efforts, little is known about the specificity of the chaperones and other factors that bind nascent polypeptides. To address this question we present an approach that systematically identifies cotranslational chaperone substrates through the mRNAs associated with ribosome-nascent chain-chaperone complexes. We here focused on two Saccharomyces cerevisiae chaperones: the Signal Recognition Particle (SRP, which acts cotranslationally to target proteins to the ER, and the Nascent chain Associated Complex (NAC, whose function has been elusive. Our results provide new insights into SRP selectivity and reveal that NAC is a general cotranslational chaperone. We found surprising differential substrate specificity for the three subunits of NAC, which appear to recognize distinct features within nascent chains. Our results also revealed a partial overlap between the sets of nascent polypeptides that interact with NAC and SRP, respectively, and showed that NAC modulates SRP specificity and fidelity in vivo. These findings give us new insight into the dynamic interplay of chaperones acting on nascent chains. The strategy we used should be generally applicable to mapping the specificity, interplay, and dynamics of the cotranslational protein homeostasis network.

  5. RNA polymerase II components and Rrn7 form a preinitiation complex on the HomolD box to promote ribosomal protein gene expression in Schizosaccharomyces pombe.

    Science.gov (United States)

    Montes, Matías; Moreira-Ramos, Sandra; Rojas, Diego A; Urbina, Fabiola; Käufer, Norbert F; Maldonado, Edio

    2017-02-01

    In Schizosaccharomyces pombe, ribosomal protein gene (RPG) promoters contain a TATA box analog, the HomolD box, which is bound by the Rrn7 protein. Despite the importance of ribosome biogenesis for cell survival, the mechanisms underlying RPG transcription remain unknown. In this study, we found that components of the RNA polymerase II (RNAPII) system, consisting of the initiation or general transcription factors (GTFs) TFIIA, IIB, IIE, TATA-binding protein (TBP) and the RNAPII holoenzyme, interacted directly with Rrn7 in vitro, and were able to form a preinitiation complex (PIC) on the HomolD box. PIC complex formation follows an ordered pathway on these promoters. The GTFs and RNAPII can also be cross-linked to HomolD-containing promoters in vivo. In an in vitro reconstituted transcription system, RNAPII components and Rrn7 were necessary for HomolD-directed transcription. The Mediator complex was required for basal transcription from those promoters in whole cell extract (WCE). The Med17 subunit of Mediator also can be cross-linked to the promoter region of HomolD-containing promoters in vivo, suggesting the presence of the Mediator complex on HomolD box-containing promoters. Together, these data show that components of the RNAPII machinery and Rrn7 participate in the PIC assembly on the HomolD box, thereby directing RPG transcription. © 2017 Federation of European Biochemical Societies.

  6. Dual binding mode of the nascent polypeptide-associated complex reveals a novel universal adapter site on the ribosome.

    Science.gov (United States)

    Pech, Markus; Spreter, Thomas; Beckmann, Roland; Beatrix, Birgitta

    2010-06-18

    Nascent polypeptide-associated complex (NAC) was identified in eukaryotes as the first cytosolic factor that contacts the nascent polypeptide chain emerging from the ribosome. NAC is present as a homodimer in archaea and as a highly conserved heterodimer in eukaryotes. Mutations in NAC cause severe embryonically lethal phenotypes in mice, Drosophila melanogaster, and Caenorhabditis elegans. In the yeast Saccharomyces cerevisiae NAC is quantitatively associated with ribosomes. Here we show that NAC contacts several ribosomal proteins. The N terminus of betaNAC, however, specifically contacts near the tunnel exit ribosomal protein Rpl31, which is unique to eukaryotes and archaea. Moreover, the first 23 amino acids of betaNAC are sufficient to direct an otherwise non-associated protein to the ribosome. In contrast, alphaNAC (Egd2p) contacts Rpl17, the direct neighbor of Rpl31 at the ribosomal tunnel exit site. Rpl31 was also recently identified as a contact site for the SRP receptor and the ribosome-associated complex. Furthermore, in Escherichia coli peptide deformylase (PDF) interacts with the corresponding surface area on the eubacterial ribosome. In addition to the previously identified universal adapter site represented by Rpl25/Rpl35, we therefore refer to Rpl31/Rpl17 as a novel universal docking site for ribosome-associated factors on the eukaryotic ribosome.

  7. Characterization of assembled MEMS

    Science.gov (United States)

    Jandric, Zoran; Randall, John N.; Saini, Rahul; Nolan, Michael; Skidmore, George

    2005-01-01

    Zyvex is developing a low-cost high-precision method for manufacturing MEMS-based three-dimensional structures/assemblies. The assembly process relies on compliant properties of the interconnecting components. The sockets and connectors are designed to benefit from their compliant nature by allowing the mechanical component to self-align, i.e. reposition themselves to their designed, stable position, independent of the initial placement of the part by the external robot. Thus, the self-aligning property guarantees the precision of the assembled structure to be very close to, or the same, as the precision of the lithography process itself. A three-dimensional (3D) structure is achieved by inserting the connectors into the sockets through the use of a passive end-effector. We have developed the automated, high-yield, assembly procedure which permits connectors to be picked up from any location within the same die, or a separate die. This general procedure allows for the possibility to assemble parts of dissimilar materials. We have built many 3D MEMS structures, including several 3D MEMS devices such as a scanning electron microscope (SEM) micro column, mass-spectrometer column, variable optical attenuator. For these 3D MEMS structures we characterize their mechanical strength through finite element simulation, dynamic properties by finite-element analysis and experimentally with UMECH"s MEMS motion analyzer (MMA), alignment accuracy by using an in-house developed dihedral angle measurement laser autocollimator, and impact properties by performing drop tests. The details of the experimental set-ups, the measurement procedures, and the experimental data are presented in this paper.

  8. 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

    RNA, and to investigate how this interaction is influenced by other ribosomal components. Complexes were characterized in both naked 23 S rRNA and ribosomes from an E. coli L11-minus strain, before and after reconstitution with L11. The protein protects 17 riboses between positions 1058 and 1085 in the naked 23 S r......The Escherichia coli ribosomal protein (r-protein) L11 and its binding site on 23 S ribosomal RNA (rRNA) are associated with ribosomal hydrolysis of guanosine 5'-triphosphate (GTP). We have used hydroxyl radical footprinting to map the contacts between L11 and the backbone riboses in 23 S r......)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....

  9. Ribosomes in the sea: a window on taxon-specific lysis

    Science.gov (United States)

    Suttle, C.; Zhong, X.; Wirth, J.

    2016-02-01

    Microbes are estimated to comprise more than 90% of the biomass in the world's oceans, are major drivers of biogeochemical cycles, and have turnover rates ranging from hours to days. Despite the central role that microbes play in marine ecosystems, there is no robust method to evaluate taxon-specific mortality rates. Here, we report a method that employs extracellular free-ribosomes as a proxy to evaluate taxon-specific microbial lysis. The method was validated with laboratory cultures of the marine heterotrophic bacterium Vibrio natriegens strain PWH3a and the photoautotroph Synechococcus strain DC2, with and without grazers or viruses, to identify the origin and fate of the extracellular free-ribosomes. Our results showed both viral lysis and programmed-cell-death (PCD) contribute to free-ribosome production. Ribosomes were not released when cells were grazed, but grazers could consume free-ribosomes. We show that extracellular free-ribosomes can be used to evaluate microbial mortality caused by viral lysis and PCD. This approach was applied to environmental samples by examining the taxonomic composition and relative abundance of free 16S-ribosomes in seawater samples collected from the Strait of Georgia and Saanich Inlet, British Columbia, Canada. Based on the presence of free ribosomes, lysis was detected in 2198 out of 4013 prokaryotic taxa, representing 22 bacterial and three archaeal phyla. Of these, lysis of 140 taxa could be detected in all nine samples. Based on the ratio of free ribosomes to cellular ribosomes, some taxa associated with specific ecological niches appeared to be subject to high rates of lysis, including the genera Achromobacter, Chryseobacterium, Clostridium, Delftia, Ferruginibacter, Lactobacillus, Marinomonas, Massilia, Microbacterium, Ochrobactrum, Paenibacillus, Phyllobacterium, Pseudomonas, Rhodobacter, and Stenotrophomonas. Our results showed high-lysis coupled with low-abundance, suggesting that taxa in lower abundance are subject

  10. Mapping contacts of the S12-S7 intercistronic region of str operon mRNA with ribosomal protein S7 of E. coli.

    Science.gov (United States)

    Golovin, Andrey; Spiridonova, Vera; Kopylov, Alexei

    2006-10-30

    In E. coli, S7 initiates 30S ribosome assembly by binding to 16S rRNA. It also regulates translation of the S12 and S7 cistrons of the 'streptomycin' operon transcript by binding to the S12-S7 intercistronic region. Here, we describe the contacts of N-terminally His(6)-tagged S7 with this region as mapped by UV-induced cross-linking. The cross-links are located at U(-34), U(-35), quite distant from the start codons of the two cistrons. In order to explain the mechanism of translational repression of S12-S7, we consider a possible conformational rearrangement of the intercistronic RNA structure induced by S7 binding.

  11. Patterns of major divergence between the internal transcribed spacers of ribosomal DNA in Xenopus borealis and Xenopus laevis, and of minimal divergence within ribosomal coding regions.

    Science.gov (United States)

    Furlong, J C; Maden, B E

    1983-01-01

    We have determined the nucleotide sequences of the two internal transcribed spacers, the adjacent ribosomal coding sequences and the boundary between the external transcribed spacer and the 18S coding sequence in a cloned ribosomal transcription unit from Xenopus borealis. The transcribed spacers differ very extensively from those of X. laevis. Nevertheless, embedded in the internal transcribed spacers are several short sequence elements which are identical between the two species. These conserved elements are laterally displaced by substantial distances in the X. borealis sequence with respect to that of X. laevis. These relative displacements imply that insertions and deletions have played a major role in transcribed spacer divergence in Xenopus. This in turn implies that large regions of the transcribed spacers do not play a sequence-specific role in ribosome maturation. In contrast, the sequenced parts of the ribosomal coding regions, which encompass 670 nucleotides, differ at only three points from the corresponding sequences in X. laevis, each by a single substitution. These substitutions are readily accommodated by current models for rRNA higher order structure.

  12. Cryo-EM structure of the archaeal 50S ribosomal subunit in complex with initiation factor 6 and implications for ribosome evolution

    DEFF Research Database (Denmark)

    Greber, Basil J; Boehringer, Daniel; Godinic-Mikulcic, Vlatka

    2012-01-01

    Translation of mRNA into proteins by the ribosome is universally conserved in all cellular life. The composition and complexity of the translation machinery differ markedly between the three domains of life. Organisms from the domain Archaea show an intermediate level of complexity, sharing sever...

  13. X-Ray Assembler Data

    Data.gov (United States)

    U.S. Department of Health & Human Services — Federal regulations require that an assembler who installs one or more certified components of a diagnostic x-ray system submit a report of assembly. This database...

  14. Assembling large, complex environmental metagenomes

    Energy Technology Data Exchange (ETDEWEB)

    Howe, A. C. [Michigan State Univ., East Lansing, MI (United States). Microbiology and Molecular Genetics, Plant Soil and Microbial Sciences; Jansson, J. [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Earth Sciences Division; Malfatti, S. A. [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Tringe, S. G. [USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Tiedje, J. M. [Michigan State Univ., East Lansing, MI (United States). Microbiology and Molecular Genetics, Plant Soil and Microbial Sciences; Brown, C. T. [Michigan State Univ., East Lansing, MI (United States). Microbiology and Molecular Genetics, Computer Science and Engineering

    2012-12-28

    The large volumes of sequencing data required to sample complex environments deeply pose new challenges to sequence analysis approaches. De novo metagenomic assembly effectively reduces the total amount of data to be analyzed but requires significant computational resources. We apply two pre-assembly filtering approaches, digital normalization and partitioning, to make large metagenome assemblies more computationaly tractable. Using a human gut mock community dataset, we demonstrate that these methods result in assemblies nearly identical to assemblies from unprocessed data. We then assemble two large soil metagenomes from matched Iowa corn and native prairie soils. The predicted functional content and phylogenetic origin of the assembled contigs indicate significant taxonomic differences despite similar function. The assembly strategies presented are generic and can be extended to any metagenome; full source code is freely available under a BSD license.

  15. Self-assembly of nanocomponents into composite structures: Derivation and simulation of Langevin equations

    Science.gov (United States)

    Pankavich, S.; Shreif, Z.; Miao, Y.; Ortoleva, P.

    2009-05-01

    The kinetics of the self-assembly of nanocomponents into a virus, nanocapsule, or other composite structure is analyzed via a multiscale approach. The objective is to achieve predictability and to preserve key atomic-scale features that underlie the formation and stability of the composite structures. We start with an all-atom description, the Liouville equation, and the order parameters characterizing nanoscale features of the system. An equation of Smoluchowski type for the stochastic dynamics of the order parameters is derived from the Liouville equation via a multiscale perturbation technique. The self-assembly of composite structures from nanocomponents with internal atomic structure is analyzed and growth rates are derived. Applications include the assembly of a viral capsid from capsomers, a ribosome from its major subunits, and composite materials from fibers and nanoparticles. Our approach overcomes errors in other coarse-graining methods, which neglect the influence of the nanoscale configuration on the atomistic fluctuations. We account for the effect of order parameters on the statistics of the atomistic fluctuations, which contribute to the entropic and average forces driving order parameter evolution. This approach enables an efficient algorithm for computer simulation of self-assembly, whereas other methods severely limit the timestep due to the separation of diffusional and complexing characteristic times. Given that our approach does not require recalibration with each new application, it provides a way to estimate assembly rates and thereby facilitate the discovery of self-assembly pathways and kinetic dead-end structures.

  16. Appearance detection device for fuel assembly

    International Nuclear Information System (INIS)

    Matsuoka, Toshihiro

    1998-01-01

    The prevent invention provides an appearance detection device which improves accuracy of images on a display and facilitates editing and selection of images upon detection of appearance of a reactor fuel assembly. Namely, the device of the present invention comprises (1) television cameras movable along fuel assemblies of a reactor, (2) a detection means for detecting the positions of the television cameras, (3) a convertor for converting analog image signals of the television cameras to digital image signals, (4) a memory means for sampling a predetermined portion of the images of the television camera and storing it together with the position signal obtained by the detection means and (5) a computer for selecting a plurality of images and positions from the above-mentioned means and joining them to one or a plurality of static images of the fuel assembly. At least two television cameras are disposed oppositely with each other. Then, position signals of the television cameras are designated by the stored sampling signals, and the fuel assembly at the position can be displayed quickly. It is scrolled, compressed or enlarged and formed into images. (I.S.)

  17. Novel mRNA-specific effects of ribosome drop-off on translation rate and polysome profile.

    Directory of Open Access Journals (Sweden)

    Pierre Bonnin

    2017-05-01

    Full Text Available The well established phenomenon of ribosome drop-off plays crucial roles in translational accuracy and nutrient starvation responses during protein translation. When cells are under stress conditions, such as amino acid starvation or aminoacyl-tRNA depletion due to a high level of recombinant protein expression, ribosome drop-off can substantially affect the efficiency of protein expression. Here we introduce a mathematical model that describes the effects of ribosome drop-off on the ribosome density along the mRNA and on the concomitant protein synthesis rate. Our results show that ribosome premature termination may lead to non-intuitive ribosome density profiles, such as a ribosome density which increases from the 5' to the 3' end. Importantly, the model predicts that the effects of ribosome drop-off on the translation rate are mRNA-specific, and we quantify their resilience to drop-off, showing that the mRNAs which present ribosome queues are much less affected by ribosome drop-off than those which do not. Moreover, among those mRNAs that do not present ribosome queues, resilience to drop-off correlates positively with the elongation rate, so that sequences using fast codons are expected to be less affected by ribosome drop-off. This result is consistent with a genome-wide analysis of S. cerevisiae, which reveals that under favourable growth conditions mRNAs coding for proteins involved in the translation machinery, known to be highly codon biased and using preferentially fast codons, are highly resilient to ribosome drop-off. Moreover, in physiological conditions, the translation rate of mRNAs coding for regulatory, stress-related proteins, is less resilient to ribosome drop-off. This model therefore allows analysis of variations in the translational efficiency of individual mRNAs by accounting for the full range of known ribosome behaviours, as well as explaining mRNA-specific variations in ribosome density emerging from ribosome profiling

  18. Hypophosphorylation of ribosomal protein S6 is a molecular mechanism underlying ischemic tolerance induced by either hibernation or preconditioning.

    Science.gov (United States)

    Miyake, Shin-ichi; Wakita, Hideaki; Bernstock, Joshua D; Castri, Paola; Ruetzler, Christl; Miyake, Junko; Lee, Yang-Ja; Hallenbeck, John M

    2015-12-01

    Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) have an extraordinary capacity to withstand prolonged and profound reductions in blood flow and oxygen delivery to the brain without incurring any cellular damage. As such, the hibernation torpor of I. tridecemlineatus provides a valuable model of tolerance to ischemic stress. Herein, we report that during hibernation torpor, a marked reduction in the phosphorylation of the ribosomal protein S6 (rpS6) occurs within the brains of I. tridecemlineatus. Of note, rpS6 phosphorylation was shown to increase in the brains of rats that underwent an occlusion of the middle cerebral artery. However, such an increase was attenuated after the implementation of an ischemic preconditioning paradigm. In addition, cultured cortical neurons treated with the rpS6 kinase (S6K) inhibitors, D-glucosamine or PF4708671, displayed a decrease in rpS6 phosphorylation and a subsequent increase in tolerance to oxygen/glucose deprivation, an in vitro model of ischemic stroke. Collectively, such evidence suggests that the down-regulation of rpS6 signal transduction may account for a substantial part of the observed increase in cellular tolerance to brain ischemia that occurs during hibernation torpor and after ischemic preconditioning. Further identification and characterization of the mechanisms used by hibernating species to increase ischemic tolerance may eventually clarify how the loss of homeostatic control that occurs during and after cerebral ischemia in the clinic can ultimately be minimized and/or prevented. Mammalian hibernation provides a valuable model of tolerance to ischemic stress. Herein, we demonstrate that marked reductions in the phosphorylation of ribosomal protein S6 (rpS6), extracellular signal-regulated kinase family of mitogen-activated protein (MAP) kinase p44/42 (p44/42MAPK) and ribosomal protein S6 kinase (S6K) occur within the brains of both hibernating squirrels and rats, which have undergone an ischemic

  19. Reflector-moderated critical assemblies

    International Nuclear Information System (INIS)

    Paxton, H.C.; Jarvis, G.A.; Byers, C.C.

    1975-07-01

    Experiments with reflector-moderated critical assemblies were part of the Rover Program at the Los Alamos Scientific Laboratory (LASL). These assemblies were characterized by thick D 2 O or beryllium reflectors surrounding large cavities that contained highly enriched uranium at low average densities. Because interest in this type of system has been revived by LASL Plasma Cavity Assembly studies, more detailed descriptions of the early assemblies than had been available in the unclassified literature are provided. (U.S.)

  20. Small-molecule inhibitor leads of ribosome-inactivating proteins developed using the doorstop approach.

    Directory of Open Access Journals (Sweden)

    Yuan-Ping Pang

    2011-03-01

    Full Text Available Ribosome-inactivating proteins (RIPs are toxic because they bind to 28S rRNA and depurinate a specific adenine residue from the α-sarcin/ricin loop (SRL, thereby inhibiting protein synthesis. Shiga-like toxins (Stx1 and Stx2, produced by Escherichia coli, are RIPs that cause outbreaks of foodborne diseases with significant morbidity and mortality. Ricin, produced by the castor bean plant, is another RIP lethal to mammals. Currently, no US Food and Drug Administration-approved vaccines nor therapeutics exist to protect against ricin, Shiga-like toxins, or other RIPs. Development of effective small-molecule RIP inhibitors as therapeutics is challenging because strong electrostatic interactions at the RIP•SRL interface make drug-like molecules ineffective in competing with the rRNA for binding to RIPs. Herein, we report small molecules that show up to 20% cell protection against ricin or Stx2 at a drug concentration of 300 nM. These molecules were discovered using the doorstop approach, a new approach to protein•polynucleotide inhibitors that identifies small molecules as doorstops to prevent an active-site residue of an RIP (e.g., Tyr80 of ricin or Tyr77 of Stx2 from adopting an active conformation thereby blocking the function of the protein rather than contenders in the competition for binding to the RIP. This work offers promising leads for developing RIP therapeutics. The results suggest that the doorstop approach might also be applicable in the development of other protein•polynucleotide inhibitors as antiviral agents such as inhibitors of the Z-DNA binding proteins in poxviruses. This work also calls for careful chemical and biological characterization of drug leads obtained from chemical screens to avoid the identification of irrelevant chemical structures and to avoid the interference caused by direct interactions between the chemicals being screened and the luciferase reporter used in screening assays.

  1. The Insect Pathogen Serratia marcescens Db10 Uses a Hybrid Non-Ribosomal Peptide Synthetase-Polyketide Synthase to Produce the Antibiotic Althiomycin

    Science.gov (United States)

    Challis, Gregory L.; Stanley-Wall, Nicola R.; Coulthurst, Sarah J.

    2012-01-01

    There is a continuing need to discover new bioactive natural products, such as antibiotics, in genetically-amenable micro-organisms. We observed that the enteric insect pathogen, Serratia marcescens Db10, produced a diffusible compound that inhibited the growth of Bacillis subtilis and Staphyloccocus aureus. Mapping the genetic locus required for this activity revealed a putative natural product biosynthetic gene cluster, further defined to a six-gene operon named alb1–alb6. Bioinformatic analysis of the proteins encoded by alb1–6 predicted a hybrid non-ribosomal peptide synthetase-polyketide synthase (NRPS-PKS) assembly line (Alb4/5/6), tailoring enzymes (Alb2/3) and an export/resistance protein (Alb1), and suggested that the machinery assembled althiomycin or a related molecule. Althiomycin is a ribosome-inhibiting antibiotic whose biosynthetic machinery had been elusive for decades. Chromatographic and spectroscopic analyses confirmed that wild type S. marcescens produced althiomycin and that production was eliminated on disruption of the alb gene cluster. Construction of mutants with in-frame deletions of specific alb genes demonstrated that Alb2–Alb5 were essential for althiomycin production, whereas Alb6 was required for maximal production of the antibiotic. A phosphopantetheinyl transferase enzyme required for althiomycin biosynthesis was also identified. Expression of Alb1, a predicted major facilitator superfamily efflux pump, conferred althiomycin resistance on another, sensitive, strain of S. marcescens. This is the first report of althiomycin production outside of the Myxobacteria or Streptomyces and paves the way for future exploitation of the biosynthetic machinery, since S. marcescens represents a convenient and tractable producing organism. PMID:23028578

  2. Wellpoint assembly and method of installing a wellpoint assembly

    Energy Technology Data Exchange (ETDEWEB)

    Share, J.H.; Share, S.

    1988-12-13

    This patent describes a wellpoint assembly comprising in combination: an elongated flexible header pipe; coupling joints attached to the flexible header, each coupling joint being adapted for attaching a well point assembly; a flexible header pipe support for supporting the flexible header pipe; and means to align portions of a continuous flexible header pipe for attaching wellpoint assemblies, the means to align portions of the flexible header pipe including an alignment tool for aligning each flexible header coupling joint into a predetermined position for attaching a well point assembly line whereby a wellpoint assembly can be laid out around an excavation site in a wide variety of patterns.

  3. Initiation factor 2, tRNA, and 50S subunits cooperatively stabilize mRNAs on the ribosome during initiation

    Science.gov (United States)

    Masuda, Tomoaki; Petrov, Alexey N.; Iizuka, Ryo; Funatsu, Takashi; Puglisi, Joseph D.; Uemura, Sotaro

    2012-01-01

    Initiation factor 2 (IF2) is a key factor in initiation of bacterial protein synthesis. It recruits initiator tRNA to the small ribosomal subunit and facilitates joining of the large ribosomal subunit. Using reconstituted translation system of Escherichia coli and optical tweezers, we directly measure the rupture force between single ribosomal complexes and mRNAs for initiation complexes in the presence and the absence of IF2. We demonstrate that IF2 together with codon recognition by initiator tRNA increases the force required to dislocate mRNA from the ribosome complexes; mRNA stabilization by IF2 required the presence of a joined 50S subunit, and was independent of bound guanine nucleotide. IF2 thus helps lock the 70S ribosome over the start codon during initiation, thus maintaining reading frame. Our results show how mRNA is progressively stabilized on the ribosome through distinct steps of initiation. PMID:22411833

  4. FLUORINE CELL ANODE ASSEMBLY

    Science.gov (United States)

    Cable, R.E.; Goode, W.B. Jr.; Henderson, W.K.; Montillon, G.H.

    1962-06-26

    An improved anode assembly is deslgned for use in electrolytlc cells ln the productlon of hydrogen and fluorlne from a moIten electrolyte. The anode assembly comprises a copper post, a copper hanger supported by the post, a plurality of carbon anode members, and bolt means for clamplng half of the anode members to one slde of the hanger and for clamplng the other half of the anode members to the other slde of the hanger. The heads of the clamplng bolts are recessed withln the anode members and carbon plugs are inserted ln the recesses above the bolt heads to protect the boIts agalnst corroslon. A copper washer is provided under the head of each clamplng boIt such that the anode members can be tightly clamped to the hanger with a resultant low anode jolnt resistance. (AEC)

  5. Gamma counter shutter assembly

    International Nuclear Information System (INIS)

    Aday, R.W. Jr.; Barber, D.G.

    1976-01-01

    A shutter assembly for a radioactivity measuring apparatus is described having a sample counting chamber, the assembly having a bulky solid lead cylinder with a sample access port extending therethrough for alignment with the sample chamber. The cylinder is rotated by a Geneva wheel arrangement having a drive wheel with a plurality of equi-angularly disposed pins perpendicular to the surface thereof engaging radially extending open-ended slots in a driven wheel secured to the lead cylinder for concurrent rotation therewith. The drive wheel is rotated at a constant speed with the driven wheel accelerating as a pin traverses the slot from the open end toward the driven wheel center and then decelerating as the pin traverses the reverse direction to provide precise positioning with adjacent pins engaging the open ends of adjacent slots in the stop position of the cylinder. 8 Claims, 3 Drawing Figures

  6. Nuclear reactor fuel assembly

    International Nuclear Information System (INIS)

    1975-01-01

    A description is given of a nuclear reactor fuel assembly comprising a cluster of fuel elements supported by transversal grids so that their axes are parallel to and at a distance from each other, in order to establish interstices for the axial flow of a coolant. At least one of the interstices is occupied by an axial duct reserved for an auxiliary cooling fluid and is fitted with side holes through which the auxiliary cooling fluid is sprayed into the cluster. Deflectors extend as from a transversal grid in a position opposite the holes to deflect the cooling fluid jet towards those parts of the fuel elements that are not accessible to the auxiliary coolant. This assembly is intended for reactors cooled by light or heavy water [fr

  7. Fuel nozzle assembly

    Science.gov (United States)

    Johnson, Thomas Edward [Greer, SC; Ziminsky, Willy Steve [Simpsonville, SC; Lacey, Benjamin Paul [Greer, SC; York, William David [Greer, SC; Stevenson, Christian Xavier [Inman, SC

    2011-08-30

    A fuel nozzle assembly is provided. The assembly includes an outer nozzle body having a first end and a second end and at least one inner nozzle tube having a first end and a second end. One of the nozzle body or nozzle tube includes a fuel plenum and a fuel passage extending therefrom, while the other of the nozzle body or nozzle tube includes a fuel injection hole slidably aligned with the fuel passage to form a fuel flow path therebetween at an interface between the body and the tube. The nozzle body and the nozzle tube are fixed against relative movement at the first ends of the nozzle body and nozzle tube, enabling the fuel flow path to close at the interface due to thermal growth after a flame enters the nozzle tube.

  8. Turbine seal assembly

    Science.gov (United States)

    Little, David A.

    2013-04-16

    A seal assembly that limits gas leakage from a hot gas path to one or more disc cavities in a turbine engine. The seal assembly includes a seal apparatus that limits gas leakage from the hot gas path to a respective one of the disc cavities. The seal apparatus comprises a plurality of blade members rotatable with a blade structure. The blade members are associated with the blade structure and extend toward adjacent stationary components. Each blade member includes a leading edge and a trailing edge, the leading edge of each blade member being located circumferentially in front of the blade member's corresponding trailing edge in a direction of rotation of the turbine rotor. The blade members are arranged such that a space having a component in a circumferential direction is defined between adjacent circumferentially spaced blade members.

  9. Mechanical Seal Assembly

    Energy Technology Data Exchange (ETDEWEB)

    Kotlyar, Oleg M.

    1999-06-18

    An improved mechanical seal assembly is provided for sealing rotating shafts with respect to their shaft housings, wherein the rotating shafts are subject to substantial axial vibrations. The mechanical seal assembly generally includes a rotating sealing ring fixed to the shaft, a non-rotating sealing ring adjacent to and in close contact with the rotating sealing ring for forming an annular seal about the shaft, and a mechanical diode element that applies a biasing force to the non-rotating sealing ring by means of hemispherical joint. The alignment of the mechanical diode with respect to the sealing rings is maintained by a series of linear bearings positioned axially along a desired length of the mechanical diode. Alternative embodiments include mechanical or hydraulic amplification components for amplifying axial displacement of the non-rotating sealing ring and transferring it to the mechanical diode.

  10. Solution deposition assembly

    Science.gov (United States)

    Roussillon, Yann; Scholz, Jeremy H; Shelton, Addison; Green, Geoff T; Utthachoo, Piyaphant

    2014-01-21

    Methods and devices are provided for improved deposition systems. In one embodiment of the present invention, a deposition system is provided for use with a solution and a substrate. The system comprises of a solution deposition apparatus; at least one heating chamber, at least one assembly for holding a solution over the substrate; and a substrate curling apparatus for curling at least one edge of the substrate to define a zone capable of containing a volume of the solution over the substrate. In another embodiment of the present invention, a deposition system for use with a substrate, the system comprising a solution deposition apparatus; at heating chamber; and at least assembly for holding solution over the substrate to allow for a depth of at least about 0.5 microns to 10 mm.

  11. Mechanical seal assembly

    Science.gov (United States)

    Kotlyar, Oleg M.

    2001-01-01

    An improved mechanical seal assembly is provided for sealing rotating shafts with respect to their shaft housings, wherein the rotating shafts are subject to substantial axial vibrations. The mechanical seal assembly generally includes a rotating sealing ring fixed to the shaft, a non-rotating sealing ring adjacent to and in close contact with the rotating sealing ring for forming an annular seal about the shaft, and a mechanical diode element that applies a biasing force to the non-rotating sealing ring by means of hemispherical joint. The alignment of the mechanical diode with respect to the sealing rings is maintained by a series of linear bearings positioned axially along a desired length of the mechanical diode. Alternative embodiments include mechanical or hydraulic amplification components for amplifying axial displacement of the non-rotating sealing ring and transferring it to the mechanical diode.

  12. Dosage sensitivity of RPL9 and concerted evolution of ribosomal protein genes in plants

    Directory of Open Access Journals (Sweden)

    Deborah eDevis

    2015-12-01

    Full Text Available The ribosome in higher eukaryotes is a large macromolecular complex composed of four rRNAs and eighty different ribosomal proteins. In plants, each ribosomal protein is encoded by multiple genes. Duplicate genes within a family are often necessary to provide a threshold dose of a ribosomal protein but in some instances appear to have non-redundant functions. Here, we addressed whether divergent members of the RPL9 gene family are dosage sensitive or whether these genes have non-overlapping functions. The RPL9 family in A. thaliana comprises two nearly identical members, RPL9B and RPL9C, and a more divergent member, RPL9D. Mutations in RPL9C and RPL9D genes leads to delayed growth early in development, and loss of both genes is embryo lethal, indicating that these are dosage-sensitive and redundant genes. Phylogenetic analysis of RPL9 as well as RPL4, RPL5, RPL27a, RPL36a and RPS6 family genes in the Brassicaceae indicated that multicopy ribosomal protein genes have been largely retained following whole genome duplication. However, these gene families also show instances of tandem duplication, small scale deletion and evidence of gene conversion. Furthermore, phylogenetic analysis of RPL9 genes in angiosperm species showed that genes within a species are more closely related to each other than to RPL9 genes in other species, suggesting ribosomal protein genes undergo convergent evolution. Our analysis indicates that ribosomal protein gene retention following whole genome duplication contributes to the number of genes in a family. However, small scale rearrangements influence copy number and likely drive concerted evolution of these dosage-sensitive genes.

  13. The 70S ribosome modulates the ATPase activity of Escherichia coli YchF.

    Science.gov (United States)

    Becker, Marion; Gzyl, Katherine E; Altamirano, Alvin M; Vuong, Anthony; Urban, Kirstin; Wieden, Hans-Joachim

    2012-10-01

    YchF is one of two universally conserved GTPases with unknown cellular function. As a first step toward elucidating YchF's cellular role, we performed a detailed biochemical characterization of the protein from Escherichia coli. Our data from fluorescence titrations not only confirmed the surprising finding that YchFE.coli binds adenine nucleotides more efficiently than guanine nucleotides, but also provides the first evidence suggesting that YchF assumes two distinct conformational states (ATP- and ADP-bound) consistent with the functional cycle of a typical GTPase. Based on an in vivo pull-down experiment using a His-tagged variant of YchF from E. coli (YchFE.coli), we were able to isolate a megadalton complex containing the 70S ribosome. Based on this finding, we report the successful reconstitution of a YchF•70S complex in vitro, revealing an affinity (KD) of the YchFE.coli•ADPNP complex for 70S ribosomes of 3 μM. The in vitro reconstitution data also suggests that the identity of the nucleotide-bound state of YchF (ADP or ATP) modulates its affinity for 70S ribosomes. A detailed Michaelis-Menten analysis of YchF's catalytic activity in the presence and the absence of the 70S ribosome and its subunits revealed for the first time that the 70S ribosome is able to stimulate YchF's ATPase activity (~10-fold), confirming the ribosome as part of the functional cycle of YchF. Our findings taken together with previously reported data for the human homolog of YchF (hOLA1) indicate a high level of evolutionary conservation in the enzymatic properties of YchF and suggest that the ribosome is the main functional partner of YchF not only in bacteria.

  14. Ribosome profiling reveals translational regulation of mammalian cells in response to hypoxic stress.

    Science.gov (United States)

    Jiang, Zhiwen; Yang, Jiaqi; Dai, Aimei; Wang, Yuming; Li, Wei; Xie, Zhi

    2017-08-21

    Retinal pigment epithelium (RPE) cells transfer oxygen and nutrients from choroid to the neural retina. Reduced oxygen to RPE perturbs development and functions of blood vessels in retina. Previous efforts of genome-wide studies have been largely focused on transcriptional changes of cells in response to hypoxia. Recently developed ribosome profiling provides an opportunity to study genome-wide translational changes. To gain systemic insights into the transcriptional and translational regulation of cellular in response to hypoxic stress, we used simultaneous RNA sequencing and ribosome profiling on an RPE cells line, ARPE-19, under hypoxia condition. Both HIF-1α and EPAS1 (HIF-2α) proteins were stabilized in ARPE-19 under hypoxic stress treatment at 1 h, 2 h and 4 h. Analysis of simultaneous RNA sequencing and ribosome profiling data showed genome-wide gene expression changes at both transcriptional and translational levels. Comparative analysis of ribosome profiling and RNA-seq data revealed that hypoxia induced changes of more genes at the translational than the transcriptional levels. Ribosomes densities at 5' untranslated region (UTR) significantly increased under hypoxic stress. Interestingly, the increase in ribosome densities at 5' UTR is positively correlated with the presence of upstream open reading frames (uORFs) in the 5' UTR of mRNAs. Our results characterized translational profiles of mRNAs for a RPE cell line in response to hypoxia. In particular, uORFs play important roles in the regulation of translation efficiency by affecting ribosomes loading onto mRNAs. This study provides the first attempt to understand translational response of mammalian cells under hypoxic condition.

  15. Optimization of ribosome structure and function by rRNA base modification.

    Directory of Open Access Journals (Sweden)

    Jennifer L Baxter-Roshek

    2007-01-01

    Full Text Available Translating mRNA sequences into functional proteins is a fundamental process necessary for the viability of organisms throughout all kingdoms of life. The ribosome carries out this process with a delicate balance between speed and accuracy. This work investigates how ribosome structure and function are affected by rRNA base modification. The prevailing view is that rRNA base modifications serve to fine tune ribosome structure and function.To test this hypothesis, yeast strains deficient in rRNA modifications in the ribosomal peptidyltransferase center were monitored for changes in and translational fidelity. These studies revealed allele-specific sensitivity to translational inhibitors, changes in reading frame maintenance, nonsense suppression and aa-tRNA selection. Ribosomes isolated from two mutants with the most pronounced phenotypic changes had increased affinities for aa-tRNA, and surprisingly, increased rates of peptidyltransfer as monitored by the puromycin assay. rRNA chemical analyses of one of these mutants identified structural changes in five specific bases associated with the ribosomal A-site.Together, the data suggest that modification of these bases fine tune the structure of the A-site region of the large subunit so as to assure correct positioning of critical rRNA bases involved in aa-tRNA accommodation into the PTC, of the eEF-1A.aa-tRNA.GTP ternary complex with the GTPase associated center, and of the aa-tRNA in the A-site. These findings represent a direct demonstration in support of the prevailing hypothesis that rRNA modifications serve to optimize rRNA structure for production of accurate and efficient ribosomes.

  16. An Assembly Funnel Makes Biomolecular Complex Assembly Efficient

    Science.gov (United States)

    Zenk, John; Schulman, Rebecca

    2014-01-01

    Like protein folding and crystallization, the self-assembly of complexes is a fundamental form of biomolecular organization. While the number of methods for creating synthetic complexes is growing rapidly, most require empirical tuning of assembly conditions and/or produce low yields. We use coarse-grained simulations of the assembly kinetics of complexes to identify generic limitations on yields that arise because of the many simultaneous interactions allowed between the components and intermediates of a complex. Efficient assembly occurs when nucleation is fast and growth pathways are few, i.e. when there is an assembly “funnel”. For typical complexes, an assembly funnel occurs in a narrow window of conditions whose location is highly complex specific. However, by redesigning the components this window can be drastically broadened, so that complexes can form quickly across many conditions. The generality of this approach suggests assembly funnel design as a foundational strategy for robust biomolecular complex synthesis. PMID:25360818

  17. Ordinary General Assembly

    CERN Multimedia

    Association du personnel

    2010-01-01

    Tuesday 20 April at 10.00 Council Chamber, Bldg 503 In conformity with the Statutes of the Staff Association, an ordinary General Assembly is organized once a year (article IV.2.1). Agenda Adoption of the Agenda Approval of the Draft Minutes of the Ordinary General Assembly of 12 May 2009 Presentation and approval of the Activity Report 2009 Presentation and approval of the Financial Report 2009 Presentation and approval of the Auditors Report 2009 Programme for 2010 Presentation et and approval of the draft budget and subscription rate 2010 Modifications to the statutes of the association Election of the Election Committee Election of the Board of Auditors Miscellaneous We remind members of article IV.3.4 in the Statutes of the Association which reads: “After having dealt with all the items on the agenda, the members may, with the consent of the Assembly, have other matters discussed, but decisions may be taken only on the items listed on the agenda...

  18. SCT Barrel Assembly Complete

    CERN Multimedia

    L. Batchelor

    As reported in the April 2005 issue of the ATLAS eNews, the first of the four Semiconductor Tracker (SCT) barrels, complete with modules and services, arrived safely at CERN in January of 2005. In the months since January, the other three completed barrels arrived as well, and integration of the four barrels into the entire barrel assembly commenced at CERN, in the SR1 building on the ATLAS experimental site, in July. Assembly was completed on schedule in September, with the addition of the innermost layer to the 4-barrel assembly. Work is now underway to seal the barrel thermal enclosure. This is necessary in order to enclose the silicon tracker in a nitrogen atmosphere and provide it with faraday-cage protection, and is a delicate and complicated task: 352 silicon module powertapes, 352 readout-fibre bundles, and over 400 Detector Control System sensors must be carefully sealed into the thermal enclosure bulkhead. The team is currently verifying the integrity of the low mass cooling system, which must be d...

  19. IAHS Third Scientific Assembly

    Science.gov (United States)

    The International Association of Hydrological Sciences (IAHS) convened its Third Scientific Assembly in Baltimore, Md., May 10-19, 1989. The Assembly was attended by about 450 scientists and engineers. The attendance was highest from the U.S., as could be expected; 37 were from Canada; 22 each, Netherlands and United Kingdom; 14, Italy; 12, China; 10, Federal Republic of Germany; 8 each from France, the Republic of South Africa, and Switzerland; 7, Austria; 6 each, Finland and Japan; others were scattered among the remainder of 48 countries total.one of the cosponsors and also handled business matters for the Assembly. Other cosponsors included the International Association of Meteorology and Atmospheric Physics (IAMAP), United Nations Environmental Program (UNEP), United Nations Educational, Scientific, and Cultural Organization (UNESCO), World Meteorological Organization (WMO), and U.K. Overseas Development Authority (ODA). U.S. federal agencies serving as cosponsors included the Environmental Protection Agency, National Aeronautics and Space Administration, National Science Foundation, National Weather Service, Department of Agriculture, Department of State, and U.S. Geological Survey.

  20. Fourth Doctoral Student Assembly

    CERN Multimedia

    Ingrid Haug

    2016-01-01

    On 10 May, over 130 PhD students and their supervisors, from both CERN and partner universities, gathered for the 4th Doctoral Student Assembly in the Council Chamber.   The assembly was followed by a poster session, at which eighteen doctoral students presented the outcome of their scientific work. The CERN Doctoral Student Programme currently hosts just over 200 students in applied physics, engineering, computing and science communication/education. The programme has been in place since 1985. It enables students to do their research at CERN for a maximum of three years and to work on a PhD thesis, which they defend at their University. The programme is steered by the TSC committee, which holds two selection committees per year, in June and December. The Doctoral Student Assembly was opened by the Director-General, Fabiola Gianotti, who stressed the importance of the programme in the scientific environment at CERN, emphasising that there is no more rewarding activity than lear...

  1. Assembly of the membrane domain of ATP synthase in human mitochondria.

    Science.gov (United States)

    He, Jiuya; Ford, Holly C; Carroll, Joe; Douglas, Corsten; Gonzales, Evvia; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2018-03-20

    The ATP synthase in human mitochondria is a membrane-bound assembly of 29 proteins of 18 kinds. All but two membrane components are encoded in nuclear genes, synthesized on cytoplasmic ribosomes, and imported into the matrix of the organelle, where they are assembled into the complex with ATP6 and ATP8, the products of overlapping genes in mitochondrial DNA. Disruption of individual human genes for the nuclear-encoded subunits in the membrane portion of the enzyme leads to the formation of intermediate vestigial ATPase complexes that provide a description of the pathway of assembly of the membrane domain. The key intermediate complex consists of the F 1 -c 8 complex inhibited by the ATPase inhibitor protein IF 1 and attached to the peripheral stalk, with subunits e, f, and g associated with the membrane domain of the peripheral stalk. This intermediate provides the template for insertion of ATP6 and ATP8, which are synthesized on mitochondrial ribosomes. Their association with the complex is stabilized by addition of the 6.8 proteolipid, and the complex is coupled to ATP synthesis at this point. A structure of the dimeric yeast F o membrane domain is consistent with this model of assembly. The human 6.8 proteolipid (yeast j subunit) locks ATP6 and ATP8 into the membrane assembly, and the monomeric complexes then dimerize via interactions between ATP6 subunits and between 6.8 proteolipids (j subunits). The dimers are linked together back-to-face by DAPIT (diabetes-associated protein in insulin-sensitive tissue; yeast subunit k), forming long oligomers along the edges of the cristae.

  2. Chromatin dynamics of plant telomeres and ribosomal genes

    Czech Academy of Sciences Publication Activity Database

    Dvořáčková, Martina; Fojtová, Miloslava; Fajkus, Jiří

    2015-01-01

    Roč. 83, č. 1 (2015), s. 18-37 ISSN 0960-7412 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0068; GA ČR(CZ) GA13-06943S; GA ČR(CZ) GP13-11563P Grant - others:GA ČR(CZ) GAP501/11/0289 Institutional support: RVO:68081707 Keywords : HISTONE VARIANT H3.3 * ASSEMBLY FACTOR-I * REPLICATION FORK PROGRESSION Subject RIV: BO - Biophysics Impact factor: 5.468, year: 2015

  3. Synthesis and methylation of ribosomal RNA in HeLa cells infected with the herpes virus pseudorabies virus

    International Nuclear Information System (INIS)

    Furlong, J.C.; Kyriakidis, S.; Stevely, W.S.

    1982-01-01

    The effects of infection with the herpes virus pseudorabies virus on the metabolism of HeLa cell ribosomal RNA were examined. There is a decline both in the synthesis of nucleolar 45S ribosomal precursor RNA and in its processing to mature cytoplasmic RNA. The methylated oligonucleotides in the ribosomal RNA species were studied. The methylation of cytoplasmic ribosomal RNA was essentially unchanged. However there was some undermethylation of the nucleolar precursor. If undermethylated RNA does not mature then this may partly explain the reduced processing in the infected cells. (Author)

  4. Coupling of mRNA Structure Rearrangement to Ribosome Movement during Bypassing of Non-coding Regions.

    Science.gov (United States)

    Chen, Jin; Coakley, Arthur; O'Connor, Michelle; Petrov, Alexey; O'Leary, Seán E; Atkins, John F; Puglisi, Joseph D

    2015-11-19

    Nearly half of the ribosomes translating a particular bacteriophage T4 mRNA bypass a region of 50 nt, resuming translation 3' of this gap. How this large-scale, specific hop occurs and what determines whether a ribosome bypasses remain unclear. We apply single-molecule fluorescence with zero-mode waveguides to track individual Escherichia coli ribosomes during translation of T4's gene 60 mRNA. Ribosomes that bypass are characterized by a 10- to 20-fold longer pause in a non-canonical rotated state at the take-off codon. During the pause, mRNA secondary structure rearrangements are coupled to ribosome forward movement, facilitated by nascent peptide interactions that disengage the ribosome anticodon-codon interactions for slippage. Close to the landing site, the ribosome then scans mRNA in search of optimal base-pairing interactions. Our results provide a mechanistic and conformational framework for bypassing, highlighting a non-canonical ribosomal state to allow for mRNA structure refolding to drive large-scale ribosome movements. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Thermus Thermophilus as a Model System for the Study of Ribosomal Antibiotic Resistance

    Science.gov (United States)

    Gregory, Steven T.

    2018-03-01

    Ribosomes are the intracellular ribonucleoprotein machines responsible for the translation of mRNA sequence into protein sequence. As an essential cell component, the ribosome is the target of numerous antibiotics that bind to critical functional sites to impair protein synthesis. Mutations causing resistance to antibiotics arise in antibiotic binding sites, and an understanding of the basis of resistance will be an essential component of efforts to develop new antibiotics by rational drug design. We have identified a number of antibiotic-resistance mutations in ribosomal genes of the thermophilic bacterium Thermus thermophilus. This species offers two primary advantages for examining the structural basis of antibiotic-resistance, in particular, its potential for genetic manipulation and the suitability of its ribosomes for analysis by X-ray crystallography. Mutations we have identified in this organism are in many instances identical to those found in other bacterial species, including important pathogens, a result of the extreme conservation of ribosome functional sites. Here I summarize the advantages of this organism as a model system to study antibiotic-resistance mechanisms at the molecular level.

  6. Structural Insight into Interaction between C20 Phenylalanyl Derivative of Tylosin and Ribosomal Tunnel.

    Science.gov (United States)

    Makarov, G I; Sumbatyan, N V; Bogdanov, A A

    2017-08-01

    Macrolides are clinically important antibiotics that inhibit protein biosynthesis on ribosomes by binding to ribosomal tunnel. Tylosin belongs to the group of 16-membered macrolides. It is a potent inhibitor of translation whose activity is largely due to reversible covalent binding of its aldehyde group with the base of A2062 in 23S ribosomal RNA. It is known that the conversion of the aldehyde group of tylosin to methyl or carbinol groups dramatically reduces its inhibitory activity. However, earlier we obtained several derivatives of tylosin having comparable activity in spite of the fact that the aldehyde group of tylosin in these compounds was substituted with an amino acid or a peptide residue. Details of the interaction of these compounds with the ribosome that underlies their high inhibitory activity were not known. In the present work, the structure of the complex of tylosin derivative containing in position 20 the residue of ethyl ester of 2-imino(oxy)acetylphenylalanine with the tunnel of the E. coli ribosome was identified by means of molecular dynamics simulations, which could explain high biological activity of this compound.

  7. Pseudoknot and translational control in the expression of the S15 ribosomal protein.

    Science.gov (United States)

    Bénard, L; Philippe, C; Ehresmann, B; Ehresmann, C; Portier, C

    1996-01-01

    Translational autocontrol of the expression of the ribosomal protein S15 proceeds through the transitory formation of a pseudoknot. A synopsis of the known data is used to propose a molecular model of the mechanism involved and for the role of the pseudoknot. This latter structure is able to recruit 30S ribosomal subunits to initiate translation, but also to bind S15 and to stop translation by trapping the ribosome on its loading site. Information on the S15 protein recognition of the messenger RNA site was deduced from mutational analyses and chemical probing. A comparison of this messenger site with the S15 ribosomal binding site was conducted by analysing hydroxyl radical footprintings of these two sites. The existence of two subsites in 16S RNA suggests that the ribosomal protein S15 might present either two different binding sites or at least one common subsite. Clues for the presence of a common site between the messenger and 16S RNA are given which cannot rule out that recognition specificity is linked to a few other determinants. Whether these determinants are different or not remains an open question.

  8. The nuclear import of ribosomal proteins is regulated by mTOR

    Science.gov (United States)

    Kazyken, Dubek; Kaz, Yelimbek; Kiyan, Vladimir; Zhylkibayev, Assylbek A.; Chen, Chien-Hung; Agarwal, Nitin K.; Sarbassov, Dos D.

    2014-01-01

    Mechanistic target of rapamycin (mTOR) is a central component of the essential signaling pathway that regulates cell growth and proliferation by controlling anabolic processes in cells. mTOR exists in two distinct mTOR complexes known as mTORC1 and mTORC2 that reside mostly in cytoplasm. In our study, the biochemical characterization of mTOR led to discovery of its novel localization on nuclear envelope where it associates with a critical regulator of nuclear import Ran Binding Protein 2 (RanBP2). We show that association of mTOR with RanBP2 is dependent on the mTOR kinase activity that regulates the nuclear import of ribosomal proteins. The mTOR kinase inhibitors within thirty minutes caused a substantial decrease of ribosomal proteins in the nuclear but not cytoplasmic fraction. Detection of a nuclear accumulation of the GFP-tagged ribosomal protein rpL7a also indicated its dependence on the mTOR kinase activity. The nuclear abundance of ribosomal proteins was not affected by inhibition of mTOR Complex 1 (mTORC1) by rapamycin or deficiency of mTORC2, suggesting a distinctive role of the nuclear envelope mTOR complex in the nuclear import. Thus, we identified that mTOR in association with RanBP2 mediates the active nuclear import of ribosomal proteins. PMID:25294810

  9. Diamond Blackfan Anemia at the Crossroad between Ribosome Biogenesis and Heme Metabolism

    Directory of Open Access Journals (Sweden)

    Deborah Chiabrando

    2010-01-01

    Full Text Available Diamond-Blackfan anemia (DBA is a rare, pure red-cell aplasia that presents during infancy. Approximately 40% of cases are associated with other congenital defects, particularly malformations of the upper limb or craniofacial region. Mutations in the gene coding for the ribosomal protein RPS19 have been identified in 25% of patients with DBA, with resulting impairment of 18S rRNA processing and 40S ribosomal subunit formation. Moreover, mutations in other ribosomal protein coding genes account for about 25% of other DBA cases. Recently, the analysis of mice from which the gene coding for the heme exporter Feline Leukemia Virus subgroup C Receptor (FLVCR1 is deleted suggested that this gene may be involved in the pathogenesis of DBA. FLVCR1-null mice show a phenotype resembling that of DBA patients, including erythroid failure and malformations. Interestingly, some DBA patients have disease linkage to chromosome 1q31, where FLVCR1 is mapped. Moreover, it has been reported that cells from DBA patients express alternatively spliced isoforms of FLVCR1 which encode non-functional proteins. Herein, we review the known roles of RPS19 and FLVCR1 in ribosome function and heme metabolism respectively, and discuss how the deficiency of a ribosomal protein or of a heme exporter may result in the same phenotype.

  10. Ribosome Levels Selectively Regulate Translation and Lineage Commitment in Human Hematopoiesis.

    Science.gov (United States)

    Khajuria, Rajiv K; Munschauer, Mathias; Ulirsch, Jacob C; Fiorini, Claudia; Ludwig, Leif S; McFarland, Sean K; Abdulhay, Nour J; Specht, Harrison; Keshishian, Hasmik; Mani, D R; Jovanovic, Marko; Ellis, Steven R; Fulco, Charles P; Engreitz, Jesse M; Schütz, Sabina; Lian, John; Gripp, Karen W; Weinberg, Olga K; Pinkus, Geraldine S; Gehrke, Lee; Regev, Aviv; Lander, Eric S; Gazda, Hanna T; Lee, Winston Y; Panse, Vikram G; Carr, Steven A; Sankaran, Vijay G

    2018-03-22

    Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation. Copyright © 2018 Elsevier Inc. All rights reserved.

  11. The ribosomal DNA transcription unit of the house cricket, Acheta domesticus.

    Science.gov (United States)

    Ware, J L; Sharp, Z D; Cave, M D

    1987-06-01

    A composite map representing a single ribosomal DNA repeat unit of the house cricket, Acheta domesticus, was constructed from overlapping cloned fragments. Sites in the repeat unit for nine restriction enzymes were mapped. R-loop mapping of sequences coding for 18 S and 28 S RNA demonstrates that the 58-kb ribosomal DNA repeat unit contains a novel-sized internal transcribed spacer of 8.4 kb. The existence of this large spacer was confirmed in genomic DNA, most if not all of the genomic repeat units containing such a spacer. A 15- to 17-kb ribosomal RNA precursor transcript is synthesized as predicted on the basis of the size of the internal transcribed spacer. The 5.8 S RNA gene is localized to a 1-kb sequence immediately 5' to the 28 S gene. The coding regions examined contain no intervening sequences analogous to those described within ribosomal DNA of other eukaryotes. Only 11% of the repeat unit codes for mature ribosomal RNA, while the remainder is nontranscribed (71%) and transcribed (18%) spacer DNA.

  12. Ribosomal RNA and protein transcripts persist in the cysts of Entamoeba invadens.

    Science.gov (United States)

    Ojha, Sandeep; Ahamad, Jamaluddin; Bhattacharya, Alok; Bhattacharya, Sudha

    2014-06-01

    In most organisms rDNA transcription ceases under conditions of growth stress. However, we have earlier shown that pre-rRNA accumulates during encystation in Entamoeba invadens. We labeled newly-synthesized rRNA during encystation, with [methyl-(3)H] methionine in the presence of chitinase to enable uptake of isotope. Incorporation rate reduced after 24h, and then increased to reach levels comparable with normal cells. The label was rapidly chased to the ribosomal pellet in dividing cells, while at late stages of encystation the ratio of counts going to the pellet dropped 3-fold. The transcript levels of selected ribosomal protein genes also went down initially but went up again at later stages of encystation. This suggested that rRNA and ribosomal protein transcription may be coordinately regulated. Our data shows that encysting E. invadens cells accumulate transcripts of both the RNA and protein components of the ribosome, which may ensure rapid synthesis of new ribosomes when growth resumes. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. 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

  14. Rape prevention

    Science.gov (United States)

    Date rape - prevention; Sexual assault - prevention ... Centers for Disease Control and Prevention website. Sexual assault and abuse and STDs. In: 2015 sexually transmitted diseases treatment guidelines 2015. www.cdc.gov/std/tg2015/sexual- ...

  15. Dengue Prevention

    Science.gov (United States)

    ... Address What's this? Submit What's this? Submit Button Prevention Recommend on Facebook Tweet Share Compartir This photograph ... medications to treat a dengue infection. This makes prevention the most important step, and prevention means avoiding ...

  16. Most advanced HTP fuel assembly design for EPR

    International Nuclear Information System (INIS)

    Francillon, Eric; Kiehlmann, Horst-Dieter

    2006-01-01

    End 2003, the Finnish electricity utility Teollisuuden Voima Oy (TVO) signed the contract for building an EPR in Olkiluoto (Finland). Mid 2004, the French electricity utility EDF selected an EPR to be built in France. In 2005, Framatome ANP, an AREVA and Siemens company, announced that they will be pursuing a design certification in the U.S. The EPR development is based on the latest PWR product lines of former Framatome (N4) and Siemens Nuklear (Konvoi). As an introductory part, different aspects of the EPR core characteristics connected to fuel assembly design are presented. It includes means of ensuring reactivity control like hybrid AIC/B4C control rod absorbers and gadolinium as burnable absorber integrated in fuel rods, and specific options for in-core instrumentation, such as Aeroball type instrumentation. Then the design requirements for the EPR fuel assembly are presented in term of very high burnup capacity, rod cladding and fuel assembly reliability. Framatome ANP fuel assembly product characteristics meeting these requirements are then described. EPR fuel assembly design characteristics benefit from the experience feedback of the latest fuel assembly products designed within Framatome ANP, leading to resistance to assembly deformation, high fuel rod restraint and prevention of handling hazards. EPR fuel assembly design features the best components composing the cornerstones of the upgraded family of fuel assemblies that FRAMATOME ANP proposes today. This family is based on a set of common characteristics and associated features, which include the HMP grid as bottom end spacer, the MONOBLOC guide tube and the Robust FUELGUARD as lower tie plate, the use of the M5 Alloy, as cladding and structure material. This fully re-crystallized, ternary Zr-Nb-O alloy produces radically improved in-reactor corrosion, very low hydrogen uptake and growth and an excellent creep behavior, which are described there. EPR fuel assembly description also includes fuel rod

  17. Nuclear reactor core assembly

    International Nuclear Information System (INIS)

    Baxi, C.B.

    1978-01-01

    The object of the present invention is to provide a fast reactor core assembly design for use with a fluid coolant such as liquid sodium or carbon monoxide incorporating a method of increasing the percentage of coolant flow though the blanket elements relative to the total coolant flow through the blanket and fuel elements during shutdown conditions without using moving parts. It is claimed that deterioration due to reactor radiation or temperature conditions is avoided and ready modification or replacement is possible. (U.K.)

  18. America's Assembly Line

    DEFF Research Database (Denmark)

    Nye, David Edwin

    A social history of the assembly line, invented in 1913. Both praised as a boon to consumers and as a curse for workers, it has been satirized, imitated, and celebrated for 100 years. It has inspired fiction, comedy, cafeteria layouts, and suburban housing. It transformed industrial labor...... and provoked strikes and union drives in the 1930s, but became a symbol of victory in the Second World War and Cold War. Reinvented by Japan as "lean production" and then increasingly automated after 1990, it remains a cornerstone of production but no longer employs many workers, even as it evolves toward...

  19. FORTRAN and ASSEMBLER programs

    International Nuclear Information System (INIS)

    Moldovan, N.

    1980-04-01

    A collection of programs written in FORTRAN and ASSEMBLER programming languages used in DOS-IBM is presented. The problems solved are of different sorts: linear programming, integration, matrix calculus, computation of absorbed doses in teletherapy, data sets (files) on magnetic tapes and disks, completion of DOS operating system etc. For reasons of space no details are given on the numerical methods or supplements and devices developed in order to achieve superior programs as to computation time and accuracy of result, although these might have been of use. All the programs in the collection have been checked up on an IBM 370/135 computer. (author)

  20. Purification and properties of a ribosomal casein kinase from rabbit reticulocytes

    DEFF Research Database (Denmark)

    Issinger, O G

    1977-01-01

    A casein kinase was isolated and purifed from rabbit reticulocytes. About 90% of the enzyme activity co-sedimented with the ribosomal fraction, whereas about 10% of the enzyme activity was found in the ribosome-free supernatant. Both casein kinases (the ribosome-bound enzyme as well as the free...... enzyme) showed identical activity and the same molecular weight. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis a single band of about 70 000 mol.wt. was observed. Sucrose-gradient analysis, however, showed that the enzyme activity sedimented with a s20,w of approx. 7.5S. This observation...... suggested that the casein kinase is a dimer composed of subunits of identical molecular weight. The enzyme utilizes GTP as well as ATP as a phosphoryl donor. It preferentially phosphorylates acidic proteins, in particular the model substrates casein and phosvitin. Casein kinase is cyclic AMP...