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Sample records for aminoacyl trna synthetases

  1. MD Simulations of tRNA and Aminoacyl-tRNA Synthetases: Dynamics, Folding, Binding, and Allostery

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    Rongzhong Li

    2015-07-01

    Full Text Available While tRNA and aminoacyl-tRNA synthetases are classes of biomolecules that have been extensively studied for decades, the finer details of how they carry out their fundamental biological functions in protein synthesis remain a challenge. Recent molecular dynamics (MD simulations are verifying experimental observations and providing new insight that cannot be addressed from experiments alone. Throughout the review, we briefly discuss important historical events to provide a context for how far the field has progressed over the past few decades. We then review the background of tRNA molecules, aminoacyl-tRNA synthetases, and current state of the art MD simulation techniques for those who may be unfamiliar with any of those fields. Recent MD simulations of tRNA dynamics and folding and of aminoacyl-tRNA synthetase dynamics and mechanistic characterizations are discussed. We highlight the recent successes and discuss how important questions can be addressed using current MD simulations techniques. We also outline several natural next steps for computational studies of AARS:tRNA complexes.

  2. The Roles of Compensatory Evolution and Constraint in Aminoacyl tRNA Synthetase Evolution.

    Science.gov (United States)

    Adrion, Jeffrey R; White, P Signe; Montooth, Kristi L

    2016-01-01

    Mitochondrial protein translation requires interactions between transfer RNAs encoded by the mitochondrial genome (mt-tRNAs) and mitochondrial aminoacyl tRNA synthetase proteins (mt-aaRS) encoded by the nuclear genome. It has been argued that animal mt-tRNAs have higher deleterious substitution rates relative to their nuclear-encoded counterparts, the cytoplasmic tRNAs (cyt-tRNAs). This dynamic predicts elevated rates of compensatory evolution of mt-aaRS that interact with mt-tRNAs, relative to aaRS that interact with cyt-tRNAs (cyt-aaRS). We find that mt-aaRS do evolve at significantly higher rates (exemplified by higher dN and dN/dS) relative to cyt-aaRS, across mammals, birds, and Drosophila. While this pattern supports a model of compensatory evolution, the level at which a gene is expressed is a more general predictor of protein evolutionary rate. We find that gene expression level explains 10-56% of the variance in aaRS dN/dS, and that cyt-aaRS are more highly expressed in addition to having lower dN/dS values relative to mt-aaRS, consistent with more highly expressed genes being more evolutionarily constrained. Furthermore, we find no evidence of positive selection acting on either class of aaRS protein, as would be expected under a model of compensatory evolution. Nevertheless, the signature of faster mt-aaRS evolution persists in mammalian, but not bird or Drosophila, lineages after controlling for gene expression, suggesting some additional effect of compensatory evolution for mammalian mt-aaRS. We conclude that gene expression is the strongest factor governing differential amino acid substitution rates in proteins interacting with mitochondrial versus cytoplasmic factors, with important differences in mt-aaRS molecular evolution among taxonomic groups. PMID:26416980

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

    Institute of Scientific and Technical Information of China (English)

    WEI; Haibin; QI; Ji; HAO; Bailin

    2004-01-01

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

  4. Molecular Evolution of Aminoacyl tRNA Synthetase Proteins in the Early History of Life

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    Fournier, Gregory P.; Andam, Cheryl P.; Alm, Eric J.; Gogarten, J. Peter

    2011-12-01

    Aminoacyl-tRNA synthetases (aaRS) consist of several families of functionally conserved proteins essential for translation and protein synthesis. Like nearly all components of the translation machinery, most aaRS families are universally distributed across cellular life, being inherited from the time of the Last Universal Common Ancestor (LUCA). However, unlike the rest of the translation machinery, aaRS have undergone numerous ancient horizontal gene transfers, with several independent events detected between domains, and some possibly involving lineages diverging before the time of LUCA. These transfers reveal the complexity of molecular evolution at this early time, and the chimeric nature of genomes within cells that gave rise to the major domains. Additionally, given the role of these protein families in defining the amino acids used for protein synthesis, sequence reconstruction of their pre-LUCA ancestors can reveal the evolutionary processes at work in the origin of the genetic code. In particular, sequence reconstructions of the paralog ancestors of isoleucyl- and valyl- RS provide strong empirical evidence that at least for this divergence, the genetic code did not co-evolve with the aaRSs; rather, both amino acids were already part of the genetic code before their cognate aaRSs diverged from their common ancestor. The implications of this observation for the early evolution of RNA-directed protein biosynthesis are discussed.

  5. Anticodon recognition in evolution: switching tRNA specificity of an aminoacyl-tRNA synthetase by site-directed peptide transplantation.

    Science.gov (United States)

    Brevet, Annie; Chen, Josiane; Commans, Stéphane; Lazennec, Christine; Blanquet, Sylvain; Plateau, Pierre

    2003-08-15

    The highly conserved aspartyl-, asparaginyl-, and lysyl-tRNA synthetases compose one subclass of aminoacyl-tRNA synthetases, called IIb. The three enzymes possess an OB-folded extension at their N terminus. The function of this extension is to specifically recognize the anticodon triplet of the tRNA. Three-dimensional models of bacterial aspartyl- and lysyl-tRNA synthetases complexed to tRNA indicate that a rigid scaffold of amino acid residues along the five beta-strands of the OB-fold accommodates the base U at the center of the anticodon. The binding of the adjacent anticodon bases occurs through interactions with a flexible loop joining strands 4 and 5 (L45). As a result, a switching of the specificity of lysyl-tRNA synthetase from tRNALys (anticodon UUU) toward tRNAAsp (GUC) could be attempted by transplanting the small loop L45 of aspartyl-tRNA synthetase inside lysyl-tRNA synthetase. Upon this transplantation, lysyl-tRNA synthetase loses its capacity to aminoacylate tRNALys. In exchange, the chimeric enzyme acquires the capacity to charge tRNAAsp with lysine. Upon giving the tRNAAsp substrate the discriminator base of tRNALys, the specificity shift is improved. The change of specificity was also established in vivo. Indeed, the transplanted lysyl-tRNA synthetase succeeds in suppressing a missense Lys --> Asp mutation inserted into the beta-lactamase gene. These results functionally establish that sequence variation in a small peptide region of subclass IIb aminoacyl-tRNA synthetases contributes to specification of nucleic acid recognition. Because this peptide element is not part of the core catalytic structure, it may have evolved independently of the active sites of these synthetases. PMID:12766171

  6. Methods and compositions for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs

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    Schultz, Peter G.; Wang, Lei; Anderson, John Christopher; Chin, Jason W.; Liu, David R.; Magliery, Thomas J.; Meggers, Eric L.; Mehl, Ryan Aaron; Pastrnak, Miro; Santoro, Stephen William; Zhang, Zhiwen

    2015-10-20

    This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

  7. Structure and Activity of an Aminoacyl-tRNA Synthetase that Charges tRNA with Nitro-Tryptophan

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    Buddha,M.; Crane, B.

    2005-01-01

    The most divergent of two tryptophanyl tRNA synthetases (TrpRS II) found in Deinococcus radiodurans interacts with a nitric oxide synthase protein that produces 4-nitro-tryptophan (4-NRP). TrpRS II efficiently charges transfer RNATrp with 4-NRP and 5-hydroxy-tryptophan (5-HRP). The crystal structures of TrpRS II bound to tryptophan and 5-HRP reveal residue substitutions that accommodate modified indoles. A class of auxiliary bacterial TrpRSs conserve this capacity to charge tRNA with nonstandard amino acids.

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

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    Iyer Lakshminarayan M

    2010-08-01

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

  9. Crystallogenesis in tRNA aminoacylation systems: how packing accounts for crystallization drawbacks with yeast aspartyl-tRNA synthetase

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    Sauter, C.; Lorber, B.; Théobald-Dietrich, A.; Giegé, R.

    2001-11-01

    Two active forms of homodimeric aspartyl-tRNA synthetase from Saccharomyces cerevisiae differing in length at their N-terminus crystallize in the same orthorhombic space group (P4 12 12) with identical cell parameters. Initial studies were hampered by the poor and anisotropic diffraction of the crystals of enzyme extracted from yeast cells. Isotropic diffraction at higher resolution was obtained when crystals were grown from an engineered protein deprived of its 70 N-terminal amino acids. The present work describes the packing contacts in crystals of the shortened protein whose structure was solved at 2.3 Å resolution. Each subunit of the enzyme develops two lattice interactions covering a surface of 670 Å 2, about 7-fold smaller than that of the interface between monomers. The smallest lattice interaction, covering 150 Å 2, brings the anticodon binding domain adjacent to the N-terminus of one monomer in contact with a loop from the active-site domain of a neighboring monomer. Modeling of the extension in the solvent channels shows that the 150 Å 2 intermolecular contact is perturbed in protein molecules possessing a floppy appendix while their second and larger 520 Å 2 contact area is unaffected. Altogether the packing organization explains the poor diffraction properties of the native enzyme crystals and the enhanced diffraction of the crystals of shortened synthetase.

  10. An archaeal tRNA-synthetase complex that enhances aminoacylation under extreme conditions

    DEFF Research Database (Denmark)

    Godinic-Mikulcic, Vlatka; Jaric, Jelena; Hausmann, Corinne D;

    2011-01-01

    Aminoacyl-tRNA synthetases (aaRSs) play an integral role in protein synthesis, functioning to attach the correct amino acid with its cognate tRNA molecule. AaRSs are known to associate into higher-order multi-aminoacyl-tRNA synthetase complexes (MSC) involved in archaeal and eukaryotic translation...... the catalytic efficiency of serine attachment to tRNA, but had no effect on the activity of MtArgRS. Further, the most pronounced improvements in the aminoacylation activity of MtSerRS induced by MtArgRS were observed under conditions of elevated temperature and osmolarity. These data indicate that......, although the precise biological role remains largely unknown. To gain further insights into archaeal MSCs, possible protein-protein interactions with the atypical Methanothermobacter thermautotrophicus seryl-tRNA synthetase (MtSerRS) were investigated. Yeast two-hybrid analysis revealed arginyl-tRNA...

  11. Evidence that the mitochondrial leucyl tRNA synthetase (LARS2) gene represents a novel type 2 diabetes susceptibility gene

    DEFF Research Database (Denmark)

    hart, Leen M; Hansen, Torben; Rietveld, Ingrid;

    2005-01-01

    Previously, we have shown that a mutation in the mitochondrial DNA-encoded tRNA(Leu(UUR)) gene is associated with type 2 diabetes. One of the consequences of this mutation is a reduced aminoacylation of tRNA(Leu(UUR)). In this study, we have examined whether variants in the leucyl tRNA synthetase...... first report of association between an aminoacyl tRNA synthetase gene and disease. Our results further highlight the important role of mitochondria in glucose homeostasis....

  12. In Silico Discovery of Aminoacyl-tRNA Synthetase Inhibitors

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    Yaxue Zhao

    2014-01-01

    Full Text Available Aminoacyl-tRNA synthetases (aaRSs are enzymes that catalyze the transfer of amino acids to their cognate tRNA. They play a pivotal role in protein synthesis and are essential for cell growth and survival. The aaRSs are one of the leading targets for development of antibiotic agents. In this review, we mainly focused on aaRS inhibitor discovery and development using in silico methods including virtual screening and structure-based drug design. These computational methods are relatively fast and cheap, and are proving to be of great benefit for the rational development of more potent aaRS inhibitors and other pharmaceutical agents that may usher in a much needed generation of new antibiotics.

  13. Phosphorylation of eukaryotic aminoacyl-tRNA synthetases

    International Nuclear Information System (INIS)

    The phosphorylation of the highly purified aminoacyl-tRNA synthetase complex from rabbit reticulocytes was examined. The synthetase complex contained, in addition to eight aminoacyl-tRNA synthetases, three unidentified proteins and was free of endogenous protein kinase activity. Incubation of the complex with casein kinase I in the presence of ATP resulted in the phosphorylation of four synthetases, the glutamyl-, isoleucyl-, methionyl-, and lysyl-tRNA synthetases. Phosphorylation by casein kinase I altered binding to tRNA-Sepharose such that the phosphorylated complex eluted at 190 mM NaCl instead of the 275 mM salt observed for the nonphosphorylated form. Phosphorylation by casein kinase I resulted in a significant inhibition of aminoacylation with the four synthetases; the activities of the nonphosphorylated synthetases were unchanged. One of the unidentified proteins in the complex (M/sub r/ 37,000) was also an excellent substrate for casein kinase I. A comparison of the properties and two-dimensional phosphopeptide pattern of this protein with that of casein kinase I suggest that the 37,000 dalton protein in the synthetase complex is an inactive form of casein kinase I. Two other protein kinases were shown to phosphorylate aminoacyl-tRNA synthetases in the complex. The phosphorylation of threonyl-tRNA synthetase was also investigated. Five aminoacyl-tRNA synthetases in the high molecular weight complex were shown to be phosphorylated in rabbit reticulocytes following labeling with (32P)orthophosphate

  14. Phosphorylation of eukaryotic aminoacyl-tRNA synthetases

    Energy Technology Data Exchange (ETDEWEB)

    Pendergast, A.M.

    1986-01-01

    The phosphorylation of the highly purified aminoacyl-tRNA synthetase complex from rabbit reticulocytes was examined. The synthetase complex contained, in addition to eight aminoacyl-tRNA synthetases, three unidentified proteins and was free of endogenous protein kinase activity. Incubation of the complex with casein kinase I in the presence of ATP resulted in the phosphorylation of four synthetases, the glutamyl-, isoleucyl-, methionyl-, and lysyl-tRNA synthetases. Phosphorylation by casein kinase I altered binding to tRNA-Sepharose such that the phosphorylated complex eluted at 190 mM NaCl instead of the 275 mM salt observed for the nonphosphorylated form. Phosphorylation by casein kinase I resulted in a significant inhibition of aminoacylation with the four synthetases; the activities of the nonphosphorylated synthetases were unchanged. One of the unidentified proteins in the complex (M/sub r/ 37,000) was also an excellent substrate for casein kinase I. A comparison of the properties and two-dimensional phosphopeptide pattern of this protein with that of casein kinase I suggest that the 37,000 dalton protein in the synthetase complex is an inactive form of casein kinase I. Two other protein kinases were shown to phosphorylate aminoacyl-tRNA synthetases in the complex. The phosphorylation of threonyl-tRNA synthetase was also investigated. Five aminoacyl-tRNA synthetases in the high molecular weight complex were shown to be phosphorylated in rabbit reticulocytes following labeling with (/sup 32/P)orthophosphate.

  15. Continuous spectrophotometric assay for aminoacyl-tRNA synthetases.

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    Chang, G G; Pan, F; Lin, Y H; Wang, H Y

    1984-11-01

    A simple, continuous assay for aminoacyl-tRNA synthetases utilizing a commercially available pyrophosphate assay reagent kit was demonstrated. The method coupled aminoacyl-tRNA synthetase activity with pyrophosphate-dependent fructose-6-phosphate kinase, aldolase, triosephosphate isomerase, and glycerophosphate dehydrogenase. PPi formation was correlated with the oxidation of NADH, and was monitored continuously by the decrease of absorbance at 340 nm. PMID:6099060

  16. Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases

    OpenAIRE

    Niehues, Sven; Bussmann, Julia; Steffes, Georg; Erdmann, Ines; Sun, Litao; Wagner, Marina; Wang, Guangxia; Koerdt, Sophia N.; Stum, Morgane; Rajbhandary, Uttam L.; Thomas, Ulrich; Aberle, Hermann; Burgess, Robert W.; Yang, Xiang-Lei; Dieterich, Daniela

    2014-01-01

    Dominant mutations in five tRNA synthetases cause Charcot–Marie–Tooth (CMT) neuropathy, suggesting that altered aminoacylation function underlies the disease. However, previous studies showed that loss of aminoacylation activity is not required to cause CMT. Here we present a Drosophila model for CMT with mutations in glycyl-tRNA synthetase (GARS). Expression of three CMT-mutant GARS proteins induces defects in motor performance and motor and sensory neuron morphology, and shortens lifespan. ...

  17. The aminoacyl-tRNA synthetases of Drosophila melanogaster

    OpenAIRE

    Lu, Jiongming; Marygold, Steven J; Gharib, Walid H; Suter, Beat

    2015-01-01

    Aminoacyl-tRNA synthetases (aaRSs) ligate amino acids to their cognate tRNAs, allowing them to decode the triplet code during translation. Through different mechanisms aaRSs also perform several non-canonical functions in transcription, translation, apoptosis, angiogenesis and inflammation. Drosophila has become a preferred system to model human diseases caused by mutations in aaRS, to dissect effects of reduced translation or non-canonical activities, and to study aminoacylation and translat...

  18. Recognition of tRNALeu by Aquifex aeolicus leucyl-tRNA synthetase during the aminoacylation and editing steps

    OpenAIRE

    Yao, Peng; Zhu, Bin; Jaeger, Sophie; Eriani, Gilbert; Wang, En-Duo

    2008-01-01

    Recognition of tRNA by the cognate aminoacyl-tRNA synthetase during translation is crucial to ensure the correct expression of the genetic code. To understand tRNALeu recognition sets and their evolution, the recognition of tRNALeu by the leucyl-tRNA synthetase (LeuRS) from the primitive hyperthermophilic bacterium Aquifex aeolicus was studied by RNA probing and mutagenesis. The results show that the base A73; the core structure of tRNA formed by the tertiary interactions U8–A14, G18–U55 and ...

  19. Recognition of tRNAs with a long variable arm by aminoacyl-tRNA synthetases

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    Tukalo M. A.

    2013-07-01

    Full Text Available In prokaryotic cells three tRNA species, tRNASer, tRNALeu and tRNATyr, possess a long variable arm of 11–20 nucleotides (type 2 tRNA rather than usual 4 or 5 nucleotides (type 1 tRNA. In this review we have summarized the results of our research on the structural basis for recognition and discrimination of type 2 tRNAs by Thermus thermophilus seryl-, tyrosyl- and leucyl-tRNA synthetases (SerRS, TyrRS and LeuRS obtained by X-ray crystallography and chemical probing tRNA in solution. Crystal structures are now known of all three aminoacyl-tRNA synthetases complexed with type 2 tRNAs and the different modes of tRNA recognition represented by these structures will be discussed. In particular, emphasis will be given to the results on recognition of characteristic shape of type 2 tRNAs by cognate synthetases. In tRNASer, tRNATyr and tRNALeu the orientation of the long variable arm with respect to the body of the tRNA is different and is controlled by different packing of the core. In the case of SerRS the N-terminal domain and in the case of TyrRS, the C-terminal domain, bind to the characteristic long variable arm of the cognate RNA, thus recognizing the unique shape of the tRNA. The core of T. thermophilus tRNALeu has several layers of unusual base-pairs, which are revealed by the crystal structure of tRNALeu complexed with T. thermophilus LeuRS and by probing a ligand-free tRNA by specific chemical reagents in solution. In the crystal structure of the LeuRS-tRNALeu complex the unique D-stem structure is recognized by the C-terminal domain of LeuRS and these data are in good agreement with those obtained in solution. LeuRS has canonical class I mode of tRNA recognition, approaching the tRNA acceptor stem from the D-stem and minor groove of the acceptor stem side. SerRS also has canonical class II mode of tRNA recognition and approaches tRNASer from opposite, variable stem and major groove of acceptor stem site. And finally, TyrRS in strong

  20. let-65 is cytoplasmic methionyl tRNA synthetase in C. elegans

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    Maha Z. Alriyami

    2014-12-01

    Full Text Available Cytoplasmic methionyl tRNA synthetase (MetRS is one of more than 20 cytoplasmic aminoacyl tRNA synthetase enzymes (ARS. This family of enzymes catalyzes a process fundamental for protein translation. Using a combination of genetic mapping, oligonucleotide array comparative genomic hybridization, and phenotypic correlation, we show that mutations in the essential gene, let-65, reside within the predicted Caenorhabditis elegans homologue of MetRS, which we have named mars-1. We demonstrate that the lethality associated with alleles of let-65 is fully rescued by a transgenic array that spans the mars-1 genomic region. Furthermore, sequence analysis reveals that six let-65 alleles lead to the alteration of highly conserved amino acids.

  1. A Multiple Aminoacyl-tRNA Synthetase Complex That Enhances tRNA-Aminoacylation in African Trypanosomes

    OpenAIRE

    Cestari, Igor; Kalidas, Savitha; Monnerat, Severine; Anupama, Atashi; Phillips, Margaret A.; Stuart, Kenneth

    2013-01-01

    The genes for all cytoplasmic and potentially all mitochondrial aminoacyl-tRNA synthetases (aaRSs) were identified, and all those tested by RNA interference were found to be essential for the growth of Trypanosoma brucei. Some of these enzymes were localized to the cytoplasm or mitochondrion, but most were dually localized to both cellular compartments. Cytoplasmic T. brucei aaRSs were organized in a multiprotein complex in both bloodstream and procyclic forms. The multiple aminoacyl-tRNA syn...

  2. Activity of aminoacyl-tRNA synthetases in experimental hyperthyroidism in muscle tissue of the rabbit

    International Nuclear Information System (INIS)

    In cardial and femoral muscles of rabbits specific activities of aminoacyl-tRNA synthetases for twenty amino acids were generally similar, namely the activities towards amino acids and their amides, leucine, isoleucine, histidine, tyrosine, proline and serine were considerably lower than towards the remaining amino acids. Specific activities of most aminoacyl-tRNA synthetases were higher in hyperthyroidism than in euthyreosis, and were higher in femoral muscle than in heart. The response to thyroxine treatment of individual aminoacyl-tRNA synthetases in both kinds of muscles varied with respect to most of the amino acids. (author). 22 refs, 1 fig., 1 tab

  3. Regulation of Angiogenesis by Aminoacyl-tRNA Synthetases

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    Adam C. Mirando

    2014-12-01

    Full Text Available In addition to their canonical roles in translation the aminoacyl-tRNA synthetases (ARSs have developed secondary functions over the course of evolution. Many of these activities are associated with cellular survival and nutritional stress responses essential for homeostatic processes in higher eukaryotes. In particular, six ARSs and one associated factor have documented functions in angiogenesis. However, despite their connection to this process, the ARSs are mechanistically distinct and exhibit a range of positive or negative effects on aspects of endothelial cell migration, proliferation, and survival. This variability is achieved through the appearance of appended domains and interplay with inflammatory pathways not found in prokaryotic systems. Complete knowledge of the non-canonical functions of ARSs is necessary to understand the mechanisms underlying the physiological regulation of angiogenesis.

  4. The aminoacyl-tRNA synthetases of Drosophila melanogaster.

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    Lu, Jiongming; Marygold, Steven J; Gharib, Walid H; Suter, Beat

    2015-01-01

    Aminoacyl-tRNA synthetases (aaRSs) ligate amino acids to their cognate tRNAs, allowing them to decode the triplet code during translation. Through different mechanisms aaRSs also perform several non-canonical functions in transcription, translation, apoptosis, angiogenesis and inflammation. Drosophila has become a preferred system to model human diseases caused by mutations in aaRS genes, to dissect effects of reduced translation or non-canonical activities, and to study aminoacylation and translational fidelity. However, the lack of a systematic annotation of this gene family has hampered such studies. Here, we report the identification of the entire set of aaRS genes in the fly genome and we predict their roles based on experimental evidence and/or orthology. Further, we propose a new, systematic and logical nomenclature for aaRSs. We also review the research conducted on Drosophila aaRSs to date. Together, our work provides the foundation for further research in the fly aaRS field. PMID:26761199

  5. Structural Insights into the Polyphyletic Origins of Glycyl tRNA Synthetases*♦

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    Valencia-Sánchez, Marco Igor; Rodríguez-Hernández, Annia; Ferreira, Ruben; Santamaría-Suárez, Hugo Aníbal; Arciniega, Marcelino; Dock-Bregeon, Anne-Catherine; Moras, Dino; Beinsteiner, Brice; Brieba, Luis G.; Grøtli, Morten

    2016-01-01

    Glycyl tRNA synthetase (GlyRS) provides a unique case among class II aminoacyl tRNA synthetases, with two clearly widespread types of enzymes: a dimeric (α2) species present in some bacteria, archaea, and eukaryotes; and a heterotetrameric form (α2β2) present in most bacteria. Although the differences between both types of GlyRS at the anticodon binding domain level are evident, the extent and implications of the variations in the catalytic domain have not been described, and it is unclear whether the mechanism of amino acid recognition is also dissimilar. Here, we show that the α-subunit of the α2β2 GlyRS from the bacterium Aquifex aeolicus is able to perform the first step of the aminoacylation reaction, which involves the activation of the amino acid with ATP. The crystal structure of the α-subunit in the complex with an analog of glycyl adenylate at 2.8 Å resolution presents a conformational arrangement that properly positions the cognate amino acid. This work shows that glycine is recognized by a subset of different residues in the two types of GlyRS. A structural and sequence analysis of class II catalytic domains shows that bacterial GlyRS is closely related to alanyl tRNA synthetase, which led us to define a new subclassification of these ancient enzymes and to propose an evolutionary path of α2β2 GlyRS, convergent with α2 GlyRS and divergent from AlaRS, thus providing a possible explanation for the puzzling existence of two proteins sharing the same fold and function but not a common ancestor. PMID:27226617

  6. Structural Insights into the Polyphyletic Origins of Glycyl tRNA Synthetases.

    Science.gov (United States)

    Valencia-Sánchez, Marco Igor; Rodríguez-Hernández, Annia; Ferreira, Ruben; Santamaría-Suárez, Hugo Aníbal; Arciniega, Marcelino; Dock-Bregeon, Anne-Catherine; Moras, Dino; Beinsteiner, Brice; Mertens, Haydyn; Svergun, Dmitri; Brieba, Luis G; Grøtli, Morten; Torres-Larios, Alfredo

    2016-07-01

    Glycyl tRNA synthetase (GlyRS) provides a unique case among class II aminoacyl tRNA synthetases, with two clearly widespread types of enzymes: a dimeric (α2) species present in some bacteria, archaea, and eukaryotes; and a heterotetrameric form (α2β2) present in most bacteria. Although the differences between both types of GlyRS at the anticodon binding domain level are evident, the extent and implications of the variations in the catalytic domain have not been described, and it is unclear whether the mechanism of amino acid recognition is also dissimilar. Here, we show that the α-subunit of the α2β2 GlyRS from the bacterium Aquifex aeolicus is able to perform the first step of the aminoacylation reaction, which involves the activation of the amino acid with ATP. The crystal structure of the α-subunit in the complex with an analog of glycyl adenylate at 2.8 Å resolution presents a conformational arrangement that properly positions the cognate amino acid. This work shows that glycine is recognized by a subset of different residues in the two types of GlyRS. A structural and sequence analysis of class II catalytic domains shows that bacterial GlyRS is closely related to alanyl tRNA synthetase, which led us to define a new subclassification of these ancient enzymes and to propose an evolutionary path of α2β2 GlyRS, convergent with α2 GlyRS and divergent from AlaRS, thus providing a possible explanation for the puzzling existence of two proteins sharing the same fold and function but not a common ancestor. PMID:27226617

  7. A multiple aminoacyl-tRNA synthetase complex that enhances tRNA-aminoacylation in African trypanosomes.

    Science.gov (United States)

    Cestari, Igor; Kalidas, Savitha; Monnerat, Severine; Anupama, Atashi; Phillips, Margaret A; Stuart, Kenneth

    2013-12-01

    The genes for all cytoplasmic and potentially all mitochondrial aminoacyl-tRNA synthetases (aaRSs) were identified, and all those tested by RNA interference were found to be essential for the growth of Trypanosoma brucei. Some of these enzymes were localized to the cytoplasm or mitochondrion, but most were dually localized to both cellular compartments. Cytoplasmic T. brucei aaRSs were organized in a multiprotein complex in both bloodstream and procyclic forms. The multiple aminoacyl-tRNA synthetase (MARS) complex contained at least six aaRS enzymes and three additional non-aaRS proteins. Steady-state kinetic studies showed that association in the MARS complex enhances tRNA-aminoacylation efficiency, which is in part dependent on a MARS complex-associated protein (MCP), named MCP2, that binds tRNAs and increases their aminoacylation by the complex. Conditional repression of MCP2 in T. brucei bloodstream forms resulted in reduced parasite growth and infectivity in mice. Thus, association in a MARS complex enhances tRNA-aminoacylation and contributes to parasite fitness. The MARS complex may be part of a cellular regulatory system and a target for drug development. PMID:24126051

  8. Modulation of Aminoacylation and Editing Properties of Leucyl-tRNA Synthetase by a Conserved Structural Module.

    Science.gov (United States)

    Yan, Wei; Ye, Qing; Tan, Min; Chen, Xi; Eriani, Gilbert; Wang, En-Duo

    2015-05-01

    A conserved structural module following the KMSKS catalytic loop exhibits α-α-β-α topology in class Ia and Ib aminoacyl-tRNA synthetases. However, the function of this domain has received little attention. Here, we describe the effect this module has on the aminoacylation and editing capacities of leucyl-tRNA synthetases (LeuRSs) by characterizing the key residues from various species. Mutation of highly conserved basic residues on the third α-helix of this domain impairs the affinity of LeuRS for the anticodon stem of tRNA(Leu), which decreases both aminoacylation and editing activities. Two glycine residues on this α-helix contribute to flexibility, leucine activation, and editing of LeuRS from Escherichia coli (EcLeuRS). Acidic residues on the β-strand enhance the editing activity of EcLeuRS and sense the size of the tRNA(Leu) D-loop. Incorporation of these residues stimulates the tRNA-dependent editing activity of the chimeric minimalist enzyme Mycoplasma mobile LeuRS fused to the connective polypeptide 1 editing domain and leucine-specific domain from EcLeuRS. Together, these results reveal the stem contact-fold to be a functional as well as a structural linker between the catalytic site and the tRNA binding domain. Sequence comparison of the EcLeuRS stem contact-fold domain with editing-deficient enzymes suggests that key residues of this module have evolved an adaptive strategy to follow the editing functions of LeuRS. PMID:25817995

  9. A naturally occurring nonapeptide functionally compensates for the CP1 domain of leucyl-tRNA synthetase to modulate aminoacylation activity.

    Science.gov (United States)

    Tan, Min; Yan, Wei; Liu, Ru-Juan; Wang, Meng; Chen, Xin; Zhou, Xiao-Long; Wang, En-Duo

    2012-04-15

    aaRSs (aminoacyl-tRNA synthetases) establish the rules of the genetic code by catalysing the formation of aminoacyl-tRNA. The quality control for aminoacylation is achieved by editing activity, which is usually carried out by a discrete editing domain. For LeuRS (leucyl-tRNA synthetase), the CP1 (connective peptide 1) domain is the editing domain responsible for hydrolysing mischarged tRNA. The CP1 domain is universally present in LeuRSs, except MmLeuRS (Mycoplasma mobile LeuRS). The substitute of CP1 in MmLeuRS is a nonapeptide (MmLinker). In the present study, we show that the MmLinker, which is critical for the aminoacylation activity of MmLeuRS, could confer remarkable tRNA-charging activity on the inactive CP1-deleted LeuRS from Escherichia coli (EcLeuRS) and Aquifex aeolicus (AaLeuRS). Furthermore, CP1 from EcLeuRS could functionally compensate for the MmLinker and endow MmLeuRS with post-transfer editing capability. These investigations provide a mechanistic framework for the modular construction of aaRSs and their co-ordination to achieve catalytic efficiency and fidelity. These results also show that the pre-transfer editing function of LeuRS originates from its conserved synthetic domain and shed light on future study of the mechanism. PMID:22292813

  10. Genetic Validation of Aminoacyl-tRNA Synthetases as Drug Targets in Trypanosoma brucei

    OpenAIRE

    Kalidas, Savitha; Cestari, Igor; Monnerat, Severine; Li, Qiong; Regmi, Sandesh; Hasle, Nicholas; Labaied, Mehdi; Parsons, Marilyn; Stuart, Kenneth; Phillips, Margaret A.

    2014-01-01

    Human African trypanosomiasis (HAT) is an important public health threat in sub-Saharan Africa. Current drugs are unsatisfactory, and new drugs are being sought. Few validated enzyme targets are available to support drug discovery efforts, so our goal was to obtain essentiality data on genes with proven utility as drug targets. Aminoacyl-tRNA synthetases (aaRSs) are known drug targets for bacterial and fungal pathogens and are required for protein synthesis. Here we survey the essentiality of...

  11. Aminoacyl-tRNA Synthetases, the Genetic Code, and the Evolutionary Process

    OpenAIRE

    Woese, Carl R.; Olsen, Gary J; Ibba, Michael; Söll, Dieter

    2000-01-01

    The aminoacyl-tRNA synthetases (AARSs) and their relationship to the genetic code are examined from the evolutionary perspective. Despite a loose correlation between codon assignments and AARS evolutionary relationships, the code is far too highly structured to have been ordered merely through the evolutionary wanderings of these enzymes. Nevertheless, the AARSs are very informative about the evolutionary process. Examination of the phylogenetic trees for each of the AARSs reveals the followi...

  12. Yeast mitochondrial threonyl-tRNA synthetase recognizes tRNA isoacceptors by distinct mechanisms and promotes CUN codon reassignment

    Energy Technology Data Exchange (ETDEWEB)

    Ling, Jiqiang; Peterson, Kaitlyn M.; Simonovic, Ivana; Cho, Chris; Soll, Dieter; Simonovic, Miljan (Yale); (UIC)

    2014-03-12

    Aminoacyl-tRNA synthetases (aaRSs) ensure faithful translation of mRNA into protein by coupling an amino acid to a set of tRNAs with conserved anticodon sequences. Here, we show that in mitochondria of Saccharomyces cerevisiae, a single aaRS (MST1) recognizes and aminoacylates two natural tRNAs that contain anticodon loops of different size and sequence. Besides a regular ?? with a threonine (Thr) anticodon, MST1 also recognizes an unusual ??, which contains an enlarged anticodon loop and an anticodon triplet that reassigns the CUN codons from leucine to threonine. Our data show that MST1 recognizes the anticodon loop in both tRNAs, but employs distinct recognition mechanisms. The size but not the sequence of the anticodon loop is critical for ?? recognition, whereas the anticodon sequence is essential for aminoacylation of ??. The crystal structure of MST1 reveals that, while lacking the N-terminal editing domain, the enzyme closely resembles the bacterial threonyl-tRNA synthetase (ThrRS). A detailed structural comparison with Escherichia coli ThrRS, which is unable to aminoacylate ??, reveals differences in the anticodon-binding domain that probably allow recognition of the distinct anticodon loops. Finally, our mutational and modeling analyses identify the structural elements in MST1 (e.g., helix {alpha}11) that define tRNA selectivity. Thus, MTS1 exemplifies that a single aaRS can recognize completely divergent anticodon loops of natural isoacceptor tRNAs and that in doing so it facilitates the reassignment of the genetic code in yeast mitochondria.

  13. Genetic validation of aminoacyl-tRNA synthetases as drug targets in Trypanosoma brucei.

    Science.gov (United States)

    Kalidas, Savitha; Cestari, Igor; Monnerat, Severine; Li, Qiong; Regmi, Sandesh; Hasle, Nicholas; Labaied, Mehdi; Parsons, Marilyn; Stuart, Kenneth; Phillips, Margaret A

    2014-04-01

    Human African trypanosomiasis (HAT) is an important public health threat in sub-Saharan Africa. Current drugs are unsatisfactory, and new drugs are being sought. Few validated enzyme targets are available to support drug discovery efforts, so our goal was to obtain essentiality data on genes with proven utility as drug targets. Aminoacyl-tRNA synthetases (aaRSs) are known drug targets for bacterial and fungal pathogens and are required for protein synthesis. Here we survey the essentiality of eight Trypanosoma brucei aaRSs by RNA interference (RNAi) gene expression knockdown, covering an enzyme from each major aaRS class: valyl-tRNA synthetase (ValRS) (class Ia), tryptophanyl-tRNA synthetase (TrpRS-1) (class Ib), arginyl-tRNA synthetase (ArgRS) (class Ic), glutamyl-tRNA synthetase (GluRS) (class 1c), threonyl-tRNA synthetase (ThrRS) (class IIa), asparaginyl-tRNA synthetase (AsnRS) (class IIb), and phenylalanyl-tRNA synthetase (α and β) (PheRS) (class IIc). Knockdown of mRNA encoding these enzymes in T. brucei mammalian stage parasites showed that all were essential for parasite growth and survival in vitro. The reduced expression resulted in growth, morphological, cell cycle, and DNA content abnormalities. ThrRS was characterized in greater detail, showing that the purified recombinant enzyme displayed ThrRS activity and that the protein localized to both the cytosol and mitochondrion. Borrelidin, a known inhibitor of ThrRS, was an inhibitor of T. brucei ThrRS and showed antitrypanosomal activity. The data show that aaRSs are essential for T. brucei survival and are likely to be excellent targets for drug discovery efforts. PMID:24562907

  14. A novel therapeutic target for peripheral nerve injury-related diseases: aminoacyl-tRNA synthetases

    Directory of Open Access Journals (Sweden)

    Byung Sun Park

    2015-01-01

    Full Text Available Aminoacyl-tRNA synthetases (AminoARSs are essential enzymes that perform the first step of protein synthesis. Beyond their original roles, AminoARSs possess non-canonical functions, such as cell cycle regulation and signal transduction. Therefore, AminoARSs represent a powerful pharmaceutical target if their non-canonical functions can be controlled. Using AminoARSs-specific primers, we screened mRNA expression in the spinal cord dorsal horn of rats with peripheral nerve injury created by sciatic nerve axotomy. Of 20 AminoARSs, we found that phenylalanyl-tRNA synthetase beta chain (FARSB, isoleucyl-tRNA synthetase (IARS and methionyl-tRNA synthetase (MARS mRNA expression was increased in spinal dorsal horn neurons on the injured side, but not in glial cells. These findings suggest the possibility that FARSB, IARS and MARS, as a neurotransmitter, may transfer abnormal sensory signals after peripheral nerve damage and become a new target for drug treatment.

  15. Compositions of orthogonal lysyl-tRNA and aminoacyl-tRNA synthetase pairs and uses thereof

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, J. Christopher; Wu, Ning; Santoro, Stephen; Schultz, Peter G

    2014-03-11

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal lysyl-tRNAs, orthogonal lysyl-aminoacyl-tRNA synthetases, and orthogonal pairs of lysyl-tRNAs/synthetases, which incorporate homoglutamines into proteins are provided in response to a four base codon. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with homoglutamines using these orthogonal pairs.

  16. Calcium regulates the expression of a Dictyostelium discoideum asparaginyl tRNA synthetase gene

    Indian Academy of Sciences (India)

    Jyoti K Jaiswal; Vidyanand Nanjundiah

    2003-12-01

    In a screen for calcium-regulated gene expression during growth and development of Dictyostelium discoideum we have identified an asparaginyl tRNA synthetase (ddAsnRS) gene, the second tRNA synthetase gene identified in this organism. The ddAsnRS gene shows many unique features. One, it is repressed by lowering cellular calcium, making it the first known calcium-regulated tRNA synthetase. Two, despite the calcium-dependence, its expression is unaltered during the cell cycle, making this the first D. discoideum gene to show a calcium-dependent but cell cycle phase-independent expression. Finally, the N-terminal domain of the predicted ddAsnRS protein shows higher sequence similarity to Glutaminyl tRNA synthetases than to other Asn tRNA synthetases. These unique features of the AsnRS from this primitive eukaryote not only point to a novel mechanism regulating the components of translation machinery and gene expression by calcium, but also hint at a link between the evolution of GlnRS and AsnRS in eukaryotes.

  17. Phosphorylation of five aminoacyl-tRNA synthetases in reticulocytes and identification of the protein kinases phosphorylating threonyl-tRNA synthetase from rat liver

    International Nuclear Information System (INIS)

    Five aminoacyl-tRNA synthetases in the high molecular weight complex were phosphorylated in rabbit reticulocytes following labeling with 32P. The five synthetases phosphorylated were the glutamyl-, glutaminyl-, lysyl-, aspartyl- and methionyl-tRNA synthetases. In addition, a 37,000 dalton protein, associated with the synthetase complex and tentatively identified as casein kinase I, was also phosphorylated in intact cells. Phosphoamino acid analysis of the proteins indicated all of the phosphate was on seryl residues. Incubation of reticulocytes with 32P in the presence of 8-bromo-cAMP and o, the 3-isobutyl-1-methylxanthine resulted in a six-fold increase in phosphorylation of the glutaminyl-tRNA synthetase, a two-fold increase in phosphorylation of the aspartyl-tRNA synthetase, and a 50 to 60% decrease in phosphorylation of the glutamyl-, methionyl- and lysyl-tRNA synthetases and the M/sub r/ 37,000 protein. When the site(s) on the glutaminyl-tRNA synthetase phosphorylated in response to 8-bromo-cAMP was analyzed by two-dimensional tryptic phosphopeptide mapping, a single phosphopeptide was observed which was identical to that obtained in vitro upon phosphorylation with the cAMP-dependent protein kinase. Also, the authors identify here, the protein kinases phosphorylating threonyl-tRNA synthetase from rat liver. They are protease activated kinase I, the cAMP-dependent protein kinase and protein kinase C

  18. Phosphorylation of five aminoacyl-tRNA synthetases in reticulocytes and identification of the protein kinases phosphorylating threonyl-tRNA synthetase from rat liver

    Energy Technology Data Exchange (ETDEWEB)

    Pendergast, A.M.; Traugh, J.A.

    1986-05-01

    Five aminoacyl-tRNA synthetases in the high molecular weight complex were phosphorylated in rabbit reticulocytes following labeling with /sup 32/P. The five synthetases phosphorylated were the glutamyl-, glutaminyl-, lysyl-, aspartyl- and methionyl-tRNA synthetases. In addition, a 37,000 dalton protein, associated with the synthetase complex and tentatively identified as casein kinase I, was also phosphorylated in intact cells. Phosphoamino acid analysis of the proteins indicated all of the phosphate was on seryl residues. Incubation of reticulocytes with /sup 32/P in the presence of 8-bromo-cAMP and o, the 3-isobutyl-1-methylxanthine resulted in a six-fold increase in phosphorylation of the glutaminyl-tRNA synthetase, a two-fold increase in phosphorylation of the aspartyl-tRNA synthetase, and a 50 to 60% decrease in phosphorylation of the glutamyl-, methionyl- and lysyl-tRNA synthetases and the M/sub r/ 37,000 protein. When the site(s) on the glutaminyl-tRNA synthetase phosphorylated in response to 8-bromo-cAMP was analyzed by two-dimensional tryptic phosphopeptide mapping, a single phosphopeptide was observed which was identical to that obtained in vitro upon phosphorylation with the cAMP-dependent protein kinase. Also, the authors identify here, the protein kinases phosphorylating threonyl-tRNA synthetase from rat liver. They are protease activated kinase I, the cAMP-dependent protein kinase and protein kinase C.

  19. Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivo.

    Science.gov (United States)

    Novoa, Eva Maria; Camacho, Noelia; Tor, Anna; Wilkinson, Barrie; Moss, Steven; Marín-García, Patricia; Azcárate, Isabel G; Bautista, José M; Mirando, Adam C; Francklyn, Christopher S; Varon, Sònia; Royo, Miriam; Cortés, Alfred; Ribas de Pouplana, Lluís

    2014-12-23

    Malaria remains a major global health problem. Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Here we explore the potential of the aminoacyl-tRNA synthetase (ARS) family as a source of antimalarial drug targets. First, a battery of known and novel ARS inhibitors was tested against Plasmodium falciparum cultures, and their activities were compared. Borrelidin, a natural inhibitor of threonyl-tRNA synthetase (ThrRS), stands out for its potent antimalarial effect. However, it also inhibits human ThrRS and is highly toxic to human cells. To circumvent this problem, we tested a library of bioengineered and semisynthetic borrelidin analogs for their antimalarial activity and toxicity. We found that some analogs effectively lose their toxicity against human cells while retaining a potent antiparasitic activity both in vitro and in vivo and cleared malaria from Plasmodium yoelii-infected mice, resulting in 100% mice survival rates. Our work identifies borrelidin analogs as potent, selective, and unexplored scaffolds that efficiently clear malaria both in vitro and in vivo. PMID:25489076

  20. Evolutionary Limitation and Opportunities for Developing tRNA Synthetase Inhibitors with 5-Binding-Mode Classification

    Directory of Open Access Journals (Sweden)

    Pengfei Fang

    2015-12-01

    Full Text Available Aminoacyl-tRNA synthetases (aaRSs are enzymes that catalyze the transfer of amino acids to their cognate tRNAs as building blocks for translation. Each of the aaRS families plays a pivotal role in protein biosynthesis and is indispensable for cell growth and survival. In addition, aaRSs in higher species have evolved important non-translational functions. These translational and non-translational functions of aaRS are attractive for developing antibacterial, antifungal, and antiparasitic agents and for treating other human diseases. The interplay between amino acids, tRNA, ATP, EF-Tu and non-canonical binding partners, had shaped each family with distinct pattern of key sites for regulation, with characters varying among species across the path of evolution. These sporadic variations in the aaRSs offer great opportunity to target these essential enzymes for therapy. Up to this day, growing numbers of aaRS inhibitors have been discovered and developed. Here, we summarize the latest developments and structural studies of aaRS inhibitors, and classify them with distinct binding modes into five categories.

  1. A genomic glimpse of aminoacyl-tRNA synthetases in malaria parasite Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Santoni Daniele

    2009-12-01

    Full Text Available Abstract Background Plasmodium parasites are causative agents of malaria which affects >500 million people and claims ~2 million lives annually. The completion of Plasmodium genome sequencing and availability of PlasmoDB database has provided a platform for systematic study of parasite genome. Aminoacyl-tRNA synthetases (aaRSs are pivotal enzymes for protein translation and other vital cellular processes. We report an extensive analysis of the Plasmodium falciparum genome to identify and classify aaRSs in this organism. Results Using various computational and bioinformatics tools, we have identified 37 aaRSs in P. falciparum. Our key observations are: (i fraction of proteome dedicated to aaRSs in P. falciparum is very high compared to many other organisms; (ii 23 out of 37 Pf-aaRS sequences contain signal peptides possibly directing them to different cellular organelles; (iii expression profiles of Pf-aaRSs vary considerably at various life cycle stages of the parasite; (iv several PfaaRSs posses very unusual domain architectures; (v phylogenetic analyses reveal evolutionary relatedness of several parasite aaRSs to bacterial and plants aaRSs; (vi three dimensional structural modelling has provided insights which could be exploited in inhibitor discovery against parasite aaRSs. Conclusion We have identified 37 Pf-aaRSs based on our bioinformatics analysis. Our data reveal several unique attributes in this protein family. We have annotated all 37 Pf-aaRSs based on predicted localization, phylogenetics, domain architectures and their overall protein expression profiles. The sets of distinct features elaborated in this work will provide a platform for experimental dissection of this family of enzymes, possibly for the discovery of novel drugs against malaria.

  2. Two distinct domains of the β subunit of Aquifex aeolicus leucyl-tRNA synthetase are involved in tRNA binding as revealed by a three-hybrid selection

    OpenAIRE

    Zheng, Yong-Gang; Wei, Hui; Ling, Chen; Martin, Franck; Eriani, Gilbert; Wang, En-Duo

    2004-01-01

    The Aquifex aeolicus αβ-LeuRS is the only known heterodimeric class Ia aminoacyl-tRNA synthetase. In this study, we investigated the function of the β subunit which is believed to bind tRNALeu. A yeast three-hybrid system was constructed on the basis of the interaction of the β subunit with its cognate tRNALeu. Then, seven mutated β subunits exhibiting impaired tRNA binding capacities were selected out from a randomly mutated library. Two mutations were identified in the class Ia-helix-bundle...

  3. Probing the global and local dynamics of aminoacyl-tRNA synthetases using all-atom and coarse-grained simulations

    OpenAIRE

    Strom, Alexander M.; Fehling, Samuel C.; Bhattacharyya, Sudeep; Hati, Sanchita

    2014-01-01

    Coarse-grained simulations have emerged as invaluable tools for studying conformational changes in biomolecules. To evaluate the effectiveness of computationally inexpensive coarse-grained models in studying global and local dynamics of large protein systems like aminoacyl-tRNA synthetases, we have performed coarse-grained normal mode analysis, as well as principle component analysis on trajectories of all-atom and coarse-grained molecular dynamics simulations for three aminoacyl-tRNA synthet...

  4. Evidence that the mitochondrial leucyl tRNA synthetase (LARS2) gene represents a novel type 2 diabetes susceptibility gene

    NARCIS (Netherlands)

    L.M. 't Hart (Leen); H.A.P. Pols (Huib); T. Hansen (Torben); I. Rietveld (Ingrid); J.M. Dekker (Jacqueline); J.A. Maassen (Johannes); M.G.A.A.M. Nijpels (Giel); G.M.C. Janssen (George); P.P. Arp (Pascal); R.J. Heine (Robert); A.G. Uitterlinden (André); T. Jorgensen (Torben); C.M. van Duijn (Cock); K. Borch-Johnsen; O. Pedersen (Oluf)

    2005-01-01

    textabstractPreviously, we have shown that a mutation in the mitochondrial DNA-encoded tRNA(Leu(UUR)) gene is associated with type 2 diabetes. One of the consequences of this mutation is a reduced aminoacylation of tRNA(Leu(UUR)). In this study, we have examined whether variants in the leucyl tRNA s

  5. Rational design and directed evolution of a bacterial-type glutaminyl-tRNA synthetase precursor

    OpenAIRE

    Guo, Li-Tao; Helgadóttir, Sunna; Söll, Dieter; Ling, Jiqiang

    2012-01-01

    Protein biosynthesis requires aminoacyl-transfer RNA (tRNA) synthetases to provide aminoacyl-tRNA substrates for the ribosome. Most bacteria and all archaea lack a glutaminyl-tRNA synthetase (GlnRS); instead, Gln-tRNAGln is produced via an indirect pathway: a glutamyl-tRNA synthetase (GluRS) first attaches glutamate (Glu) to tRNAGln, and an amidotransferase converts Glu-tRNAGln to Gln-tRNAGln. The human pathogen Helicobacter pylori encodes two GluRS enzymes, with GluRS2 specifically aminoacyl...

  6. The tRNA synthetase paralog PoxA modifies elongation factor-P with (R)-ß-lysine

    DEFF Research Database (Denmark)

    Roy, Hervé; Zou, S Betty; Bullwinkle, Tammy J;

    2011-01-01

    The lysyl-tRNA synthetase paralog PoxA modifies elongation factor P (EF-P) with a-lysine at low efficiency. Cell-free extracts containing non-a-lysine substrates of PoxA modified EF-P with a change in mass consistent with addition of ß-lysine, a substrate also predicted by genomic analyses. EF......-P was efficiently functionally modified with (R)-ß-lysine but not (S)-ß-lysine or genetically encoded a-amino acids, indicating that PoxA has evolved an activity orthogonal to that of the canonical aminoacyl-tRNA synthetases....

  7. Phosphorolytic activity of Escherichia coli glycyl-tRNA synthetase towards its cognate aminoacyl adenylate detected by 31P-NMR spectroscopy and thin-layer chromatography

    DEFF Research Database (Denmark)

    Led, Jens Jørgen; Switon, Werner K.; Jensen, Kaj Frank

    1983-01-01

    The catalytic activity of highly purified Escherichia coli glycyl-tRNA synthetase has been studied by 31P-NMR spectroscopy and thin-layer chromatography on poly(ethyleneimine)-cellulose. It was found that this synthetase, besides the activation of its cognate amino acid and the syntheses of...... adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')triphospho(5')adenosine (Ap3A), also catalyzes the formation of ADP from inorganic phosphate and the enzyme-bound glycyl adenylate. Accordingly it was shown that E. coli glycyl-tRNA synthetase, in the presence of inorganic phosphate, glycine...... remaining catalytic activities of aminoacyl-tRNA synthetases is discussed, as well as the biological significance of the reaction....

  8. An example of non-conservation of oligomeric structure in prokaryotic aminoacyl-tRNA synthetases. Biochemical and structural properties of glycyl-tRNA synthetase from Thermus thermophilus.

    Science.gov (United States)

    Mazauric, M H; Reinbolt, J; Lorber, B; Ebel, C; Keith, G; Giegé, R; Kern, D

    1996-11-01

    Glycyl-tRNA synthetase (Gly-tRNA synthetase) from Thermus thermophilus was purified to homogeneity and with high yield using a five-step purification procedure in amounts sufficient to solve its crystallographic structure [Logan, D.T., Mazauric, M.-H., Kern, D. & Moras, D. (1995) EMBO J. 14, 4156-4167]. Molecular-mass determinations of the native and denatured protein indicate an oligomeric structure of the alpha 2 type consistent with that found for eukaryotic Gly-tRNA synthetases (yeast and Bombyx mori), but different from that of Gly-tRNA synthetases from mesophilic prokaryotes (Escherichia coli and Bacillus brevis) which are alpha 2 beta 2 tetramers. N-terminal sequencing of the polypeptide chain reveals significant identity, reaching 50% with those of the eukaryotic enzymes (B. mori, Homo sapiens, yeast and Caenorhabditis elegans) but no significant identity was found with both alpha and beta chains of the prokaryotic enzymes (E. coli, Haemophilus influenzae and Coxiella burnetii) albeit the enzyme is deprived of the N-terminal extension characterizing eukaryotic synthetases. Thus, the thermophilic Gly-tRNA synthetase combines strong structural homologies of eukaryotic Gly-tRNA synthetases with a feature of prokaryotic synthetases. Heat-stability measurements show that this synthetase keeps its ATP-PPi exchange and aminoacylation activities up to 70 degrees C. Glycyladenylate strongly protects the enzyme against thermal inactivation at higher temperatures. Unexpectedly, tRNA(Gly) does not induce protection. Cross-aminoacylations reveal that the thermophilic Gly-tRNA synthetase charges heterologous E. coli tRNA(gly(GCC)) and tRNA(Gly(GCC)) and yeast tRNA(Gly(GCC)) as efficiently as T. thermophilus tRNA(Gly). All these aminoacylation reactions are characterized by similar activation energies as deduced from Arrhenius plots. Therefore, contrary to the E. coli and H. sapiens Gly-tRNA synthetases, the prokaryotic thermophilic enzyme does not possess a strict

  9. N-alkylated aminoacyl sulfamoyladenosines as potential inhibitors of aminoacylation reactions and microcin C analogues containing D-amino acids.

    Directory of Open Access Journals (Sweden)

    Gaston H Vondenhoff

    Full Text Available Microcin C analogues were recently envisaged as important compounds for the development of novel antibiotics. Two issues that may pose problems to these potential antibiotics are possible acquisition of resistance through acetylation and in vivo instability of the peptide chain. N-methylated aminoacyl sulfamoyladenosines were synthesized to investigate their potential as aminoacyl tRNA synthetase inhibitors and to establish whether these N-alkylated analogues would escape the natural inactivation mechanism via acetylation of the alpha amine. It was shown however, that these compounds are not able to effectively inhibit their respective aminoacyl tRNA synthetase. In addition, we showed that (D-aspartyl-sulfamoyladenosine (i.e. with a (D-configuration for the aspartyl moiety, is a potent inhibitor of aspartyl tRNA synthetase. However, we also showed that the inhibitory effect of (D- aspartyl-sulfamoyladenosine is relatively short-lasting. Microcin C analogues with (D-amino acids throughout from positions two to six proved inactive. They were shown to be resistant against metabolism by the different peptidases and therefore not able to release the active moiety. This observation could not be reversed by incorporation of (L-amino acids at position six, showing that none of the available peptidases exhibit endopeptidase activity.

  10. Decreased aminoacylation in pathology-related mutants of mitochondrial tRNATyr is associated with structural perturbations in tRNA architecture

    OpenAIRE

    Bonnefond, Luc; Florentz, Catherine; GIEGÉ, RICHARD; Rudinger-Thirion, Joëlle

    2008-01-01

    A growing number of human pathologies are ascribed to mutations in mitochondrial tRNA genes. Here, we report biochemical investigations on three mt-tRNATyr molecules with point substitutions associated with diseases. The mutations occur in the atypical T- and D-loops at positions homologous to those involved in the tertiary interaction network of canonical tRNAs. They do not correspond to tyrosine identity positions and likely do not contact the mitochondrial tyrosyl-tRNA synthetase during th...

  11. Gamma-irradiation effect on the acceptor activity of phenylalanine-tRNA and aminoacyl-tRNA-synthetases of cotton seeds

    International Nuclear Information System (INIS)

    Changes in the activity of phenylalanine-tRNA-synthetases in the reaction of addition of C14-phenylalanine to tRNA, as well as in the acceptor activity of phenylalanine-tRNA itself and the substrate specificity of phenylalanine--tRNA-synthetases have been studied in two-day-old cotton seedlings grown from seeds irradiated with doses of 6, 25 and 50 kR. Phenylalanine-tRNA and its synthetase, and the isoacceptor tRNAs were shown to be differently sensitive to the action of ionizing radiation. The activity of enzyme was inhibited more markedly than the corresponding tRNA. Study of the substrate specificity of each fraction showed that enzyme E1 acylated isoacceptor phenylalanine-tRNA 1 and 2; the enzyme E2, isoacceptor form 3

  12. Kinetic evidence for half-of-the-sites reactivity in tRNA/sup Trp/ aminoacylation by tryptophanyl-tRNA synthetase from beef pancreas

    International Nuclear Information System (INIS)

    The aminoacylation reaction catalyzed by the dimeric tryptophanyl-tRNA synthetase from beef pancreas was studied under pre-steady-state conditions by the quenched-flowed method. The transfer of tryptophan to tRNA/sup Trp/ was monitored by using preformed enzyme-bis(tryptophanyl adenylate) complex. Combinations of either unlabeled or L-[14C]tryptophan-labeled tryptophanyl adenylate and of aminoacylation incubation mixtures containing either unlabeled tryptophan or L-[14C]tryptophan were used. The authors measured either the formation of a single labeled aminoacyl-tRNA/sup Trp/ per enzyme subunit or the turnover of labeled aminoacyl-tRNA/sup Trp/ synthesis. Four models were proposed to analyze the experimental data: (A) two independent and nonequivalent subunits; (B) a single active subunit (subunits presenting absolute half-of-the-sites reactivity); (C) alternate functioning of the subunits (flip-flop mechanism); (D) random functioning of the subunits with half-of-the-sites reactivity. The equations corresponding to the formation of labeled tryptophanyl-tRNA/sup Trp/ under each labeling conditions were derived for each model. By use of least-squares criteria, the experimental curves were fitted with the four models, and it was possible to disregard models B and C as likely mechanisms. Complementary experiments, in which there was no significant excess of ATP-Mg over the enzyme-adenylate complex, emphasized an activator effect of free L-tryptophan on the rate if aminoacylation. This result disfavored model A. Model D was in agreement with all data. The analyses showed that the transfer step was not the major limiting reaction in the overall aminoacylation process

  13. Selection of tRNA charging quality control mechanisms that increase mistranslation of the genetic code

    DEFF Research Database (Denmark)

    Yadavalli, Srujana S; Ibba, Michael

    2013-01-01

    Mistranslation can follow two events during protein synthesis: production of non-cognate amino acid:transfer RNA (tRNA) pairs by aminoacyl-tRNA synthetases (aaRSs) and inaccurate selection of aminoacyl-tRNAs by the ribosome. Many aaRSs actively edit non-cognate amino acids, but editing mechanisms...... are not evolutionarily conserved, and their physiological significance remains unclear. To address the connection between aaRSs and mistranslation, the evolutionary divergence of tyrosine editing by phenylalanyl-tRNA synthetase (PheRS) was used as a model. Certain PheRSs are naturally error prone...

  14. Assembly of the novel five-component apicomplexan multi-aminoacyl-tRNA synthetase complex is driven by the hybrid scaffold protein Tg-p43.

    Directory of Open Access Journals (Sweden)

    Jason M van Rooyen

    Full Text Available In Toxoplasma gondii, as in other eukaryotes, a subset of the amino-acyl-tRNA synthetases are arranged into an abundant cytoplasmic multi-aminoacyl-tRNA synthetase (MARS complex. Through a series of genetic pull-down assays, we have identified the enzymes of this complex as: methionyl-, glutaminyl-, glutamyl-, and tyrosyl-tRNA synthetases, and we show that the N-terminal GST-like domain of a partially disordered hybrid scaffold protein, Tg-p43, is sufficient for assembly of the intact complex. Our gel filtration studies revealed significant heterogeneity in the size and composition of isolated MARS complexes. By targeting the tyrosyl-tRNA synthetases subunit, which was found exclusively in the complete 1 MDa complex, we were able to directly visualize MARS particles in the electron microscope. Image analyses of the negative stain data revealed the observed heterogeneity and instability of these complexes to be driven by the intrinsic flexibility of the domain arrangements within the MARS complex. These studies provide unique insights into the assembly of these ubiquitous but poorly understood eukaryotic complexes.

  15. Development of a new method to identify aminoacylated RNA

    Directory of Open Access Journals (Sweden)

    Wang Ji

    2014-02-01

    Full Text Available A RT-PCR method is developed to isolate RNA aminoacylated on their 3’ end from large pools of RNA. The method is being applied in two separate projects. We are interested in isolating a new class of ribozymes that could successively catalyze the two chemical reactions leading to their own 3’ aminoacylation (ATP activation of an amino acid followed by 3' esterification of the RNA. The catalysis of each of the two reactions has independently been demonstrated for some RNA isolated with the SELEX methodology [1-2]. However, the coupling of both reactions on a same molecule has not been achieved yet. The identification of these still hypothetical ribozymes may help understand how the former translation system started in the absence of the aminoacyltRNA Synthetase, which catalyzes the above two reactions on tRNA in modern cells. In another project, we would like to identify the whole repertoire of aminoacylated RNA (the “aminoacylome” in cells. There are strong indications that other RNA besides tRNA and tmRNA may be aminoacylated for biological purposes [3-4].

  16. Aminoacetylation Reaction Catalyzed by Leucyl-tRNA Synthetase Operates via a Self-Assisted Mechanism Using a Conserved Residue and the Aminoacyl Substrate.

    Science.gov (United States)

    Aleksandrov, Alexey; Palencia, Andrés; Cusack, Stephen; Field, Martin

    2016-05-19

    Leucyl-tRNA synthetase catalyzes attachment of leucine amino acid to its cognate tRNA. During the second, aminoacetylation, step of the reaction, the leucyl moiety is transferred from leucyl-adenylate to the terminal A76 adenosine of tRNA. In this work, we have investigated the aminoacetylation step catalyzed by leucyl-tRNA synthase, using ab initio quantum chemical/molecular mechanical hybrid potentials in conjunction with reaction-path-location algorithms and molecular dynamics free energy simulations. We have modeled reaction mechanisms arising from both crystallographic studies and computational work. We invoke various groups as potential proton acceptors-namely, the phosphate and leucyl amino groups of leucyl-adenylate, the A76 base of tRNA, and the Asp80 and Glu532 residues of the protein-and consider both metal-assisted and metal-free reactions. Free energy calculations indicate that both the phosphate group of leucyl adenylate and Glu532 are not strong bases. This agrees with the results of the quantum chemical/molecular mechanical reaction path calculations which give high free energy barriers for the studied pathways involving these groups. A self-assisted mechanism with the leucyl amino group and Asp80 as proton acceptors is the most likely. Furthermore, in this mechanism the presence of a metal ion coordinated by the phosphate group and Glu532 strongly activates the reaction. PMID:27115861

  17. A Drosophila model for mito-nuclear diseases generated by an incompatible interaction between tRNA and tRNA synthetase.

    Science.gov (United States)

    Holmbeck, Marissa A; Donner, Julia R; Villa-Cuesta, Eugenia; Rand, David M

    2015-08-01

    Communication between the mitochondrial and nuclear genomes is vital for cellular function. The assembly of mitochondrial enzyme complexes, which produce the majority of cellular energy, requires the coordinated expression and translation of both mitochondrially and nuclear-encoded proteins. The joint genetic architecture of this system complicates the basis of mitochondrial diseases, and mutations both in mitochondrial DNA (mtDNA)- and nuclear-encoded genes have been implicated in mitochondrial dysfunction. Previously, in a set of mitochondrial-nuclear introgression strains, we characterized a dual genome epistasis in which a naturally occurring mutation in the Drosophila simulans simw(501) mtDNA-encoded transfer RNA (tRNA) for tyrosine (tRNA(Tyr)) interacts with a mutation in the nuclear-encoded mitochondrially localized tyrosyl-tRNA synthetase from Drosophila melanogaster. Here, we show that the incompatible mitochondrial-nuclear combination results in locomotor defects, reduced mitochondrial respiratory capacity, decreased oxidative phosphorylation (OXPHOS) enzyme activity and severe alterations in mitochondrial morphology. Transgenic rescue strains containing nuclear variants of the tyrosyl-tRNA synthetase are sufficient to rescue many of the deleterious phenotypes identified when paired with the simw(501) mtDNA. However, the severity of this defective mito-nuclear interaction varies across traits and genetic backgrounds, suggesting that the impact of mitochondrial dysfunction might be tissue specific. Because mutations in mitochondrial tRNA(Tyr) are associated with exercise intolerance in humans, this mitochondrial-nuclear introgression model in Drosophila provides a means to dissect the molecular basis of these, and other, mitochondrial diseases that are a consequence of the joint genetic architecture of mitochondrial function. PMID:26035388

  18. Aminoacylation of hypomodified tRNAGlu in vivo

    DEFF Research Database (Denmark)

    Krüger, Malene Kappen; Sørensen, M.A.

    1998-01-01

    The highly specific interaction of each aminoacyl-tRNA synthetase and its substrate tRNAs constitutes an intriguing problem in protein-RNA recognition. All tRNAs have the same overall three-dimensional structure in order to fit interchangeably into the translational apparatus. Thus, the recognition...... by aminoacyl-tRNA synthetase must be more or less limited to discrimination between bases at specific positions within the tRNA. The hypermodified nucleotide 5-methylaminomethyl-2-thiouridine (mnm5s2U) present at the wobble position of bacterial tRNAs specific for glutamic acid, lysine and possibly...... glutamine has been shown to be important in the recognition of these tRNAs by their synthetases in vitro. Here, we have determined the aminoacylation level in vivo of tRNAGlu, tRNALys, and tRNA1GIn in Escherichia coli strains containing undermodified derivatives of mnm5s2U34. Lack of the 5-methylaminomethyl...

  19. Origins and Early Evolution of the tRNA Molecule

    Directory of Open Access Journals (Sweden)

    Koji Tamura

    2015-12-01

    Full Text Available Modern transfer RNAs (tRNAs are composed of ~76 nucleotides and play an important role as “adaptor” molecules that mediate the translation of information from messenger RNAs (mRNAs. Many studies suggest that the contemporary full-length tRNA was formed by the ligation of half-sized hairpin-like RNAs. A minihelix (a coaxial stack of the acceptor stem on the T-stem of tRNA can function both in aminoacylation by aminoacyl tRNA synthetases and in peptide bond formation on the ribosome, indicating that it may be a vestige of the ancestral tRNA. The universal CCA-3′ terminus of tRNA is also a typical characteristic of the molecule. “Why CCA?” is the fundamental unanswered question, but several findings give a comprehensive picture of its origin. Here, the origins and early evolution of tRNA are discussed in terms of various perspectives, including nucleotide ligation, chiral selectivity of amino acids, genetic code evolution, and the organization of the ribosomal peptidyl transferase center (PTC. The proto-tRNA molecules may have evolved not only as adaptors but also as contributors to the composition of the ribosome.

  20. Amino acid modifications on tRNA

    Institute of Scientific and Technical Information of China (English)

    Jing Yuan; Kelly Sheppard; Dieter S(o)ll

    2008-01-01

    The accurate formation of cognate aminoacyl-transfer RNAs (aa-tRNAs) is essential for the fidelity of translation.Most amino acids are esterified onto their cognate tRNA isoacceptors directly by aa.tRNA synthetases.However,in the case of four amino acids (Gin,Asn,Cys and Sec),aminoacyl-tRNAs are made through indirect pathways in many organisms across all three domains of life.The process begins with the charging ofnoncognate amino acids to tRNAs by a specialized synthetase in the case of Cys-tRNAcys formation or by synthetases with relaxed specificity,such as the non-discriminating glutamyl-tRNA,non-discriminating aspartyl-tRNA and seryl-tRNA synthetases.The resulting misacylated tRNAs are then converted to cognate pairs through transformation of the amino acids on the tRNA,which is catalyzed by a group of tRNA-dependent modifying enzymes,such as tRNA-dependent amidotransferases,Sep-tRNA:Cys-tRNA synthase,O-phosphoseryi-tRNA kinase and Sep-tRNA:Sec-tRNA synthase.The majority of these indirect pathways are widely spread in all domains of life and thought to be part of the evolutionary process.

  1. Identity elements for the aminoacylation of metazoan mitochondrial tRNAArg have been widely conserved throughout evolution and ensure the fidelity of the AGR codon reassignment

    OpenAIRE

    Igloi, Gabor L; Leisinger, Anne-Katrin

    2015-01-01

    Eumetazoan mitochondrial tRNAs possess structures (identity elements) that require the specific recognition by their cognate nuclear-encoded aminoacyl-tRNA synthetases. The AGA (arginine) codon of the standard genetic code has been reassigned to serine/glycine/termination in eumetazoan organelles and is translated in some organisms by a mitochondrially encoded tRNASer UCU. One mechanism to prevent mistranslation of the AGA codon as arginine would require a set of tRNA identity elements distin...

  2. The multicenter study of a new assay for simultaneous detection of multiple anti-aminoacyl-tRNA synthetases in myositis and interstitial pneumonia.

    Directory of Open Access Journals (Sweden)

    Ran Nakashima

    Full Text Available OBJECTIVE: Autoantibodies to aminoacyl-tRNA synthetases (ARSs are useful in the diagnosis of idiopathic inflammatory myopathy (IIM with interstitial pneumonia (IP. We developed an enzyme-linked immunosorbent assay (ELISA system using a mixture of recombinant ARS antigens and tested its utility in a multicenter study. METHODS: We prepared six recombinant ARSs: GST-Jo-1, His-PL-12, His-EJ and GST-KS expressed in Escherichia coli, and His-PL-7 and His-OJ expressed in Hi-5 cells. After confirming their antigenic activity, with the exception of His-OJ, we developed our ELISA system in which the five recombinant ARSs (without His-OJ were mixed. Efficiency was confirmed using the sera from 526 Japanese patients with connective tissue disease (CTD (IIM n = 250, systemic lupus erythematosus n = 91, systemic sclerosis n = 70, rheumatoid arthritis n = 75, Sjögren's syndrome n = 27 and other diseases n = 13, 168 with idiopathic interstitial pneumonia (IIP and 30 healthy controls collected from eight institutes. IIPs were classified into two groups; idiopathic pulmonary fibrosis (IPF (n = 38 and non-IPF (n = 130. RESULTS were compared with those of RNA immunoprecipitation. RESULTS: Sensitivity and specificity of the ELISA were 97.1% and 99.8%, respectively when compared with the RNA immunoprecipitation assay. Anti-ARS antibodies were detected in 30.8% of IIM, 2.5% of non-myositis CTD, and 10.7% of IIP (5.3% of IPF and 12.3% of non-IPF. Anti-ARS-positive non-IPF patients were younger and more frequently treated with glucocorticoids and/or immunosuppressants than anti-ARS-negative patients. CONCLUSION: A newly established ELISA detected anti-ARS antibodies as efficiently as RNA immunoprecipitation. This system will enable easier and wider use in the detection of anti-ARS antibodies in patients with IIM and IIP.

  3. Mitochondrial phenylalanyl-tRNA synthetase mutations underlie fatal infantile Alpers encephalopathy

    DEFF Research Database (Denmark)

    Elo, Jenni M; Yadavalli, Srujana S; Euro, Liliya;

    2012-01-01

    Next-generation sequencing has turned out to be a powerful tool to uncover genetic basis of childhood mitochondrial disorders. We utilized whole-exome analysis and discovered novel compound heterozygous mutations in FARS2 (mitochondrial phenylalanyl transfer RNA synthetase), encoding the mitochon...... aminoacyl-tRNA synthetase as a cause of mitochondrial disease.......Next-generation sequencing has turned out to be a powerful tool to uncover genetic basis of childhood mitochondrial disorders. We utilized whole-exome analysis and discovered novel compound heterozygous mutations in FARS2 (mitochondrial phenylalanyl transfer RNA synthetase), encoding the...... mitochondrial phenylalanyl transfer RNA (tRNA) synthetase (mtPheRS) in two patients with fatal epileptic mitochondrial encephalopathy. The mutations affected highly conserved amino acids, p.I329T and p.D391V. Recently, a homozygous FARS2 variant p.Y144C was reported in a Saudi girl with mitochondrial...

  4. Covalent coupling of 4-thiouridine in the initiator methionine tRNA to specific lysine residues in Escherichia coli methionyl-tRNA synthetase

    International Nuclear Information System (INIS)

    A new method has been developed to couple a lysine-reactive cross-linker to the 4-thiouridine residue at position 8 in the primary structure of the Escherichia coli initiator methionine tRNA (tRNA/sup fMet/). Incubation of the affinity-labeling tRNA/sup fMet/ derivative with E. coli methionyl-tRNA synthetase (MetRS) yielded a covalent complex of the protein and nucleic acid and resulted in loss of amino acid acceptor activity of the enzyme. A stoichiometric relationship (1:1) was observed between the amount of cross-linked tRNA and the amount of enzyme inactivated. Cross-linking was effectively inhibited by unmodified tRNA/sup fMet/, but not by noncognate tRNA/sup Phe/. The covalent complex was digested with trypsin, and the resulting tRNA-bound peptides were purified from excess free peptides by anion-exchange chromatography. The tRNA was then degraded with T1 ribonuclease, and the peptides bound to the 4-thiouridine-containing dinucleotide were purified by high-pressure liquid chromatography. Two major peptide products were isolated plus several minor peptides. N-Terminal sequencing of the peptides obtained in highest yield revealed that the 4-thiouridine was cross-linked to lysine residues 402 and 439 in the primary sequence of MetRS. Since many prokaryotic tRNAs contain 4-thiouridine, the procedures described here should prove useful for identification of peptide sequences near this modified base when a variety of tRNAs are bound to specific proteins

  5. Paths of lateral gene transfer of lysyl-aminoacyl-tRNA synthetases with a unique evolutionary transition stage of prokaryotes coding for class I and II varieties by the same organisms

    Directory of Open Access Journals (Sweden)

    Nussinov Ruth

    2006-03-01

    Full Text Available Abstract Background While the premise that lateral gene transfer (LGT is a dominant evolutionary force is still in considerable dispute, the case for widespread LGT in the family of aminoacyl-tRNA synthetases (aaRS is no longer contentious. aaRSs are ancient enzymes, guarding the fidelity of the genetic code. They are clustered in two structurally unrelated classes. Only lysine aminoacyl-tRNA synthetase (LysRS is found both as a class 1 and a class 2 enzyme (LysRS1-2. Remarkably, in several extant prokaryotes both classes of the enzyme coexist, a unique phenomenon that has yet to receive its due attention. Results We applied a phylogenetic approach for determining the extent and origin of LGT in prokaryotic LysRS. Reconstructing species trees for Archaea and Bacteria, and inferring that their last common ancestors encoded LysRS1 and LysRS2, respectively, we studied the gains and losses of both classes. A complex pattern of LGT events emerged. In specific groups of organisms LysRS1 was replaced by LysRS2 (and vice versa. In one occasion, within the alpha proteobacteria, a LysRS2 to LysRS1 LGT was followed by reversal to LysRS2. After establishing the most likely LGT paths, we studied the possible origins of the laterally transferred genes. To this end, we reconstructed LysRS gene trees and evaluated the likely origins of the laterally transferred genes. While the sources of LysRS1 LGTs were readily identified, those for LysRS2 remain, for now, uncertain. The replacement of one LysRS by another apparently transits through a stage simultaneously coding for both synthetases, probably conferring a selective advantage to the affected organisms. Conclusion The family of LysRSs features complex LGT events. The currently available data were sufficient for identifying unambiguously the origins of LysRS1 but not of LysRS2 gene transfers. A selective advantage is suggested to organisms encoding simultaneously LysRS1-2.

  6. Expression, purification, crystallization and preliminary phasing of the heteromerization domain of the tRNA-export and aminoacylation cofactor Arc1p from yeast

    International Nuclear Information System (INIS)

    The heteromerization domain of an aminoacyl-tRNA synthetase cofactor from yeast was crystallized, complete selenomethionine MAD data were collected to 2.8 Å resolution and preliminary phasing reveals the presence of 20 monomers in the asymmetric unit. Eukaryotic aminoacyl-tRNA synthetases (aaRSs) must be integrated into an efficient tRNA-export and shuttling machinery. This is reflected by the presence of additional protein–protein interaction domains and a correspondingly higher degree of complex formation in eukaryotic aaRSs. However, the structural basis of interaction between eukaryotic aaRSs and associated protein cofactors has remained elusive. The N-terminal heteromerization domain of the tRNA aminoacylation and export cofactor Arc1p has been cloned from yeast, expressed and purified. Crystals have been obtained belonging to space group C2, with unit-cell parameters a = 222.32, b = 89.46, c = 126.79 Å, β = 99.39°. Calculated Matthews coefficients are compatible with the presence of 10–25 monomers in the asymmetric unit. A complete multiple-wavelength anomalous dispersion data set has been collected from a selenomethionine-substituted crystal at 2.8 Å resolution. Preliminary phasing reveals the presence of 20 monomers organized in five tetramers per asymmetric unit

  7. Mapping Escherichia coli elongation factor Tu residues involved in binding of aminoacyl-tRNA

    DEFF Research Database (Denmark)

    Wiborg, Ove; Andersen, C; Knudsen, Charlotte Rohde;

    1996-01-01

    were characterized with respect to thermal and chemical stability, GTPase activity, tRNA affinity, and activity in an in vitro translation assay. Most conspicuously tRNA affinities were reduced for all mutants. The results verify our structural analysis of elongation factor Tu in complex with aminoacyl....... Their functional roles are discussed in relation to the structure of elongation factor Tu in complex with aminoacyl-tRNA. Udgivelsesdato: 1996-Aug-23...

  8. Transfer RNA: From pioneering crystallographic studies to contemporary tRNA biology.

    Science.gov (United States)

    Fernández-Millán, Pablo; Schelcher, Cédric; Chihade, Joseph; Masquida, Benoît; Giegé, Philippe; Sauter, Claude

    2016-07-15

    Transfer RNAs (tRNAs) play a key role in protein synthesis as adaptor molecules between messenger RNA and protein sequences on the ribosome. Their discovery in the early sixties provoked a worldwide infatuation with the study of their architecture and their function in the decoding of genetic information. tRNAs are also emblematic molecules in crystallography: the determination of the first tRNA crystal structures represented a milestone in structural biology and tRNAs were for a long period the sole source of information on RNA folding, architecture, and post-transcriptional modifications. Crystallographic data on tRNAs in complex with aminoacyl-tRNA synthetases (aaRSs) also provided the first insight into protein:RNA interactions. Beyond the translation process and the history of structural investigations on tRNA, this review also illustrates the renewal of tRNA biology with the discovery of a growing number of tRNA partners in the cell, the involvement of tRNAs in a variety of regulatory and metabolic pathways, and emerging applications in biotechnology and synthetic biology. PMID:26968773

  9. Human tyrosyl-tRNA synthetase shares amino acid sequence homology with a putative cytokine.

    Science.gov (United States)

    Kleeman, T A; Wei, D; Simpson, K L; First, E A

    1997-05-30

    To test the hypothesis that tRNATyr recognition differs between bacterial and human tyrosyl-tRNA synthetases, we sequenced several clones identified as human tyrosyl-tRNA synthetase cDNAs by the Human Genome Project. We found that human tyrosyl-tRNA synthetase is composed of three domains: 1) an amino-terminal Rossmann fold domain that is responsible for formation of the activated E.Tyr-AMP intermediate and is conserved among bacteria, archeae, and eukaryotes; 2) a tRNA anticodon recognition domain that has not been conserved between bacteria and eukaryotes; and 3) a carboxyl-terminal domain that is unique to the human tyrosyl-tRNA synthetase and whose primary structure is 49% identical to the putative human cytokine endothelial monocyte-activating protein II, 50% identical to the carboxyl-terminal domain of methionyl-tRNA synthetase from Caenorhabditis elegans, and 43% identical to the carboxyl-terminal domain of Arc1p from Saccharomyces cerevisiae. The first two domains of the human tyrosyl-tRNA synthetase are 52, 36, and 16% identical to tyrosyl-tRNA synthetases from S. cerevisiae, Methanococcus jannaschii, and Bacillus stearothermophilus, respectively. Nine of fifteen amino acids known to be involved in the formation of the tyrosyl-adenylate complex in B. stearothermophilus are conserved across all of the organisms, whereas amino acids involved in the recognition of tRNATyr are not conserved. Kinetic analyses of recombinant human and B. stearothermophilus tyrosyl-tRNA synthetases expressed in Escherichia coli indicate that human tyrosyl-tRNA synthetase aminoacylates human but not B. stearothermophilus tRNATyr, and vice versa, supporting the original hypothesis. It is proposed that like endothelial monocyte-activating protein II and the carboxyl-terminal domain of Arc1p, the carboxyl-terminal domain of human tyrosyl-tRNA synthetase evolved from gene duplication of the carboxyl-terminal domain of methionyl-tRNA synthetase and may direct tRNA to the active site of

  10. ß-Lysine discrimination by lysyl-tRNA synthetase

    DEFF Research Database (Denmark)

    Gilreath, Marla S; Roy, Hervé; Bullwinkle, Tammy J;

    2011-01-01

    Elongation factor P is modified with (R)-ß-lysine by the lysyl-tRNA synthetase (LysRS) paralog PoxA. PoxA specificity is orthogonal to LysRS, despite their high similarity. To investigate a- and ß-lysine recognition by LysRS and PoxA, amino acid replacements were made in the LysRS active site gui...... enantiomers of ß-lysine were substrates for tRNA aminoacylation by LysRS, which, together with the relaxed specificity of the A233S variant, suggest a possible means to develop systems for in vivo co-translational insertion of ß-amino acids.......Elongation factor P is modified with (R)-ß-lysine by the lysyl-tRNA synthetase (LysRS) paralog PoxA. PoxA specificity is orthogonal to LysRS, despite their high similarity. To investigate a- and ß-lysine recognition by LysRS and PoxA, amino acid replacements were made in the LysRS active site...

  11. Identity determinants of E. coli tryptophan tRNA.

    OpenAIRE

    Himeno, H; T. Hasegawa; Asahara, H; Tamura, K.; Shimizu, M

    1991-01-01

    The first base pair of the acceptor stem A1-U72 and the discriminator base G73, as well as the anticodon nucleotides, characterize the tryptophan tRNA in E. coli. To determine the contribution of these nucleotides to the tryptophan acceptor activity, various transcripts of E. coli tryptophan tRNA mutants were constructed. Substitutions of the discriminator base G73, which is conserved within prokaryotic tryptophan tRNAs, impaired aminoacylation with tryptophan. Substitutions of other purine-p...

  12. tRNA Biology in Mitochondria

    Directory of Open Access Journals (Sweden)

    Thalia Salinas-Giegé

    2015-02-01

    Full Text Available Mitochondria are the powerhouses of eukaryotic cells. They are considered as semi-autonomous because they have retained genomes inherited from their prokaryotic ancestor and host fully functional gene expression machineries. These organelles have attracted considerable attention because they combine bacterial-like traits with novel features that evolved in the host cell. Among them, mitochondria use many specific pathways to obtain complete and functional sets of tRNAs as required for translation. In some instances, tRNA genes have been partially or entirely transferred to the nucleus and mitochondria require precise import systems to attain their pool of tRNAs. Still, tRNA genes have also often been maintained in mitochondria. Their genetic arrangement is more diverse than previously envisaged. The expression and maturation of mitochondrial tRNAs often use specific enzymes that evolved during eukaryote history. For instance many mitochondria use a eukaryote-specific RNase P enzyme devoid of RNA. The structure itself of mitochondrial encoded tRNAs is also very diverse, as e.g., in Metazoan, where tRNAs often show non canonical or truncated structures. As a result, the translational machinery in mitochondria evolved adapted strategies to accommodate the peculiarities of these tRNAs, in particular simplified identity rules for their aminoacylation. Here, we review the specific features of tRNA biology in mitochondria from model species representing the major eukaryotic groups, with an emphasis on recent research on tRNA import, maturation and aminoacylation.

  13. Quality control in aminoacyl-tRNA synthesis its role in translational fidelity

    DEFF Research Database (Denmark)

    Yadavalli, Srujana S; Ibba, Michael

    2012-01-01

    . AaRSs define the genetic code by catalyzing the formation of precise aminoacyl ester-linked tRNAs via a two-step reaction. AaRSs ensure faithful aa-tRNA synthesis via high substrate selectivity and/or by proofreading (editing) of noncognate products. About half of the aaRSs rely on proofreading......Accurate translation of mRNA into protein is vital for maintenance of cellular integrity. Translational fidelity is achieved by two key events: synthesis of correctly paired aminoacyl-tRNAs by aminoacyl-tRNA synthetases (aaRSs) and stringent selection of aminoacyl-tRNAs (aa-tRNAs) by the ribosome...

  14. RNA versatility governs tRNA function: Why tRNA flexibility is essential beyond the translation cycle.

    Science.gov (United States)

    Kuhn, Claus-D

    2016-05-01

    tRNAs undergo multiple conformational changes during the translation cycle that are required for tRNA translocation and proper communication between the ribosome and translation factors. Recent structural data on how destabilized tRNAs utilize the CCA-adding enzyme to proofread themselves put a spotlight on tRNA flexibility beyond the translation cycle. In analogy to tRNA surveillance, this review finds that other processes also exploit versatile tRNA folding to achieve, amongst others, specific aminoacylation, translational regulation by riboswitches or a block of bacterial translation. tRNA flexibility is thereby not restricted to the hinges utilized during translation. In contrast, the flexibility of tRNA is distributed all over its L-shape and is actively exploited by the tRNA-interacting partners to discriminate one tRNA from another. Since the majority of tRNA modifications also modulate tRNA flexibility it seems that cells devote enormous resources to tightly sense and regulate tRNA structure. This is likely required for error-free protein synthesis. PMID:26990636

  15. Entamoeba lysyl-tRNA synthetase contains a cytokine-like domain with chemokine activity towards human endothelial cells.

    Directory of Open Access Journals (Sweden)

    Manuel Castro de Moura

    2011-11-01

    Full Text Available Immunological pressure encountered by protozoan parasites drives the selection of strategies to modulate or avoid the immune responses of their hosts. Here we show that the parasite Entamoeba histolytica has evolved a chemokine that mimics the sequence, structure, and function of the human cytokine HsEMAPII (Homo sapiens endothelial monocyte activating polypeptide II. This Entamoeba EMAPII-like polypeptide (EELP is translated as a domain attached to two different aminoacyl-tRNA synthetases (aaRS that are overexpressed when parasites are exposed to inflammatory signals. EELP is dispensable for the tRNA aminoacylation activity of the enzymes that harbor it, and it is cleaved from them by Entamoeba proteases to generate a standalone cytokine. Isolated EELP acts as a chemoattractant for human cells, but its cell specificity is different from that of HsEMAPII. We show that cell specificity differences between HsEMAPII and EELP can be swapped by site directed mutagenesis of only two residues in the cytokines' signal sequence. Thus, Entamoeba has evolved a functional mimic of an aaRS-associated human cytokine with modified cell specificity.

  16. Effect of ionizing radiation on methylation of lysil-tRNA-synthetase from rat liver

    International Nuclear Information System (INIS)

    X-irradiation of animals with an absolutely lethal dose of 0.21 C/kg increases the incorporation of a methyl group into aminoacyl-tRNA-synthetases the radioactive label being traced in lysine, arginine, and histidine. The isolated protein methyltransferase methylated total preparaions of aminoacyl-tRNA-synthetases and lysil-tRNA-synthetase of rat liver in the in vitro experiments with the use of S-adenosylmethionine-14CH3 as a donor of methyl groups: the radioactive label was traced in arginine, lysine, histidine, aspartic and glytamic acids

  17. On origin of genetic code and tRNA before translation

    Directory of Open Access Journals (Sweden)

    Szathmáry Eörs

    2011-02-01

    Full Text Available Abstract Background Synthesis of proteins is based on the genetic code - a nearly universal assignment of codons to amino acids (aas. A major challenge to the understanding of the origins of this assignment is the archetypal "key-lock vs. frozen accident" dilemma. Here we re-examine this dilemma in light of 1 the fundamental veto on "foresight evolution", 2 modular structures of tRNAs and aminoacyl-tRNA synthetases, and 3 the updated library of aa-binding sites in RNA aptamers successfully selected in vitro for eight amino acids. Results The aa-binding sites of arginine, isoleucine and tyrosine contain both their cognate triplets, anticodons and codons. We have noticed that these cases might be associated with palindrome-dinucleotides. For example, one-base shift to the left brings arginine codons CGN, with CG at 1-2 positions, to the respective anticodons NCG, with CG at 2-3 positions. Formally, the concomitant presence of codons and anticodons is also expected in the reverse situation, with codons containing palindrome-dinucleotides at their 2-3 positions, and anticodons exhibiting them at 1-2 positions. A closer analysis reveals that, surprisingly, RNA binding sites for Arg, Ile and Tyr "prefer" (exactly as in the actual genetic code the anticodon(2-3/codon(1-2 tetramers to their anticodon(1-2/codon(2-3 counterparts, despite the seemingly perfect symmetry of the latter. However, since in vitro selection of aa-specific RNA aptamers apparently had nothing to do with translation, this striking preference provides a new strong support to the notion of the genetic code emerging before translation, in response to catalytic (and possibly other needs of ancient RNA life. Consistently with the pre-translation origin of the code, we propose here a new model of tRNA origin by the gradual, Fibonacci process-like, elongation of a tRNA molecule from a primordial coding triplet and 5'DCCA3' quadruplet (D is a base-determinator to the eventual 76 base

  18. A Deoxynivalenol-Activated Methionyl-tRNA Synthetase Gene from Wheat Encodes a Nuclear Localized Protein and Protects Plants Against Fusarium Pathogens and Mycotoxins.

    Science.gov (United States)

    Zuo, Dong-Yun; Yi, Shu-Yuan; Liu, Rong-Jing; Qu, Bo; Huang, Tao; He, Wei-Jie; Li, Cheng; Li, He-Ping; Liao, Yu-Cai

    2016-06-01

    Fusarium graminearum is the fungal pathogen that causes globally important diseases of cereals and produces mycotoxins such as deoxynivalenol (DON). Owing to the dearth of available sources of resistance to Fusarium pathogens, characterization of novel genes that confer resistance to mycotoxins and mycotoxin-producing fungi is vitally important for breeding resistant crop varieties. In this study, a wheat methionyl-tRNA synthetase (TaMetRS) gene was identified from suspension cell cultures treated with DON. It shares conserved aminoacylation catalytic and tRNA anticodon binding domains with human MetRS and with the only previously characterized plant MetRS, suggesting that it functions in aminoacylation in the cytoplasm. However, the TaMetRS comprises a typical nuclear localization signal and cellular localization studies with a TaMetRS::GFP fusion protein showed that TaMetRS is localized in the nucleus. Expression of TaMetRS was activated by DON treatment and by infection with a DON-producing F. graminearum strain in wheat spikes. No such activation was observed following infection with a non-DON-producing F. graminearum strain. Expression of TaMetRS in Arabidopsis plants conferred significant resistance to DON and F. graminearum. These results indicated that this DON-activated TaMetRS gene may encode a novel type of MetRS in plants that has a role in defense and detoxification. PMID:26882849

  19. Probe data: 4096 [RED

    Lifescience Database Archive (English)

    Full Text Available R12-025080-1AR RED AU166273 AU166274 CB0250 >(P07814) MULTIFUNCTIONAL AMINOACYL-TRNA SYNTHETASE ... (CONTAINS: GLUTAMYL-TRNA SYNTHETASE (EC ... 6.1.1.17) (GLUTAMATE--TRNA LIGASE), AND PROLYL-TRN ... A SYNTHETASE (EC ... 6.1.1.15) (PROLINE--TRNA LIGASE)). &HSGTS_1(X54326 ...

  20. Leucyl-tRNA synthetase from the ancestral bacterium Aquifex aeolicus contains relics of synthetase evolution

    OpenAIRE

    Zhao, Ming-Wei; Zhu, Bin; Hao, Rui; Xu, Min-Gang; Eriani, Gilbert; Wang, En-Duo

    2005-01-01

    The editing reactions catalyzed by aminoacyl-tRNA synthetases are critical for the faithful protein synthesis by correcting misactivated amino acids and misaminoacylated tRNAs. We report that the isolated editing domain of leucyl-tRNA synthetase from the deep-rooted bacterium Aquifex aeolicus (αβ-LeuRS) catalyzes the hydrolytic editing of both mischarged tRNALeu and minihelixLeu. Within the domain, we have identified a crucial 20-amino-acid peptide that confers editing capacity when transplan...

  1. Analysis of the Resistance Mechanism of a Benzoxaborole Inhibitor Reveals Insight into the Leucyl-tRNA Synthetase Editing Mechanism.

    Science.gov (United States)

    Zhao, Hanchao; Palencia, Andres; Seiradake, Elena; Ghaemi, Zhaleh; Cusack, Stephen; Luthey-Schulten, Zaida; Martinis, Susan

    2015-10-16

    A new class of antimicrobial benzoxaborole compounds was identified as a potent inhibitor of leucyl-tRNA synthetase (LeuRS) and therefore of protein synthesis. In a novel mechanism, AN2690 (5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole) blocks fungal cytoplasmic LeuRS by covalently trapping tRNA(Leu) in the editing site of the enzyme's CP1 domain. However, some resistant mutation sites are located outside of the CP1 hydrolytic editing active site. Thus, their mode of action that undermines drug inhibition was not understood. A combination of X-ray crystallography, molecular dynamics, metadynamics, biochemical experiments, and mutational analysis of a distal benzoxaborole-resistant mutant uncovered a eukaryote-specific tyrosine "switch" that is critical to tRNA-dependent post-transfer editing. The tyrosine "switch" has three states that shift between interactions with a lysine and the 3'-hydroxyl of the tRNA terminus, to inhibit or promote post-transfer editing. The oxaborole's mechanism of action capitalizes upon one of these editing active site states. This tunable editing mechanism in eukaryotic and archaeal LeuRSs is proposed to facilitate precise quality control of aminoacylation fidelity. These mechanistic distinctions could also be capitalized upon for development of the benzoxaboroles as a broad spectrum antibacterial. PMID:26172575

  2. InterProScan Result: FS867702 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available Threonyl/alanyl tRNA synthetase, SAD Molecular Function: ATP binding (GO:0005524)|Cellular Component: cytoplasm (GO:0005737)|Biol...ogical Process: translation (GO:0006412)|Biological Process: tRNA aminoacylation (GO:0043039) ...

  3. Metabolism of D-aminoacyl-tRNAs in Escherichia coli and Saccharomyces cerevisiae cells.

    Science.gov (United States)

    Soutourina, J; Plateau, P; Blanquet, S

    2000-10-20

    In Escherichia coli, tyrosyl-tRNA synthetase is known to esterify tRNA(Tyr) with tyrosine. Resulting d-Tyr-tRNA(Tyr) can be hydrolyzed by a d-Tyr-tRNA(Tyr) deacylase. By monitoring E. coli growth in liquid medium, we systematically searched for other d-amino acids, the toxicity of which might be exacerbated by the inactivation of the gene encoding d-Tyr-tRNA(Tyr) deacylase. In addition to the already documented case of d-tyrosine, positive responses were obtained with d-tryptophan, d-aspartate, d-serine, and d-glutamine. In agreement with this observation, production of d-Asp-tRNA(Asp) and d-Trp-tRNA(Trp) by aspartyl-tRNA synthetase and tryptophanyl-tRNA synthetase, respectively, was established in vitro. Furthermore, the two d-aminoacylated tRNAs behaved as substrates of purified E. coli d-Tyr-tRNA(Tyr) deacylase. These results indicate that an unexpected high number of d-amino acids can impair the bacterium growth through the accumulation of d-aminoacyl-tRNA molecules and that d-Tyr-tRNA(Tyr) deacylase has a specificity broad enough to recycle any of these molecules. The same strategy of screening was applied using Saccharomyces cerevisiae, the tyrosyl-tRNA synthetase of which also produces d-Tyr-tRNA(Tyr), and which, like E. coli, possesses a d-Tyr-tRNA(Tyr) deacylase activity. In this case, inhibition of growth by the various 19 d-amino acids was followed on solid medium. Two isogenic strains containing or not the deacylase were compared. Toxic effects of d-tyrosine and d-leucine were reinforced upon deprivation of the deacylase. This observation suggests that, in yeast, at least two d-amino acids succeed in being transferred onto tRNAs and that, like in E. coli, the resulting two d-aminoacyl-tRNAs are substrates of a same d-aminoacyl-tRNA deacylase. PMID:10918062

  4. RNA editing changes the identity of a mitochondrial tRNA in marsupials.

    OpenAIRE

    Börner, G V; Mörl, M.; Janke, A.; Pääbo, S

    1996-01-01

    In the mitochondrial genome of marsupials, the tRNA gene located at the position where in other mammals an aspartyl-tRNA is encoded carries the glycine anticodon GCC. Post-transcriptionally, an RNA editing mechanism affects the second position of the anticodon such that the aspartate anticodon GUC is created in approximately 50% of the mature tRNA pool. We show that the unedited version of this tRNA'Asp' (GCC) can be specifically aminoacylated with glycine in vitro, while the edited version b...

  5. Association of a multi-synthetase complex with translating ribosomes in the archaeon Thermococcus kodakarensis

    DEFF Research Database (Denmark)

    Raina, Medha; Elgamal, Sara; Santangelo, Thomas J; Ibba, Michael

    2012-01-01

    In archaea and eukaryotes aminoacyl-tRNA synthetases (aaRSs) associate in multi-synthetase complexes (MSCs), however the role of such MSCs in translation is unknown. MSC function was investigated in vivo in the archaeon Thermococcus kodakarensis, wherein six aaRSs were affinity co-purified together...... with several other factors involved in protein synthesis, suggesting that MSCs may interact directly with translating ribosomes. In support of this hypothesis, the aminoacyl-tRNA synthetase (aaRS) activities of the MSC were enriched in isolated T. kodakarensis polysome fractions. These data indicate...... that components of the archaeal protein synthesis machinery associate into macromolecular assemblies in vivo and provide the potential to increase translation efficiency by limiting substrate diffusion away from the ribosome, thus facilitating rapid recycling of tRNAs. STRUCTURED SUMMARY OF PROTEIN...

  6. Saturation of recognition elements blocks evolution of new tRNA identities

    Science.gov (United States)

    Saint-Léger, Adélaïde; Bello, Carla; Dans, Pablo D.; Torres, Adrian Gabriel; Novoa, Eva Maria; Camacho, Noelia; Orozco, Modesto; Kondrashov, Fyodor A.; Ribas de Pouplana, Lluís

    2016-01-01

    Understanding the principles that led to the current complexity of the genetic code is a central question in evolution. Expansion of the genetic code required the selection of new transfer RNAs (tRNAs) with specific recognition signals that allowed them to be matured, modified, aminoacylated, and processed by the ribosome without compromising the fidelity or efficiency of protein synthesis. We show that saturation of recognition signals blocks the emergence of new tRNA identities and that the rate of nucleotide substitutions in tRNAs is higher in species with fewer tRNA genes. We propose that the growth of the genetic code stalled because a limit was reached in the number of identity elements that can be effectively used in the tRNA structure. PMID:27386510

  7. Allosteric vs. spontaneous exit-site (E-site) tRNA dissociation early in protein synthesis.

    Science.gov (United States)

    Chen, Chunlai; Stevens, Benjamin; Kaur, Jaskiran; Smilansky, Zeev; Cooperman, Barry S; Goldman, Yale E

    2011-10-11

    During protein synthesis, deacylated transfer RNAs leave the ribosome via an exit (E) site after mRNA translocation. How the ribosome regulates tRNA dissociation and whether functional linkages between the aminoacyl (A) and E sites modulate the dynamics of protein synthesis have long been debated. Using single molecule fluorescence resonance energy transfer experiments, we find that, during early cycles of protein elongation, tRNAs are often held in the E site until being allosterically released when the next aminoacyl tRNA binds to the A site. This process is regulated by the length and sequence of the nascent peptide and by the conformational state, detected by tRNA proximity, prior to translocation. In later cycles, E-site tRNA dissociates spontaneously. Our results suggest that the distribution of pretranslocation tRNA states and posttranslocation pathways are correlated within each elongation cycle via communication between distant subdomains in the ribosome, but that this correlation between elongation cycle intermediates does not persist into succeeding cycles. PMID:21969541

  8. Allosteric vs. spontaneous exit-site (E-site) tRNA dissociation early in protein synthesis

    OpenAIRE

    Chen, Chunlai; Stevens, Benjamin; Kaur, Jaskiran; Smilansky, Zeev; Cooperman, Barry S.; Goldman, Yale E.

    2011-01-01

    During protein synthesis, deacylated transfer RNAs leave the ribosome via an exit (E) site after mRNA translocation. How the ribosome regulates tRNA dissociation and whether functional linkages between the aminoacyl (A) and E sites modulate the dynamics of protein synthesis have long been debated. Using single molecule fluorescence resonance energy transfer experiments, we find that, during early cycles of protein elongation, tRNAs are often held in the E site until being allosterically relea...

  9. Mitochondrial aminoacyl-tRNA synthetase single-nucleotide polymorphisms that lead to defects in refolding but not aminoacylation

    DEFF Research Database (Denmark)

    Banerjee, Rajat; Reynolds, Noah M; Yadavalli, Srujana S;

    2011-01-01

    Defects in organellar translation are the underlying cause of a number of mitochondrial diseases, including diabetes, deafness, encephalopathy, and other mitochondrial myopathies. The most common causes of these diseases are mutations in mitochondria-encoded tRNAs. It has recently become apparent...

  10. Antibiotic inhibition of the movement of tRNA substrates through a peptidyl transferase cavity

    DEFF Research Database (Denmark)

    Porse, B T; Rodriguez-Fonseca, C; Leviev, I;

    1996-01-01

    The present review attempts to deal with movement of tRNA substrates through the peptidyl transferase centre on the large ribosomal subunit and to explain how this movement is interrupted by antibiotics. It builds on the concept of hybrid tRNA states forming on ribosomes and on the observed...... movement of the 5' end of P-site-bound tRNA relative to the ribosome that occurs on peptide bond formation. The 3' ends of the tRNAs enter, and move through, a catalytic cavity where antibiotics are considered to act by at least three primary mechanisms: (i) they interfere with the entry of the aminoacyl...... moiety into the catalytic cavity before peptide bond formation; (ii) they inhibit movement of the nascent peptide along the peptide channel, a process that may generally involve destabilization of the peptidyl tRNA, and (iii) they prevent movement of the newly deacylated tRNA between the P/P and hybrid P...

  11. The structural basis of tRNA mimicry and conformational plasticity by a viral RNA

    Science.gov (United States)

    Colussi, Timothy M.; Costantino, David A.; Hammond, John A.; Ruehle, Grant M.; Nix, Jay C.; Kieft, Jeffrey S.

    2014-01-01

    RNA is arguably the most functionally diverse biological macromolecule. In some cases a single discrete RNA sequence performs multiple roles and this can be conferred by a complex three-dimensional structure. This multifunctionality can also be driven or enhanced by the ability of a given RNA to assume different conformational (and therefore functional) states1. Despite its biological importance, a detailed structural understanding of the paradigm of RNA structure-driven multifunctionality is lacking. Examples to address this gap are found in single-stranded positive-sense RNA viruses, a prototype being the tRNA-like structure (TLS) found at the 3′ end of the Turnip Yellow Mosaic Virus (TYMV). This TLS not only acts like a tRNA to drive aminoacylation of the viral genomic RNA (gRNA)2-4, but also interacts with other structures in the gRNA's 3′ untranslated region5, contains the promoter for negative strand synthesis, and influences several infection-critical processes6. This TLS RNA can provide a glimpse into the structural basis of RNA multifunctionality and plasticity, but for decades its high-resolution structure has remained elusive. Here, we present the crystal structure of the complete TYMV TLS to 2.0 Å resolution. Globally, the RNA adopts a shape that mimics tRNA, but it uses a very different set of intramolecular interactions to achieve this shape. These interactions also allow the TLS to readily switch conformations. In addition, the TLS structure is ‘two-faced’: one ‘face’ closely mimics tRNA and drives aminoacylation, the other ‘face’ diverges from tRNA and enables additional functionality. The TLS is thus structured to perform several functions and interact with diverse binding partners, and we demonstrate its ability to specifically bind to ribosomes. PMID:24909993

  12. Kinetics profiling of gramicidin S synthetase A, a member of nonribosomal peptide synthetases.

    Science.gov (United States)

    Sun, Xun; Li, Hao; Alfermann, Jonas; Mootz, Henning D; Yang, Haw

    2014-12-23

    Nonribosomal peptide synthetases (NRPS) incorporate assorted amino acid substrates into complex natural products. The substrate is activated via the formation of a reactive aminoacyl adenylate and is subsequently attached to the protein template via a thioester bond. The reactive nature of such intermediates, however, leads to side reactions that also break down the high-energy anhydride bond. The off-pathway kinetics or their relative weights compared to that of the on-pathway counterpart remains generally elusive. Here, we introduce multiplatform kinetics profiling to quantify the relative weights of on- and off-pathway reactions. Using the well-defined stoichiometry of thioester formation, we integrate a mass spectrometry (MS) kinetics assay, a high-performance liquid chromatography (HPLC) assay, and an ATP-pyrophosphate (PPi) exchange assay to map out a highly efficient on-pathway kinetics profile of the substrate activation and intermediate uploading (>98% relative weight) for wide-type gramicidin S synthetase A (GrsA) and a 87% rate profile for a cysteine-free GrsA mutant. Our kinetics profiling approach complements the existing enzyme-coupled byproduct-release assays, unraveling new mechanistic insights of substrate activation/channeling in NRPS enzymes. PMID:25437123

  13. Redox status affects the catalytic activity of glutamyl-tRNA synthetase

    DEFF Research Database (Denmark)

    Katz, Assaf; Banerjee, Rajat; de Armas, Merly;

    2010-01-01

    Glutamyl-tRNA synthetases (GluRS) provide Glu-tRNA for different processes including protein synthesis, glutamine transamidation and tetrapyrrole biosynthesis. Many organisms contain multiple GluRSs, but whether these duplications solely broaden tRNA specificity or also play additional roles in...... vitro, GluRS1 activity is reversibly inactivated upon oxidation by hemin and hydrogen peroxide. The targets for oxidation-based inhibition were found to be cysteines from a SWIM zinc-binding motif located in the tRNA acceptor helix-binding domain. tRNA(Glu) was able to protect GluRS1 against oxidative...

  14. Kinetics of the interactions between yeast elongation factors 1A and 1Balpha, guanine nucleotides, and aminoacyl-tRNA

    DEFF Research Database (Denmark)

    Gromadski, Kirill B; Schümmer, Tobias; Strømgaard, Anne;

    2007-01-01

    The interactions of elongation factor 1A (eEF1A) from Saccharomyces cerevisiae with elongation factor 1Balpha (eEF1Balpha), guanine nucleotides, and aminoacyl-tRNA were studied kinetically by fluorescence stopped-flow. eEF1A has similar affinities for GDP and GTP, 0.4 and 1.1 microm, respectively...... no significant binding, indicating that eEF1A has similar tRNA binding properties as its prokaryotic homolog, EF-Tu. Udgivelsesdato: 2007-Dec-7......The interactions of elongation factor 1A (eEF1A) from Saccharomyces cerevisiae with elongation factor 1Balpha (eEF1Balpha), guanine nucleotides, and aminoacyl-tRNA were studied kinetically by fluorescence stopped-flow. eEF1A has similar affinities for GDP and GTP, 0.4 and 1.1 microm, respectively...... guanine nucleotides. At the concentrations of nucleotides and factors prevailing in the cell, the overall exchange rate is expected to be in the range of 6 s(-1), which is compatible with the rate of protein synthesis in the cell. eEF1A.GTP binds Phe-tRNA(Phe) with a K(d) of 3 nm, whereas eEF1A.GDP shows...

  15. Structural studies of lysyl-tRNA synthetase: conformational changes induced by substrate binding.

    Science.gov (United States)

    Onesti, S; Desogus, G; Brevet, A; Chen, J; Plateau, P; Blanquet, S; Brick, P

    2000-10-24

    Lysyl-tRNA synthetase is a member of the class II aminoacyl-tRNA synthetases and catalyses the specific aminoacylation of tRNA(Lys). The crystal structure of the constitutive lysyl-tRNA synthetase (LysS) from Escherichia coli has been determined to 2.7 A resolution in the unliganded form and in a complex with the lysine substrate. A comparison between the unliganded and lysine-bound structures reveals major conformational changes upon lysine binding. The lysine substrate is involved in a network of hydrogen bonds. Two of these interactions, one between the alpha-amino group and the carbonyl oxygen of Gly 216 and the other between the carboxylate group and the side chain of Arg 262, trigger a subtle and complicated reorganization of the active site, involving the ordering of two loops (residues 215-217 and 444-455), a change in conformation of residues 393-409, and a rotation of a 4-helix bundle domain (located between motif 2 and 3) by 10 degrees. The result of these changes is a closing up of the active site upon lysine binding. PMID:11041850

  16. Polyspecific pyrrolysyl-tRNA synthetases from directed evolution.

    Science.gov (United States)

    Guo, Li-Tao; Wang, Yane-Shih; Nakamura, Akiyoshi; Eiler, Daniel; Kavran, Jennifer M; Wong, Margaret; Kiessling, Laura L; Steitz, Thomas A; O'Donoghue, Patrick; Söll, Dieter

    2014-11-25

    Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA(Pyl) have emerged as ideal translation components for genetic code innovation. Variants of the enzyme facilitate the incorporation >100 noncanonical amino acids (ncAAs) into proteins. PylRS variants were previously selected to acylate N(ε)-acetyl-Lys (AcK) onto tRNA(Pyl). Here, we examine an N(ε)-acetyl-lysyl-tRNA synthetase (AcKRS), which is polyspecific (i.e., active with a broad range of ncAAs) and 30-fold more efficient with Phe derivatives than it is with AcK. Structural and biochemical data reveal the molecular basis of polyspecificity in AcKRS and in a PylRS variant [iodo-phenylalanyl-tRNA synthetase (IFRS)] that displays both enhanced activity and substrate promiscuity over a chemical library of 313 ncAAs. IFRS, a product of directed evolution, has distinct binding modes for different ncAAs. These data indicate that in vivo selections do not produce optimally specific tRNA synthetases and suggest that translation fidelity will become an increasingly dominant factor in expanding the genetic code far beyond 20 amino acids. PMID:25385624

  17. Occurrence and functional compatibility within Enterobacteriaceae of a tRNA species which inserts selenocysteine into protein.

    OpenAIRE

    Heider, J; Leinfelder, W; Böck, A

    1989-01-01

    The selC gene from E. coli codes for a tRNA species (tRNA(UCASer] which is aminoacylated with L-serine and which cotranslationally inserts selenocysteine into selenoproteins. By means of Southern hybridization it was demonstrated that this gene occurs in all enterobacteria tested. To assess whether the unique primary and secondary structural features of the E. coli selC gene product are conserved in that of other organisms, the selC homologue from Proteus vulgaris was cloned and sequenced. It...

  18. Structure of the prolyl-tRNA synthetase from the eukaryotic pathogen Giardia lamblia

    Energy Technology Data Exchange (ETDEWEB)

    Larson, Eric T.; Kim, Jessica E.; Napuli, Alberto J.; Verlinde, Christophe L. M. J.; Fan, Erkang; Zucker, Frank H.; Van Voorhis, Wesley C.; Buckner, Frederick S.; Hol, Wim G. J.; Merritt, Ethan A., E-mail: merritt@u.washington.edu [Medical Structural Genomics of Pathogenic Protozoa, (United States); University of Washington, Seattle, WA 98195 (United States)

    2012-09-01

    The structure of Giardia prolyl-tRNA synthetase cocrystallized with proline and ATP shows evidence for half-of-the-sites activity, leading to a corresponding mixture of reaction substrates and product (prolyl-AMP) in the two active sites of the dimer. The genome of the human intestinal parasite Giardia lamblia contains only a single aminoacyl-tRNA synthetase gene for each amino acid. The Giardia prolyl-tRNA synthetase gene product was originally misidentified as a dual-specificity Pro/Cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-tRNA synthetases. The 2.2 Å resolution crystal structure of the G. lamblia enzyme presented here is thus the first structure determination of a prolyl-tRNA synthetase from a eukaryote. The relative occupancies of substrate (proline) and product (prolyl-AMP) in the active site are consistent with half-of-the-sites reactivity, as is the observed biphasic thermal denaturation curve for the protein in the presence of proline and MgATP. However, no corresponding induced asymmetry is evident in the structure of the protein. No thermal stabilization is observed in the presence of cysteine and ATP. The implied low affinity for the off-target activation product cysteinyl-AMP suggests that translational fidelity in Giardia is aided by the rapid release of misactivated cysteine.

  19. Structure of the prolyl-tRNA synthetase from the eukaryotic pathogen Giardia lamblia

    International Nuclear Information System (INIS)

    The structure of Giardia prolyl-tRNA synthetase cocrystallized with proline and ATP shows evidence for half-of-the-sites activity, leading to a corresponding mixture of reaction substrates and product (prolyl-AMP) in the two active sites of the dimer. The genome of the human intestinal parasite Giardia lamblia contains only a single aminoacyl-tRNA synthetase gene for each amino acid. The Giardia prolyl-tRNA synthetase gene product was originally misidentified as a dual-specificity Pro/Cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-tRNA synthetases. The 2.2 Å resolution crystal structure of the G. lamblia enzyme presented here is thus the first structure determination of a prolyl-tRNA synthetase from a eukaryote. The relative occupancies of substrate (proline) and product (prolyl-AMP) in the active site are consistent with half-of-the-sites reactivity, as is the observed biphasic thermal denaturation curve for the protein in the presence of proline and MgATP. However, no corresponding induced asymmetry is evident in the structure of the protein. No thermal stabilization is observed in the presence of cysteine and ATP. The implied low affinity for the off-target activation product cysteinyl-AMP suggests that translational fidelity in Giardia is aided by the rapid release of misactivated cysteine

  20. Cytosolic glutamine synthetase

    DEFF Research Database (Denmark)

    Thomsen, Hanne Cecilie; Eriksson, Ulf Dennis; Møller, Inge Skrumsager;

    2014-01-01

    Overexpression of the cytosolic enzyme glutamine synthetase 1 (GS1) has been investigated in numerous cases with the goal of improving crop nitrogen use efficiency. However, the outcome has generally been inconsistent. Here, we review possible reasons underlying the lack of success and conclude...

  1. Effects of Trypanosoma brucei tryptophanyl-tRNA synthetases silencing by RNA interference

    Directory of Open Access Journals (Sweden)

    Liliana Torcoroma García

    2007-09-01

    Full Text Available The kinetoplast genetic code deviates from the universal code in that 90% of mitochondrial tryptophans are specified by UGA instead of UGG codons. A single nucleus-encoded tRNA Trp(CCA is used by both nuclear and mitochondria genes, since all kinetoplast tRNAs are imported into the mitochondria from the cytoplasm. To allow decoding of the mitochondrial UGA codons as tryptophan, the tRNA Trp(CCA anticodon is changed to UCA by an editing event. Two tryptophanyl tRNA synthetases (TrpRSs have been identified in Trypanosoma brucei: TbTrpRS1 and TbTrpRS2 which localize to the cytoplasm and mitochondria respectively. We used inducible RNA interference (RNAi to assess the role of TbTrpRSs. Our data validates previous observations of TrpRS as potential drug design targets and investigates the RNAi effect on the mitochondria of the parasite.

  2. Network of tRNA Gene Sequences

    Institute of Scientific and Technical Information of China (English)

    WEI Fang-ping; LI Sheng; MA Hong-ru

    2008-01-01

    A network of 3719 tRNA gene sequences was constructed using simplest alignment. Its topology, degree distribution and clustering coefficient were studied. The behaviors of the network shift from fluctuated distribution to scale-free distribution when the similarity degree of the tRNA gene sequences increases. The tRNA gene sequences with the same anticodon identity are more self-organized than those with different anticodon identities and form local clusters in the network. Some vertices of the local cluster have a high connection with other local clusters, and the probable reason was given. Moreover, a network constructed by the same number of random tRNA sequences was used to make comparisons. The relationships between the properties of the tRNA similarity network and the characters of tRNA evolutionary history were discussed.

  3. A Hypertension-Associated tRNAAla Mutation Alters tRNA Metabolism and Mitochondrial Function

    Science.gov (United States)

    Jiang, Pingping; Wang, Meng; Xue, Ling; Xiao, Yun; Yu, Jialing; Wang, Hui; Yao, Juan; Liu, Hao; Peng, Yanyan; Liu, Hanqing; Li, Haiying; Chen, Ye

    2016-01-01

    In this report, we investigated the pathophysiology of a novel hypertension-associated mitochondrial tRNAAla 5655A → G (m.5655A → G) mutation. The destabilization of a highly conserved base pairing (A1-U72) at the aminoacyl acceptor stem by an m.5655A → G mutation altered the tRNAAla function. An in vitro processing analysis showed that the m.5655A → G mutation reduced the efficiency of tRNAAla precursor 5′ end cleavage catalyzed by RNase P. By using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from a Chinese family into mitochondrial DNA (mtDNA)-less (ρo) cells, we showed a 41% reduction in the steady-state level of tRNAAla in mutant cybrids. The mutation caused an improperly aminoacylated tRNAAla, as suggested by aberrantly aminoacylated tRNAAla and slower electrophoretic mobility of mutated tRNA. A failure in tRNAAla metabolism contributed to variable reductions in six mtDNA-encoded polypeptides in mutant cells, ranging from 21% to 37.5%, with an average of a 29.1% reduction, compared to levels of the controls. The impaired translation caused reduced activities of mitochondrial respiration chains. Furthermore, marked decreases in the levels of mitochondrial ATP and membrane potential were observed in mutant cells. These caused increases in the production of reactive oxygen species in the mutant cybrids. The data provide evidence for the association of the tRNAAla 5655A → G mutation with hypertension. PMID:27161322

  4. Splicing Defect in Mitochondrial Seryl-tRNA Synthetase Gene Causes Progressive Spastic Paresis Instead of HUPRA Syndrome.

    Science.gov (United States)

    Linnankivi, Tarja; Neupane, Nirajan; Richter, Uwe; Isohanni, Pirjo; Tyynismaa, Henna

    2016-09-01

    Mitochondrial aminoacyl-tRNA synthetases are an important group of disease genes typically underlying either a disorder affecting an isolated tissue or a distinct syndrome. Missense mutations in the mitochondrial seryl-tRNA synthetase gene, SARS2, have been identified in HUPRA syndrome (hyperuricemia, pulmonary hypertension, renal failure in infancy, and alkalosis). We report here a homozygous splicing mutation in SARS2 in a patient with progressive spastic paresis. We show that the mutation leads to diminished levels of the synthetase in patient's fibroblasts. This has a destabilizing effect on the tRNASer(AGY) isoacceptor, but to a lesser degree than in HUPRA syndrome patients. tRNASer(UCN) is largely unaffected in both phenotypes. In conclusion, the level of tRNASer(AGY) instability may be a factor in determining tissue manifestation in patients with SARS2 mutations. This finding exemplifies the sensitivity of the nervous system to partially reduced aminoacylation, which is sufficient in other tissues to maintain respiratory chain function. PMID:27279129

  5. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analyses of threonyl-tRNA synthetase editing domain from Aeropyrum pernix

    International Nuclear Information System (INIS)

    The editing domain of threonyl-tRNA synthetase from the archaeon Aeropyrum pernix has been overexpressed, purified and crystallized. The crystal diffracted to a resolution of 1.66 Å. The proofreading function of aminoacyl-tRNA synthetases is crucial in maintaining the fidelity of protein synthesis. Most archaeal threonyl-tRNA synthetases (ThrRSs) possess a unique proofreading domain unrelated to their eukaryotic/bacterial counterpart. The crystal structure of this domain from the archaeon Pyrococcus abysii in complex with its cognate and noncognate substrate analogues had given insights into its catalytic and discriminatory mechanisms. To probe further into the mechanistic and evolutionary aspects of this domain, work has been extended to another archaeon Aeropyrum pernix. The organism possesses two proteins corresponding to threonyl-tRNA synthetase, i.e. ThrRS1 and ThrRS2, encoded by two different genes, thrS1 and thrS2, respectively. ThrRS1 is responsible for aminoacylation and ThrRS2 for proofreading activity. Here the purification, crystallization and preliminary X-ray crystallographic investigation of the N-terminal proofreading domain of ThrRS2 from A. pernix is reported. The crystals belong to either the P41212 or P43212 space group and consist of one monomer per asymmetric unit

  6. Non Proteolytic Aminoacyl Derivatives (NPADs) in cheeses: Synthesis, Origin, Properties

    OpenAIRE

    Bottesini, Chiara

    2014-01-01

    La presente tesi di dottorato riguarda la caratterizzazione di derivati amminoacidici (gamma-glutamil amminoacidi, lattoil-amminoacidi, piroglutamil-amminoacidi), denominati Non Proteolyltic Aminoacyl Derivatives (NPADs) presenti nel Parmigiano Reggiano, considerando in particolare la caratterizzazione strutturale, l’origine e le proprietà biologico-funzionali. Dopo un’introduzione generale riguardante lo studio e l’evoluzione peptidica nel formaggio durante la stagionatura, i peptidi bio...

  7. Chemical modification of the tryptophan residues of leucyl-tRNA synthetase by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide

    International Nuclear Information System (INIS)

    The accessibility for modification of the tryptophan residues in leucyl-tRNA synthetase from cow mammary glands has been studied with the aid of the specific chemical reagents N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide. UV absorption and the intrinsic fluorescence of the tryptophan residues of the enzymes were used for recording the course of the modification. It has been shown that under native conditions (pH 7.8) two superficial residues in each subunit of the dimeric enzyme undergo modification. Under denaturing conditions (6 M guanidine hydrochloride), the internal tryptophan residues are also modified. When the tryptophan residues are modified, the leucyl-tRNA synthetase is inactivated both in the aminoacylation reaction and in the ATP-PP/sub i/ exchange reaction. In the specific complex of leucyl-tRNA synthetase with tRNA/sup Leu/ one of the superficial tryptophan residues is screened and does not undergo modification by the reagents used

  8. Preferential hydrophobic interactions are responsible for a preference of D-amino acids in the aminoacylation of 5'-AMP with hydrophobic amino acids

    Science.gov (United States)

    Lacey, J. C. Jr; Wickramasinghe, N. S.; Sabatini, R. S.

    1992-01-01

    We have studied the chemistry of aminoacyl AMP to model reactions at the 3' terminus of aminoacyl tRNA for the purpose of understanding the origin of protein synthesis. The present studies relate to the D, L preference in the esterification of 5'-AMP. All N-acetyl amino acids we studied showed faster reaction of the D-isomer, with a generally decreasing preference for D-isomer as the hydrophobicity of the amino acid decreased. The beta-branched amino acids, Ile and Val, showed an extreme preference for D-isomer. Ac-Leu, the gamma-branched amino acid, showed a slightly low D/L ratio relative to its hydrophobicity. The molecular basis for these preferences for D-isomer is understandable in the light of our previous studies and seems to be due to preferential hydrophobic interaction of the D-isomer with adenine. The preference for hydrophobic D-amino acids can be decreased by addition of an organic solvent to the reaction medium. Conversely, peptidylation with Ac-PhePhe shows a preference for the LL isomer over the DD isomer.

  9. Cytonuclear Interactions in the Evolution of Animal Mitochondrial tRNA Metabolism.

    Science.gov (United States)

    Pett, Walker; Lavrov, Dennis V

    2015-08-01

    The evolution of mitochondrial information processing pathways, including replication, transcription and translation, is characterized by the gradual replacement of mitochondrial-encoded proteins with nuclear-encoded counterparts of diverse evolutionary origins. Although the ancestral enzymes involved in mitochondrial transcription and replication have been replaced early in eukaryotic evolution, mitochondrial translation is still carried out by an apparatus largely inherited from the α-proteobacterial ancestor. However, variation in the complement of mitochondrial-encoded molecules involved in translation, including transfer RNAs (tRNAs), provides evidence for the ongoing evolution of mitochondrial protein synthesis. Here, we investigate the evolution of the mitochondrial translational machinery using recent genomic and transcriptomic data from animals that have experienced the loss of mt-tRNAs, including phyla Cnidaria and Ctenophora, as well as some representatives of all four classes of Porifera. We focus on four sets of mitochondrial enzymes that directly interact with tRNAs: Aminoacyl-tRNA synthetases, glutamyl-tRNA amidotransferase, tRNA(Ile) lysidine synthetase, and RNase P. Our results support the observation that the fate of nuclear-encoded mitochondrial proteins is influenced by the evolution of molecules encoded in mitochondrial DNA, but in a more complex manner than appreciated previously. The data also suggest that relaxed selection on mitochondrial translation rather than coevolution between mitochondrial and nuclear subunits is responsible for elevated rates of evolution in mitochondrial translational proteins. PMID:26116918

  10. Disrupted tRNA Genes and tRNA Fragments: A Perspective on tRNA Gene Evolution

    Directory of Open Access Journals (Sweden)

    Akio Kanai

    2015-01-01

    Full Text Available Transfer RNAs (tRNAs are small non-coding RNAs with lengths of approximately 70–100 nt. They are directly involved in protein synthesis by carrying amino acids to the ribosome. In this sense, tRNAs are key molecules that connect the RNA world and the protein world. Thus, study of the evolution of tRNA molecules may reveal the processes that led to the establishment of the central dogma: genetic information flows from DNA to RNA to protein. Thanks to the development of DNA sequencers in this century, we have determined a huge number of nucleotide sequences from complete genomes as well as from transcriptomes in many species. Recent analyses of these large data sets have shown that particular tRNA genes, especially in Archaea, are disrupted in unique ways: some tRNA genes contain multiple introns and some are split genes. Even tRNA molecules themselves are fragmented post-transcriptionally in many species. These fragmented small RNAs are known as tRNA-derived fragments (tRFs. In this review, I summarize the progress of research into the disrupted tRNA genes and the tRFs, and propose a possible model for the molecular evolution of tRNAs based on the concept of the combination of fragmented tRNA halves.

  11. Disrupted tRNA Genes and tRNA Fragments: A Perspective on tRNA Gene Evolution.

    Science.gov (United States)

    Kanai, Akio

    2015-01-01

    Transfer RNAs (tRNAs) are small non-coding RNAs with lengths of approximately 70-100 nt. They are directly involved in protein synthesis by carrying amino acids to the ribosome. In this sense, tRNAs are key molecules that connect the RNA world and the protein world. Thus, study of the evolution of tRNA molecules may reveal the processes that led to the establishment of the central dogma: genetic information flows from DNA to RNA to protein. Thanks to the development of DNA sequencers in this century, we have determined a huge number of nucleotide sequences from complete genomes as well as from transcriptomes in many species. Recent analyses of these large data sets have shown that particular tRNA genes, especially in Archaea, are disrupted in unique ways: some tRNA genes contain multiple introns and some are split genes. Even tRNA molecules themselves are fragmented post-transcriptionally in many species. These fragmented small RNAs are known as tRNA-derived fragments (tRFs). In this review, I summarize the progress of research into the disrupted tRNA genes and the tRFs, and propose a possible model for the molecular evolution of tRNAs based on the concept of the combination of fragmented tRNA halves. PMID:25629271

  12. Multistep modeling of protein structure: application towards refinement of tyr-tRNA synthetase

    Science.gov (United States)

    Srinivasan, S.; Shibata, M.; Roychoudhury, M.; Rein, R.

    1987-01-01

    The scope of multistep modeling (MSM) is expanding by adding a least-squares minimization step in the procedure to fit backbone reconstruction consistent with a set of C-alpha coordinates. The analytical solution of Phi and Psi angles, that fits a C-alpha x-ray coordinate is used for tyr-tRNA synthetase. Phi and Psi angles for the region where the above mentioned method fails, are obtained by minimizing the difference in C-alpha distances between the computed model and the crystal structure in a least-squares sense. We present a stepwise application of this part of MSM to the determination of the complete backbone geometry of the 321 N terminal residues of tyrosine tRNA synthetase to a root mean square deviation of 0.47 angstroms from the crystallographic C-alpha coordinates.

  13. Nucleotide sequence of a spinach chloroplast valine tRNA.

    OpenAIRE

    Sprouse, H M; Kashdan, M; Otis, L; Dudock, B

    1981-01-01

    The nucleotide sequence of a spinach chloroplast valine tRNA (sp. chl. tRNA Val) has been determined. This tRNA shows essentially equal homology to prokaryotic valine tRNAs (58-65% homology) and to the mitochondrial valine tRNAs of lower eukaryotes (yeast and N. crassa, 61-62% homology). Sp. chl. tRNA Val shows distinctly lower homology to mouse mitochondrial valine tRNA (53% homology) and to eukaryotic cytoplasmic valine tRNAs (47-53% homology). Sp. chl. tRNA Val, like all other chloroplast ...

  14. Functional asymmetry in the lysyl-tRNA synthetase explored by molecular dynamics, free energy calculations and experiment

    Directory of Open Access Journals (Sweden)

    Miller Andrew D

    2003-06-01

    Full Text Available Abstract Background Charging of transfer-RNA with cognate amino acid is accomplished by the aminoacyl-tRNA synthetases, and proceeds through an aminoacyl adenylate intermediate. The lysyl-tRNA synthetase has evolved an active site that specifically binds lysine and ATP. Previous molecular dynamics simulations of the heat-inducible Escherichia coli lysyl-tRNA synthetase, LysU, have revealed differences in the binding of ATP and aspects of asymmetry between the nominally equivalent active sites of this dimeric enzyme. The possibility that this asymmetry results in different binding affinities for the ligands is addressed here by a parallel computational and biochemical study. Results Biochemical experiments employing isothermal calorimetry, steady-state fluorescence and circular dichroism are used to determine the order and stoichiometries of the lysine and nucleotide binding events, and the associated thermodynamic parameters. An ordered mechanism of substrate addition is found, with lysine having to bind prior to the nucleotide in a magnesium dependent process. Two lysines are found to bind per dimer, and trigger a large conformational change. Subsequent nucleotide binding causes little structural rearrangement and crucially only occurs at a single catalytic site, in accord with the simulations. Molecular dynamics based free energy calculations of the ATP binding process are used to determine the binding affinities of each site. Significant differences in ATP binding affinities are observed, with only one active site capable of realizing the experimental binding free energy. Half-of-the-sites models in which the nucleotide is only present at one active site achieve their full binding potential irrespective of the subunit choice. This strongly suggests the involvement of an anti-cooperative mechanism. Pathways for relaying information between the two active sites are proposed. Conclusions The asymmetry uncovered here appears to be a common

  15. Selective inhibition of apicoplast tryptophanyl-tRNA synthetase causes delayed death in Plasmodium falciparum.

    Science.gov (United States)

    Pasaje, Charisse Flerida A; Cheung, Vanessa; Kennedy, Kit; Lim, Erin E; Baell, Jonathan B; Griffin, Michael D W; Ralph, Stuart A

    2016-01-01

    The malaria parasite Plasmodium falciparum relies on efficient protein translation. An essential component of translation is the tryptophanyl-tRNA synthetase (TrpRS) that charges tRNA(trp). Here we characterise two isoforms of TrpRS in Plasmodium; one eukaryotic type localises to the cytosol and a bacterial type localises to the remnant plastid (apicoplast). We show that the apicoplast TrpRS aminoacylates bacterial tRNA(trp) while the cytosolic TrpRS charges eukaryotic tRNA(trp). An inhibitor of bacterial TrpRSs, indolmycin, specifically inhibits aminoacylation by the apicoplast TrpRS in vitro, and inhibits ex vivo Plasmodium parasite growth, killing parasites with a delayed death effect characteristic of apicoplast inhibitors. Indolmycin treatment ablates apicoplast inheritance and is rescuable by addition of the apicoplast metabolite isopentenyl pyrophosphate (IPP). These data establish that inhibition of an apicoplast housekeeping enzyme leads to loss of the apicoplast and this is sufficient for delayed death. Apicoplast TrpRS is essential for protein translation and is a promising, specific antimalarial target. PMID:27277538

  16. tRNA creation by hairpin duplication.

    Science.gov (United States)

    Widmann, Jeremy; Di Giulio, Massimo; Yarus, Michael; Knight, Rob

    2005-10-01

    Many studies have suggested that the modern cloverleaf structure of tRNA may have arisen through duplication of a primordial hairpin, but the timing of this duplication event has been unclear. Here we measure the level of sequence identity between the two halves of each of a large sample of tRNAs and compare this level to that of chimeric tRNAs constructed either within or between groups defined by phylogeny and/or specificity. We find that actual tRNAs have significantly more matches between the two halves than do random sequences that can form the tRNA structure, but there is no difference in the average level of matching between the two halves of an individual tRNA and the average level of matching between the two halves of the chimeric tRNAs in any of the sets we constructed. These results support the hypothesis that the modern tRNA cloverleaf arose from a single hairpin duplication prior to the divergence of modern tRNA specificities and the three domains of life. PMID:16155749

  17. Cytosolic glutamine synthetase in barley

    DEFF Research Database (Denmark)

    Thomsen, Hanne Cecilie

    fertilizer requirement. The enzyme glutamine synthetase (GS) has been a major topic in plant nitrogen research for decades due to its central role in plant N metabolism. The cytosolic version of this enzyme (GS1) plays an important role in relation to primary N assimilation as well as in relation to N...

  18. Mitochondrial tRNA 5'-editing in Dictyostelium discoideum and Polysphondylium pallidum.

    Science.gov (United States)

    Abad, Maria G; Long, Yicheng; Kinchen, R Dimitri; Schindel, Elinor T; Gray, Michael W; Jackman, Jane E

    2014-05-30

    Mitochondrial tRNA (mt-tRNA) 5'-editing was first described more than 20 years ago; however, the first candidates for 5'-editing enzymes were only recently identified in a eukaryotic microbe (protist), the slime mold Dictyostelium discoideum. In this organism, eight of 18 mt-tRNAs are predicted to be edited based on the presence of genomically encoded mismatched nucleotides in their aminoacyl-acceptor stem sequences. Here, we demonstrate that mt-tRNA 5'-editing occurs at all predicted sites in D. discoideum as evidenced by changes in the sequences of isolated mt-tRNAs compared with the expected sequences encoded by the mitochondrial genome. We also identify two previously unpredicted editing events in which G-U base pairs are edited in the absence of any other genomically encoded mismatches. A comparison of 5'-editing in D. discoideum with 5'-editing in another slime mold, Polysphondylium pallidum, suggests organism-specific idiosyncrasies in the treatment of U-G/G-U pairs. In vitro activities of putative D. discoideum editing enzymes are consistent with the observed editing reactions and suggest an overall lack of tRNA substrate specificity exhibited by the repair component of the editing enzyme. Although the presence of terminal mismatches in mt-tRNA sequences is highly predictive of the occurrence of mt-tRNA 5'-editing, the variability in treatment of U-G/G-U base pairs observed here indicates that direct experimental evidence of 5'-editing must be obtained to understand the complete spectrum of mt-tRNA editing events in any species. PMID:24737330

  19. Elongation Factor Tu Prevents Misediting of Gly-tRNA(Gly Caused by the Design Behind the Chiral Proofreading Site of D-Aminoacyl-tRNA Deacylase.

    Directory of Open Access Journals (Sweden)

    Satya Brata Routh

    2016-05-01

    Full Text Available D-aminoacyl-tRNA deacylase (DTD removes D-amino acids mischarged on tRNAs and is thus implicated in enforcing homochirality in proteins. Previously, we proposed that selective capture of D-aminoacyl-tRNA by DTD's invariant, cross-subunit Gly-cisPro motif forms the mechanistic basis for its enantioselectivity. We now show, using nuclear magnetic resonance (NMR spectroscopy-based binding studies followed by biochemical assays with both bacterial and eukaryotic systems, that DTD effectively misedits Gly-tRNAGly. High-resolution crystal structure reveals that the architecture of DTD's chiral proofreading site is completely porous to achiral glycine. Hence, L-chiral rejection is the only design principle on which DTD functions, unlike other chiral-specific enzymes such as D-amino acid oxidases, which are specific for D-enantiomers. Competition assays with elongation factor thermo unstable (EF-Tu and DTD demonstrate that EF-Tu precludes Gly-tRNAGly misediting at normal cellular concentrations. However, even slightly higher DTD levels overcome this protection conferred by EF-Tu, thus resulting in significant depletion of Gly-tRNAGly. Our in vitro observations are substantiated by cell-based studies in Escherichia coli that show that overexpression of DTD causes cellular toxicity, which is largely rescued upon glycine supplementation. Furthermore, we provide direct evidence that DTD is an RNA-based catalyst, since it uses only the terminal 2'-OH of tRNA for catalysis without the involvement of protein side chains. The study therefore provides a unique paradigm of enzyme action for substrate selection/specificity by DTD, and thus explains the underlying cause of DTD's activity on Gly-tRNAGly. It also gives a molecular and functional basis for the necessity and the observed tight regulation of DTD levels, thereby preventing cellular toxicity due to misediting.

  20. Elongation Factor Tu Prevents Misediting of Gly-tRNA(Gly) Caused by the Design Behind the Chiral Proofreading Site of D-Aminoacyl-tRNA Deacylase.

    Science.gov (United States)

    Routh, Satya Brata; Pawar, Komal Ishwar; Ahmad, Sadeem; Singh, Swati; Suma, Katta; Kumar, Mantu; Kuncha, Santosh Kumar; Yadav, Kranthikumar; Kruparani, Shobha P; Sankaranarayanan, Rajan

    2016-05-01

    D-aminoacyl-tRNA deacylase (DTD) removes D-amino acids mischarged on tRNAs and is thus implicated in enforcing homochirality in proteins. Previously, we proposed that selective capture of D-aminoacyl-tRNA by DTD's invariant, cross-subunit Gly-cisPro motif forms the mechanistic basis for its enantioselectivity. We now show, using nuclear magnetic resonance (NMR) spectroscopy-based binding studies followed by biochemical assays with both bacterial and eukaryotic systems, that DTD effectively misedits Gly-tRNAGly. High-resolution crystal structure reveals that the architecture of DTD's chiral proofreading site is completely porous to achiral glycine. Hence, L-chiral rejection is the only design principle on which DTD functions, unlike other chiral-specific enzymes such as D-amino acid oxidases, which are specific for D-enantiomers. Competition assays with elongation factor thermo unstable (EF-Tu) and DTD demonstrate that EF-Tu precludes Gly-tRNAGly misediting at normal cellular concentrations. However, even slightly higher DTD levels overcome this protection conferred by EF-Tu, thus resulting in significant depletion of Gly-tRNAGly. Our in vitro observations are substantiated by cell-based studies in Escherichia coli that show that overexpression of DTD causes cellular toxicity, which is largely rescued upon glycine supplementation. Furthermore, we provide direct evidence that DTD is an RNA-based catalyst, since it uses only the terminal 2'-OH of tRNA for catalysis without the involvement of protein side chains. The study therefore provides a unique paradigm of enzyme action for substrate selection/specificity by DTD, and thus explains the underlying cause of DTD's activity on Gly-tRNAGly. It also gives a molecular and functional basis for the necessity and the observed tight regulation of DTD levels, thereby preventing cellular toxicity due to misediting. PMID:27224426

  1. Elongation Factor Tu Prevents Misediting of Gly-tRNA(Gly) Caused by the Design Behind the Chiral Proofreading Site of D-Aminoacyl-tRNA Deacylase

    Science.gov (United States)

    Routh, Satya Brata; Ahmad, Sadeem; Suma, Katta; Kumar, Mantu; Kuncha, Santosh Kumar; Yadav, Kranthikumar; Kruparani, Shobha P; Sankaranarayanan, Rajan

    2016-01-01

    D-aminoacyl-tRNA deacylase (DTD) removes D-amino acids mischarged on tRNAs and is thus implicated in enforcing homochirality in proteins. Previously, we proposed that selective capture of D-aminoacyl-tRNA by DTD’s invariant, cross-subunit Gly-cisPro motif forms the mechanistic basis for its enantioselectivity. We now show, using nuclear magnetic resonance (NMR) spectroscopy-based binding studies followed by biochemical assays with both bacterial and eukaryotic systems, that DTD effectively misedits Gly-tRNAGly. High-resolution crystal structure reveals that the architecture of DTD’s chiral proofreading site is completely porous to achiral glycine. Hence, L-chiral rejection is the only design principle on which DTD functions, unlike other chiral-specific enzymes such as D-amino acid oxidases, which are specific for D-enantiomers. Competition assays with elongation factor thermo unstable (EF-Tu) and DTD demonstrate that EF-Tu precludes Gly-tRNAGly misediting at normal cellular concentrations. However, even slightly higher DTD levels overcome this protection conferred by EF-Tu, thus resulting in significant depletion of Gly-tRNAGly. Our in vitro observations are substantiated by cell-based studies in Escherichia coli that show that overexpression of DTD causes cellular toxicity, which is largely rescued upon glycine supplementation. Furthermore, we provide direct evidence that DTD is an RNA-based catalyst, since it uses only the terminal 2′-OH of tRNA for catalysis without the involvement of protein side chains. The study therefore provides a unique paradigm of enzyme action for substrate selection/specificity by DTD, and thus explains the underlying cause of DTD’s activity on Gly-tRNAGly. It also gives a molecular and functional basis for the necessity and the observed tight regulation of DTD levels, thereby preventing cellular toxicity due to misediting. PMID:27224426

  2. Tertiary structure of bacterial selenocysteine tRNA.

    Science.gov (United States)

    Itoh, Yuzuru; Sekine, Shun-ichi; Suetsugu, Shiro; Yokoyama, Shigeyuki

    2013-07-01

    Selenocysteine (Sec) is translationally incorporated into proteins in response to the UGA codon. The tRNA specific to Sec (tRNA(Sec)) is first ligated with serine by seryl-tRNA synthetase (SerRS). In the present study, we determined the 3.1 Å crystal structure of the tRNA(Sec) from the bacterium Aquifex aeolicus, in complex with the heterologous SerRS from the archaeon Methanopyrus kandleri. The bacterial tRNA(Sec) assumes the L-shaped structure, from which the long extra arm protrudes. Although the D-arm conformation and the extra-arm orientation are similar to those of eukaryal/archaeal tRNA(Sec)s, A. aeolicus tRNA(Sec) has unique base triples, G14:C21:U8 and C15:G20a:G48, which occupy the positions corresponding to the U8:A14 and R15:Y48 tertiary base pairs of canonical tRNAs. Methanopyrus kandleri SerRS exhibited serine ligation activity toward A. aeolicus tRNA(Sec) in vitro. The SerRS N-terminal domain interacts with the extra-arm stem and the outer corner of tRNA(Sec). Similar interactions exist in the reported tRNA(Ser) and SerRS complex structure from the bacterium Thermus thermophilus. Although the catalytic C-terminal domain of M. kandleri SerRS lacks interactions with A. aeolicus tRNA(Sec) in the present complex structure, the conformational flexibility of SerRS is likely to allow the CCA terminal region of tRNA(Sec) to enter the SerRS catalytic site. PMID:23649835

  3. Site specific incorporation of heavy atom-containing unnatural amino acids into proteins for structure determination

    Science.gov (United States)

    Xie, Jianming; Wang, Lei; Wu, Ning; Schultz, Peter G.

    2008-07-15

    Translation systems and other compositions including orthogonal aminoacyl tRNA-synthetases that preferentially charge an orthogonal tRNA with an iodinated or brominated amino acid are provided. Nucleic acids encoding such synthetases are also described, as are methods and kits for producing proteins including heavy atom-containing amino acids, e.g., brominated or iodinated amino acids. Methods of determining the structure of a protein, e.g., a protein into which a heavy atom has been site-specifically incorporated through use of an orthogonal tRNA/aminoacyl tRNA-synthetase pair, are also described.

  4. Substrate specificity of formylglycinamidine synthetase

    Energy Technology Data Exchange (ETDEWEB)

    Schendel, F.J.; Stubbe, J.

    1986-04-22

    Formylglycinamidine ribonucleotide (FGAM) synthetase, which catalyzes the conversion of formylglycinamide ribonucleotide (FGAR), glutamine, and ATP to FGAM, ADP, glutamate, and Pi, has been purified to homogeneity (sp act. 0.20 mumol min-1 mg-1) from chicken liver by an alternative procedure to that of Buchanan et al. (Buchanan, J. M., Ohnoki, S., and Hong, B. S. (1978) Methods Enzymol. 51, 193-201) (sp act. 0.12 mumol min-1 mg-1). A variety of new analogues of formylglycinamide ribonucleotide have been prepared in which the formylglycinamide arm (R = CH/sub 2/NHCHO) has been replaced by R = CH/sub 3/, CH/sub 2/OH, CH/sub 2/Cl, CH/sub 2/NH/sub 3/, CH/sub 2/NHCOCH/sub 3/, CH/sub 2/NHCOCH/sub 2/Cl, CH/sub 2/NHCO/sub 2/CH/sub 2/Ph, and L-CHC-H/sub 3/NHCHO. These compounds have been characterized by 1H and 13C NMR spectroscopy. With compounds R = CH/sub 3/, CH/sub 2/OH, and CH/sub 2/NHCOCH/sub 3/ and ATP, in the presence or absence of glutamine, FGAM synthetase catalyzes the production of Pi at 4.5, 48, and 20%, respectively, the rate of production of Pi from formylglycinamide ribonucleotide. Only R = CH/sub 2/NHCOCH/sub 3/ causes glutaminase activity as well as ATPase activity and has been shown to be converted to the amidine analogue. Both FGAR (R = CH/sub 2/NHCHO) and the FGAR analogue (R = CH/sub 2/NHCHOCH/sub 3/) in the presence of ATP and FGAM synthetase and in the absence of glutamine form a complex isolable by Sephadex G-50 chromatography. FGAM synthetase is thus highly specific for its formylglycine side chain. (18O)-beta-FGAR was prepared biosynthetically, and FGAM synthetase was shown by 31P NMR spectroscopy to catalyze the transfer of amide 18O to inorganic phosphate.

  5. Kinetic Analysis of tRNA Methylfransferases

    Science.gov (United States)

    Hou, Ya-Ming; Masuda, Isao

    2016-01-01

    Transfer RNA (tRNA) molecules contain many chemical modifications that are introduced after transcription. A major form of these modifications is methyl transfer to bases and backbone groups, using S-adenosyl methionine (AdoMet) as the methyl donor. Each methylation confers a specific advantage to tRNA in structure or in function. A remarkable methylation is to the G37 base on the 3' side of the anticodon to generate m1G37-tRNA, which suppresses frameshift errors during protein synthesis and is therefore essential for cell growth in all three domains of life. This methylation is catalyzed by TrmD in bacteria and by Trm5 in eukaryotes and archaea. Although TrmD and Trm5 catalyze the same methylation reaction, kinetic analysis reveal that these two enzymes are unrelated to each other and are distinct in their reaction mechanism. This chapter summarizes the kinetic assays that are used to reveal the distinction between TrmD and Trm5. Three types of assays are described, the steady-state, the pre-steady-state, and the single turnover assays, which collectively provide the basis for mechanistic investigation of AdoMet-dependent methyl transfer reactions. PMID:26253967

  6. Structural Insights into tRNA Dynamics on the Ribosome

    Directory of Open Access Journals (Sweden)

    Xabier Agirrezabala

    2015-04-01

    Full Text Available High-resolution structures at different stages, as well as biochemical, single molecule and computational approaches have highlighted the elasticity of tRNA molecules when bound to the ribosome. It is well acknowledged that the inherent structural flexibility of the tRNA lies at the heart of the protein synthesis process. Here, we review the recent advances and describe considerations that the conformational changes of the tRNA molecules offer about the mechanisms grounded in translation.

  7. Structural Insights into tRNA Dynamics on the Ribosome.

    Science.gov (United States)

    Agirrezabala, Xabier; Valle, Mikel

    2015-01-01

    High-resolution structures at different stages, as well as biochemical, single molecule and computational approaches have highlighted the elasticity of tRNA molecules when bound to the ribosome. It is well acknowledged that the inherent structural flexibility of the tRNA lies at the heart of the protein synthesis process. Here, we review the recent advances and describe considerations that the conformational changes of the tRNA molecules offer about the mechanisms grounded in translation. PMID:25941930

  8. Structural Insights into tRNA Dynamics on the Ribosome

    OpenAIRE

    Xabier Agirrezabala; Mikel Valle

    2015-01-01

    High-resolution structures at different stages, as well as biochemical, single molecule and computational approaches have highlighted the elasticity of tRNA molecules when bound to the ribosome. It is well acknowledged that the inherent structural flexibility of the tRNA lies at the heart of the protein synthesis process. Here, we review the recent advances and describe considerations that the conformational changes of the tRNA molecules offer about the mechanisms grounded in translation.

  9. Mitochondrial tRNA gene translocations in highly eusocial bees

    OpenAIRE

    Daniela Silvestre; Maria Cristina Arias

    2006-01-01

    Mitochondrial gene rearrangement events, especially involving tRNA genes, have been described more frequently as more complete mitochondrial genome sequences are becoming available. In the present work, we analyzed mitochondrial tRNA gene rearrangements between two bee species belonging to the tribes Apini and Meliponini within the "corbiculate Apidae". Eleven tRNA genes are in different genome positions or strands. The molecular events responsible for each translocation are explained. Consid...

  10. Nucleolar Clustering of Dispersed tRNA Genes

    OpenAIRE

    Thompson, Martin; Haeusler, Rebecca A.; Good, Paul D.; Engelke, David R.

    2003-01-01

    Early transfer RNA (tRNA) processing events in Saccharomyces cerevisiae are coordinated in the nucleolus, the site normally associated with ribosome biosynthesis. To test whether spatial organization of the tRNA pathway begins with nucleolar clustering of the genes, we have probed the subnuclear location of five different tRNA gene families. The results show that tRNA genes, though dispersed in the linear genome, colocalize with 5S ribosomal DNA and U14 small nucleolar RNA at the nucleolus. N...

  11. Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Zhiwen; Alfonta, Lital; Schultz, Peter G

    2014-02-25

    This invention provides methods and compositions for incorporation of an unnatural amino acid into a peptide using an orthogonal aminoacyl tRNA synthetase/tRNA pair. In particular, an orthogonal pair is provided to incorporate 5-hydroxy-L-tryptophan in a position encoded by an opal mutation.

  12. Radioimmune assay of human platelet prostaglandin synthetase

    International Nuclear Information System (INIS)

    Normal platelet function depends, in part, on platelet PG synthesis. PG synthetase (cyclo-oxygenase) catalyzes the first step in PG synthesis, the formation of PGH2 from arachidonic acid. Inhibition of the enzyme by ASA results in an abnormality in the platelet release reaction. Patients with pparent congenital abnormalities in the enzyme have been described, and the effects have been referred to as ''aspirin-like'' defects of the platelet function. These patients lack platelet PG synthetase activity, but the actual content of PG synthetase protein in these individuals' platelets is unknown. Therefore an RIA for human platelet PG synthetase would provide new information, useful in assessing the aspirin-like defects of platelet function. An RIA for human platelet PG synthetase is described. The assay utilizes a rabbit antibody directed against the enzyme and [125I]-labelled sheep PG synthetase as antigen. The human platelet enzyme is assayed by its ability to inhibit precipitation of the [125I]antigen. The assay is sensitive to 1 ng of enzyme. By the immune assay, human platelets contain approximately 1200 ng of PG synethetase protein per 1.5 mg of platelet protein (approximately 109 platelets). This content corresponds to 10,000 enzyme molecules per platelet. The assay provides a rapid and convenient assay for the human platelet enzyme, and it can be applied to the assessment of patients with apparent platelet PG synthetase (cyclo-oxygenase) deficiency

  13. Heterologous expression in Escherichia coli of the first module of the nonribosomal peptide synthetase for chloroeremomycin, a vancomycin-type glycopeptide antibiotic.

    Science.gov (United States)

    Trauger, J W; Walsh, C T

    2000-03-28

    The gene cluster from Amycolotopsis orientalis responsible for biosynthesis of the vancomycin-type glycopeptide antibiotic chloroeremomycin was recently sequenced, indicating that this antibiotic derives from a seven-residue peptide synthesized by a three-subunit (CepA, CepB, and CepC) modular nonribosomal peptide synthetase. Expression of all or parts of the peptide synthetase in Escherichia coli would facilitate biochemical characterization of its substrate specificity, an important step toward the development of more potent glycopeptides by combinatorial biosynthesis. To determine whether CepA, a three-module 3,158-residue peptide synthetase expected to assemble the first three residues of the heptapeptide precursor, could be heterologously expressed in E. coli and converted to active, holo form by posttranslational priming with a phosphopantetheinyltransferase, we expressed two CepA fragments (CepA1-575 and CepA1-1596) as well as full-length CepA (CepA1-3158). All three constructs were expressed in soluble form. We find that the CepA1-575 fragment, containing adenylation and peptidyl carrier protein domains (A1-PCP1), specifically adenylates l-leucine and d-leucine in a 6:1 ratio, and it can be converted to holo form by the phosphopantetheinyltransferase Sfp; also, we find that holo-CepA1-575 can be covalently aminoacylated with l-leucine on the peptidyl carrier protein 1 domain. However, no amino acid-dependent adenylation or aminoacylation activity was detected for the larger CepA constructs with l-leucine or other expected amino acid substrates, suggesting severe folding problems in the multidomain proteins. PMID:10716695

  14. HUMAN MITOCHONDRIAL tRNA MUTATIONS IN MATERNALLY INHERITED DEAFNESS

    Institute of Scientific and Technical Information of China (English)

    ZHENG Jing; GONG Sha-sha; TANG Xiao-wen; ZHU Yi; GUAN Min-xin

    2013-01-01

    Mutations in mitochondrial tRNA genes have been shown to be associated with maternally inherited syn-dromic and non-syndromic deafness. Among those, mutations such as tRNALeu(UUR) 3243A>G associated with syndromic deafness are often present in heteroplasmy, and the non-syndromic deafness-associated tRNA mu-tations including tRNASer(UCN) 7445A>G are often in homoplasmy or in high levels of heteroplasmy. These tRNA mutations are the primary factors underlying the development of hearing loss. However, other tRNA mutations such as tRNAThr 15927G>A and tRNASer(UCN) 7444G>A are insufficient to produce a deafness phe-notype, but always act in synergy with the primary mitochondrial DNA mutations, and can modulate their phenotypic manifestation. These tRNA mutations may alter the structure and function of the corresponding mitochondrial tRNAs and cause failures in tRNAs metabolism. Thereby, the impairment of mitochondrial protein synthesis and subsequent defects in respiration caused by these tRNA mutations, results in mitochon-drial dysfunctions and eventually leads to the development of hearing loss. Here, we summarized the deaf-ness-associated mitochondrial tRNA mutations and discussed the pathophysiology of these mitochondrial tRNA mutations, and we hope these data will provide a foundation for the early diagnosis, management, and treatment of maternally inherited deafness.

  15. Nucleotide sequence of a human tRNA gene heterocluster

    International Nuclear Information System (INIS)

    Leucine tRNA from bovine liver was used as a hybridization probe to screen a human gene library harbored in Charon-4A of bacteriophage lambda. The human DNA inserts from plaque-pure clones were characterized by restriction endonuclease mapping and Southern hybridization techniques, using both [3'-32P]-labeled bovine liver leucine tRNA and total tRNA as hybridization probes. An 8-kb Hind III fragment of one of these γ-clones was subcloned into the Hind III site of pBR322. Subsequent fine restriction mapping and DNA sequence analysis of this plasmid DNA indicated the presence of four tRNA genes within the 8-kb DNA fragment. A leucine tRNA gene with an anticodon of AAG and a proline tRNA gene with an anticodon of AGG are in a 1.6-kb subfragment. A threonine tRNA gene with an anticodon of UGU and an as yet unidentified tRNA gene are located in a 1.1-kb subfragment. These two different subfragments are separated by 2.8 kb. The coding regions of the three sequenced genes contain characteristic internal split promoter sequences and do not have intervening sequences. The 3'-flanking region of these three genes have typical RNA polymerase III termination sites of at least four consecutive T residues

  16. NMR structure and dynamics of the Specifier Loop domain from the Bacillus subtilis tyrS T box leader RNA

    OpenAIRE

    Wang, Jiachen; Henkin, Tina M.; Nikonowicz, Edward P.

    2010-01-01

    Gram-positive bacteria utilize a tRNA-responsive transcription antitermination mechanism, designated the T box system, to regulate expression of many amino acid biosynthetic and aminoacyl-tRNA synthetase genes. The RNA transcripts of genes controlled by this mechanism contain 5′ untranslated regions, or leader RNAs, that specifically bind cognate tRNA molecules through pairing of nucleotides in the tRNA anticodon loop with nucleotides in the Specifier Loop domain of the leader RNA. We have de...

  17. Genetics Home Reference: glutathione synthetase deficiency

    Science.gov (United States)

    ... elevated acidity in the blood and tissues (metabolic acidosis). In addition to the features present in moderate ... Kaabachi N, Hachicha M. Hemolytic anemia and metabolic acidosis: think about glutathione synthetase deficiency. Fetal Pediatr Pathol. ...

  18. Genetics Home Reference: holocarboxylase synthetase deficiency

    Science.gov (United States)

    ... the body is unable to use the vitamin biotin effectively. This disorder is classified as a multiple ... impaired activity of certain enzymes that depend on biotin. The signs and symptoms of holocarboxylase synthetase deficiency ...

  19. Structural Aspects of Phenylalanylation and Quality Control in Three Major Forms of Phenylalanyl-tRNA Synthetase

    Directory of Open Access Journals (Sweden)

    Liron Klipcan

    2010-01-01

    Full Text Available Aminoacyl-tRNA synthetases (aaRSs are a canonical set of enzymes that specifically attach corresponding amino acids to their cognate transfer RNAs in the cytoplasm, mitochondria, and nucleus. The aaRSs display great differences in primary sequence, subunit size, and quaternary structure. Existence of three types of phenylalanyl-tRNA synthetase (PheRS—bacterial (αβ2, eukaryotic/archaeal cytosolic (αβ2, and mitochondrial α—is a prominent example of structural diversity within the aaRSs family. Although archaeal/eukaryotic and bacterial PheRSs share common topology of the core domains and the B3/B4 interface, where editing activity of heterotetrameric PheRSs is localized, the detailed investigation of the three-dimensional structures from three kingdoms revealed significant variations in the local design of their synthetic and editing sites. Moreover, as might be expected from structural data eubacterial, Thermus thermophilus and human cytoplasmic PheRSs acquire different patterns of tRNAPhe anticodon recognition.

  20. An Incompatibility between a Mitochondrial tRNA and Its Nuclear-Encoded tRNA Synthetase Compromises Development and Fitness in Drosophila

    OpenAIRE

    Meiklejohn, Colin D.; Holmbeck, Marissa A.; Siddiq, Mohammad A.; Abt, Dawn N.; Rand, David M.; Montooth, Kristi L.

    2013-01-01

    Mitochondrial transcription, translation, and respiration require interactions between genes encoded in two distinct genomes, generating the potential for mutations in nuclear and mitochondrial genomes to interact epistatically and cause incompatibilities that decrease fitness. Mitochondrial-nuclear epistasis for fitness has been documented within and between populations and species of diverse taxa, but rarely has the genetic or mechanistic basis of these mitochondrial–nuclear interactions be...

  1. A Drosophila model for mito-nuclear diseases generated by an incompatible interaction between tRNA and tRNA synthetase

    OpenAIRE

    Holmbeck, Marissa A.; Donner, Julia R.; Villa-Cuesta, Eugenia; Rand, David M.

    2015-01-01

    ABSTRACT Communication between the mitochondrial and nuclear genomes is vital for cellular function. The assembly of mitochondrial enzyme complexes, which produce the majority of cellular energy, requires the coordinated expression and translation of both mitochondrially and nuclear-encoded proteins. The joint genetic architecture of this system complicates the basis of mitochondrial diseases, and mutations both in mitochondrial DNA (mtDNA)- and nuclear-encoded genes have been implicated in m...

  2. Point mutation in mitochondrial tRNA gene is associated with polycystic ovary syndrome and insulin resistance.

    Science.gov (United States)

    Ding, Yu; Zhuo, Guangchao; Zhang, Caijuan; Leng, Jianhang

    2016-04-01

    Polycystic ovarian syndrome (PCOS) is characterized by chronic anovulation, hyperandrogenism and polycystic ovaries. To date, the molecular mechanisms underlying PCOS have remained to be fully elucidated. As recent studies have revealed a positive association between mitochondrial dysfunction and PCOS, current investigations focus on mutations in the mitochondrial genome of patients with POCS. The present study reported a Chinese patient with PCOS. Sequence analysis of the mitochondrial genome showed the presence of homoplasmic ND5 T12338C and tRNASer (UCN) C7492T mutations as well as a set of polymorphisms belonging to the human mitochondrial haplogroup F2. The T12338C mutation is known to decrease the ND5 mRNA levels and to inhibit the processing of RNA precursors. The C7492T mutation, which occurred at the highly conserved nucleotide in the anticodon stem of the tRNASer (UCN) gene, is important for the tRNA steady‑state level as well as the aminoacylation ability. Therefore, the combination of the ND5 T12338C and tRNASer (UCN) C7492T mutations may lead to mitochondrial dysfunction, and is likely to be involved in the pathogenesis of PCOS. The present study provided novel insight into the molecular mechanisms of PCOS. PMID:26935780

  3. Molecular basis of dihydrouridine formation on tRNA

    OpenAIRE

    Yu, Futao; Tanaka, Yoshikazu; Yamashita, Keitaro; Suzuki, Takeo; Nakamura, Akiyoshi; Hirano, Nagisa; Suzuki, Tsutomu; Yao, Min; Tanaka, Isao

    2011-01-01

    Dihydrouridine (D) is a highly conserved modified base found in tRNAs from all domains of life. Dihydrouridine synthase (Dus) catalyzes the D formation of tRNA through reduction of uracil base with flavin mononucleotide (FMN) as a cofactor. Here, we report the crystal structures of Thermus thermophilus Dus (TthDus), which is responsible for D formation at positions 20 and 20a, in complex with tRNA and with a short fragment of tRNA (D-loop). Dus interacts extensively with the D-arm and recogni...

  4. Aminoacylation of an unusual tRNACys from an extreme halophile

    OpenAIRE

    EVILIA, CARYN; MING, XIAOTIAN; DasSarma, Shiladitya; Hou, Ya-Ming

    2003-01-01

    The extreme halophile Halobacterium species NRC-1 overcomes external near-saturating salt concentrations by accumulating intracellular salts comparable to those of the medium. This raises the fundamental question of how halophiles can maintain the specificity of protein–nucleic acid interactions that are particularly sensitive to high salts in mesophiles. Here we address the specificity of the essential aminoacylation reaction of the halophile, by focusing on molecular recognition of tRNACys ...

  5. tRNAfeature: An algorithm for tRNA features to identify tRNA genes in DNA sequences.

    Science.gov (United States)

    Yang, Cheng-Hong; Lin, Yu-Da; Chuang, Li-Yeh

    2016-09-01

    The identification of transfer RNAs (tRNAs) is critical for a detailed understanding of the evolution of biological organisms and viruses. However, some tRNAs are difficult to recognize due to their unusual sub-structures and may result in the detection of the wrong anticodon. Therefore, the detection of unusual sub-structures of tRNA genes remains an important challenge. In this study, we propose a method to identify tRNA genes based on tRNA features. tRNAfeature attempts to refold the sequence with single-stranded regions longer than those found in the canonical and conventional structural models for tRNA. We predicted a set of 53926 archaeal, eubacterial and eukaryotic tRNA genes annotated in tRNADB-CE and scanned the tRNA genes in whole genome sequencing. The results indicate that tRNAfeature is more powerful than other existing methods for identifying tRNAs. PMID:27291467

  6. Roles of tRNA in cell wall biosynthesis

    DEFF Research Database (Denmark)

    Dare, Kiley; Ibba, Michael

    2012-01-01

    Recent research into various aspects of bacterial metabolism such as cell wall and antibiotic synthesis, degradation pathways, cellular stress, and amino acid biosynthesis has elucidated roles of aminoacyl-transfer ribonucleic acid (aa-tRNA) outside of translation. Although the two enzyme families...... specificity of this diverse enzymatic family is necessary to aid current efforts in designing potential bactericidal agents. These two enzyme families are linked only by the substrate with which they modify the cell wall, aa-tRNA; their structure, cell wall modification processes and the physiological changes...... responsible for cell wall modifications, aminoacyl-phosphatidylglycerol synthases (aaPGSs) and Fem, were discovered some time ago, they have recently become of intense interest for their roles in the antimicrobial resistance of pathogenic microorganisms. The addition of positively charged amino acids to...

  7. Crystallization and preliminary X-ray crystallographic analysis of bacterial tRNASec in complex with seryl-tRNA synthetase

    International Nuclear Information System (INIS)

    Bacterial selenocysteine tRNA was crystallized as the heterologous complex with archaeal seryl-tRNA synthetase. X-ray diffraction was improved by introducing point mutations and heavy-atom labeling, and a 3.2 Å diffraction data set for phase determination was obtained from a platinum-labeled crystal. Selenocysteine (Sec) is translationally incorporated into proteins in response to the UGA codon. The tRNA specific to Sec (tRNASec) is first ligated with serine by seryl-tRNA synthetase (SerRS). To elucidate the tertiary structure of bacterial tRNASec and its specific interaction with SerRS, the bacterial tRNASec from Aquifex aeolicus was crystallized as the heterologous complex with the archaeal SerRS from Methanopyrus kandleri. Although X-ray diffraction by crystals of tRNASec in complex with wild-type SerRS was rather poor (to 5.7 Å resolution), the resolution was improved by introducing point mutations targeting the crystal-packing interface. Heavy-atom labelling also contributed to resolution improvement. A 3.2 Å resolution diffraction data set for phase determination was obtained from a K2Pt(CN)4-soaked crystal

  8. Neural control of glutamine synthetase activity in rat skeletal muscles.

    Science.gov (United States)

    Feng, B; Konagaya, M; Konagaya, Y; Thomas, J W; Banner, C; Mill, J; Max, S R

    1990-05-01

    The mechanism of glutamine synthetase induction in rat skeletal muscle after denervation or limb immobilization was investigated. Adult male rats were subjected to midthigh section of the sciatic nerve. At 1, 2, and 5 h and 1, 2, and 7 days after denervation, rats were killed and denervated, and contralateral control soleus and plantaris muscles were excised, weighted, homogenized, and assayed for glutamine synthetase. Glutamine synthetase activity increased approximately twofold 1 h after denervation in both muscles. By 7 days postdenervation enzyme activity had increased to three times the control level in plantaris muscle and to four times the control level in soleus muscle. Increased enzyme activity after nerve section was associated with increased maximum velocity with no change in apparent Michaelis constant. Immunotitration with an antiglutamine synthetase antibody suggested that denervation caused an increase in the number of glutamine synthetase molecules in muscle. However, Northern-blot analysis revealed no increase in the steady-state level of glutamine synthetase mRNA after denervation. A mixing experiment failed to yield evidence for the presence of a soluble factor involved in regulating the activity of glutamine synthetase in denervated muscle. A combination of denervation and dexamethasone injections resulted in additive increases in glutamine synthetase. Thus the mechanism underlying increased glutamine synthetase after denervation appears to be posttranscriptional and is distinct from that of the glucocorticoid-mediated glutamine synthetase induction previously described by us. PMID:1970709

  9. Mitochondrial tRNA gene translocations in highly eusocial bees

    Directory of Open Access Journals (Sweden)

    Daniela Silvestre

    2006-01-01

    Full Text Available Mitochondrial gene rearrangement events, especially involving tRNA genes, have been described more frequently as more complete mitochondrial genome sequences are becoming available. In the present work, we analyzed mitochondrial tRNA gene rearrangements between two bee species belonging to the tribes Apini and Meliponini within the "corbiculate Apidae". Eleven tRNA genes are in different genome positions or strands. The molecular events responsible for each translocation are explained. Considering the high number of rearrangements observed, the data presented here contradict the general rule of high gene order conservation among closely related organisms, and also represent a powerful molecular tool to help solve questions about phylogeny and evolution in bees.

  10. Interaction of tRNA with MEK2 in pancreatic cancer cells

    OpenAIRE

    Xiaoyun Wang; Christina R. Chow; Kazumi Ebine; Jiyoung Lee; Marsha R Rosner; Tao Pan; Munshi, Hidayatullah G.

    2016-01-01

    Although the translational function of tRNA has long been established, extra translational functions of tRNA are still being discovered. We previously developed a computational method to systematically predict new tRNA-protein complexes and experimentally validated six candidate proteins, including the mitogen-activated protein kinase kinase 2 (MEK2), that interact with tRNA in HEK293T cells. However, consequences of the interaction between tRNA and these proteins remain to be elucidated. Her...

  11. Biosynthesis and functions of sulfur modifications in tRNA

    OpenAIRE

    Shigi, Naoki

    2014-01-01

    Sulfur is an essential element for a variety of cellular constituents in all living organisms. In tRNA molecules, there are many sulfur-containing nucleosides, such as the derivatives of 2-thiouridine (s2U), 4-thiouridine (s4U), 2-thiocytidine (s2C), and 2-methylthioadenosine (ms2A). Earlier studies established the functions of these modifications for accurate and efficient translation, including proper recognition of the codons in mRNA or stabilization of tRNA structure. In many cases, the b...

  12. Recognition of aminoacyl-tRNA: a common molecular mechanism revealed by cryo-EM

    OpenAIRE

    Li, Wen; Agirrezabala, Xabier; Lei, Jianlin; Bouakaz, Lamine; Brunelle, Julie L; Ortiz-Meoz, Rodrigo F.; Green, Rachel; Sanyal, Suparna; Ehrenberg, Måns; Frank, Joachim

    2008-01-01

    The accuracy of ribosomal translation is achieved by an initial selection and a proofreading step, mediated by EF-Tu, which forms a ternary complex with aminoacyl(aa)-tRNA. To study the binding modes of different aa-tRNAs, we compared cryo-EM maps of the kirromycin-stalled ribosome bound with ternary complexes containing Phe-tRNAPhe, Trp-tRNATrp, or Leu-tRNALeuI. The three maps suggest a common binding manner of cognate aa-tRNAs in their specific binding with both the ribosome and EF-Tu. All ...

  13. Aromatase inhibitors and anti-synthetase syndrome.

    Science.gov (United States)

    Mascella, Fabio; Gianni, Lorenzo; Affatato, Alessandra; Fantini, Manuela

    2016-09-01

    Adjuvant therapy in postmenopausal women with endocrine-responsive breast cancer (BC) is actually centered on the use of anti-aromatase inhibitors (AI). Several reports, however, are emerging in literature associating the use of this drugs to rheumatic disorders. This case report describes the first case of anti-synthetase syndrome diagnosis after treatment with anti-estrogen agents in a patient with pre-existing rheumatoid arthritis. PMID:27225465

  14. Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of glutamyl-tRNA synthetase (Xoo1504) from Xanthomonas oryzae pv. oryzae

    International Nuclear Information System (INIS)

    Glutamyl-tRNA synthetase (GluRS) is an essential enzyme for protein translation and has been considered as a target for drug development. In this paper, the cloning, expression and purification of the GluRS protein encoded by the gltX gene (Xoo1504) and its preliminarily crystallographic analysis are reported. The gltX gene from Xanthomonas oryzae pv. oryzae (Xoo1504) encodes glutamyl-tRNA synthetase (GluRS), one of the most important enzymes involved in bacterial blight (BB), which causes huge production losses of rice worldwide. GluRS is a class I-type aminoacyl-tRNA synthetase (aaRS) that is primarily responsible for the glutamylation of tRNAGlu. It plays an essential role in protein synthesis, as well as the regulation of cells, in all organisms. As it represents an important target for the development of new antibacterial drugs against BB, determination of the three-dimensional structure of GluRS is essential in order to understand its catalytic mechanism. In order to analyze its structure and function, the gltX gene was cloned and the GluRS enzyme was expressed, purified and then crystallized. A GluRS crystal belonging to the monoclinic space group C2 diffracted to 2.8 Å resolution and had unit-cell parameters a = 186.8, b = 108.4, c = 166.1 Å, β = 96.3°. The unit-cell volume of the crystal allowed the presence of six to eight monomers in the asymmetric unit, with a corresponding Matthews coefficient (VM) range of 2.70–2.02 Å3 Da−1 and a solvent-content range of 54.5–39.3%

  15. Translocation and rotation of tRNA during template-independent RNA polymerization by tRNA nucleotidyltransferase.

    Science.gov (United States)

    Yamashita, Seisuke; Takeshita, Daijiro; Tomita, Kozo

    2014-02-01

    The 3'-terminal CCA (CCA-3' at positions 74-76) of tRNA is synthesized by CCA-adding enzyme using CTP and ATP as substrates, without a nucleic acid template. In Aquifex aeolicus, CC-adding and A-adding enzymes collaboratively synthesize the CCA-3'. The mechanism of CCA-3' synthesis by these two enzymes remained obscure. We now present crystal structures representing CC addition onto tRNA by A. aeolicus CC-adding enzyme. After C₇₄ addition in an enclosed active pocket and pyrophosphate release, the tRNA translocates and rotates relative to the enzyme, and C₇₅ addition occurs in the same active pocket as C₇₄ addition. At both the C₇₄-adding and C₇₅-adding stages, CTP is selected by Watson-Crick-like hydrogen bonds between the cytosine of CTP and conserved Asp and Arg residues in the pocket. After C₇₄C₇₅ addition and pyrophosphate release, the tRNA translocates further and drops off the enzyme, and the CC-adding enzyme terminates RNA polymerization. PMID:24389024

  16. AMINOACYL FUCOSIDES AS POSSIBLE BIOCHEMICAL MARKERS OF TUMORIGENIC AND METASTATIC POTENTIAL IN HERPES SIMPLEX VIRUS TYPE 2-TRANSFORMED RAT CELLS

    Science.gov (United States)

    Two classes of aminoacyl fucosides termed F13 and F14 were studied as possible markers of tumorigenic and metastatic potential in herpes simplex virus type 2 transformed rat cells. In the present study, clonal cell lines of transformed highly tumorigenic and metastatic (t-REF-G-2...

  17. Semisynthesis and biological activity of aminoacyl triesters of squamocin, an annonaceous acetogenin.

    Science.gov (United States)

    Duval, Romain A; Duret, Philippe; Lewin, Guy; Peris, Eva; Hocquemiller, Reynald

    2005-06-01

    A number of aminoacyl triesters of squamocin 1, a cytotoxic acetogenin isolated from the seeds of Annona reticulata, have been synthesized in two to three steps from protected (l)-aminoacids and squamocin 1 using standard coupling/deprotection procedures. These semisynthetic analogs were tested on submitochondrial particles (SMP) for their complex I inhibitory activities, and against KB 3-1 cells in vitro. All triesters derivatives exhibited a complete extinction of activity at the enzymatic level, correlated to a reduced though modulated cytotoxicity in comparison with squamocin 1. This activity can apparently be considered as a function of the amphipathy of the analogs, the more amphiphilic ones being the more cytotoxic. PMID:15863004

  18. Biosynthetic engineering of nonribosomal peptide synthetases.

    Science.gov (United States)

    Kries, Hajo

    2016-09-01

    From the evolutionary melting pot of natural product synthetase genes, microorganisms elicit antibiotics, communication tools, and iron scavengers. Chemical biologists manipulate these genes to recreate similarly diverse and potent biological activities not on evolutionary time scales but within months. Enzyme engineering has progressed considerably in recent years and offers new screening, modelling, and design tools for natural product designers. Here, recent advances in enzyme engineering and their application to nonribosomal peptide synthetases are reviewed. Among the nonribosomal peptides that have been subjected to biosynthetic engineering are the antibiotics daptomycin, calcium-dependent antibiotic, and gramicidin S. With these peptides, incorporation of unnatural building blocks and modulation of bioactivities via various structural modifications have been successfully demonstrated. Natural product engineering on the biosynthetic level is not a reliable method yet. However, progress in the understanding and manipulation of biosynthetic pathways may enable the routine production of optimized peptide drugs in the near future. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:27465074

  19. Nucleotide sequence of Streptomyces griseus initiator tRNA.

    OpenAIRE

    Kuchino, Y; Yamamoto, I.; Nishimura, S.

    1982-01-01

    The primary structure of initiator tRNA from Streptomyces griseus was determined by post-labeling procedures. The nucleotide sequence is pC-G-C-G-G-G-G-U-G-G-A-G-C-A-G-C-U-C-G-G-D-A-G-C-U-C-G-C-U-G-G-G-C-U-C-A-U-A-A-C-C- C-A-G-A-G-G-U-C-G-C-A-G-G-U-psi-C-A-m1A-A-U-C-C-U-G-U-C-C-C-C-G-C-U-A-C-C-A0H. The unique feature of the sequence of this tRNA is that residue 54 is occupied by unmodified U, while ribothymidine is located in that position in most initiator tRNAs from eubacteria.

  20. Acinetobacter species identification by using tRNA spacer fingerprinting.

    OpenAIRE

    Ehrenstein, B; Bernards, A T; Dijkshoorn, L.; Gerner-Smidt, P; Towner, K. J.; Bouvet, P J; Daschner, F D; Grundmann, H

    1996-01-01

    Identification of Acinetobacter spp. to the DNA group level by phenotypic techniques is problematic, and there is a need for an alternative identification method for routine use. The present study validated the suitability of a rapid identification technique based on tRNA spacer (tDNA) fingerprinting in comparison with that of a commercially available assay involving carbon source utilization tests (Biolog MicroStation System) for identifying the 21 DNA-DNA hybridization groups belonging to t...

  1. Photoaffinity labelling of t-RNA binding sites

    International Nuclear Information System (INIS)

    For the photoaffinity labelling of E.coli ribosomes in the region of peptidyl transferase, an analogue to the substrate peptidyl-tRNA-ethyl-2-diazomalalonyl-Phe-tRNAsup(Phe) was synthesized. UV irradiation of the reversible complex with 70S ribosomes and poly(U) led to the formation of a covalent bond between N-acyl-Phe-tRNA and 23S-rRNA. The irreversibly bound N-acyl-phenylalanyl group may be transferred to puromycin in a reaction catalyzed by peptidyl transferase, in the presence of the Phe-tRNA, it forms products of a peptide synthesis covalently bound to 23S-RNA. The 23S-rRNA sequence thus labelled, which has not yet been identified, should therefore be in the active centre of the peptidyl transferase or in its near neighbourhood. An analysis of the reaction product showed that the N-acyl-Phe-tRNA is bound specifically to one or more sites of a 3'-terminal 18S fragment of the 23S-RNA. An attempt to prove the existence of further tRNA interaction with ribosonal substrate binding sites led to the discovery of a poly(U2,G)-stimulated, UV-inducible irreversible binding of valin-specific tRNA (E.coli) to 16S-rRNA in one or several tRNA decoding sites. A preliminary analysis of the T1 fragments of tRNAsup(Val) after binding to 16S-rRNA indicates that the DHU loop of tRNA takes part in this photoreaction. (orig.)

  2. tRNA modifications regulate translation during cellular stress

    OpenAIRE

    Gu, Chen; Thomas J Begley; Peter C. Dedon

    2014-01-01

    The regulation of gene expression in response to stress is an essential cellular protection mechanism. Recent advances in tRNA modification analysis and genome-based codon bias analytics have facilitated studies that lead to a novel model for translational control, with translation elongation dynamically regulated during stress responses. Stress-induced increases in specific anticodon wobble bases are required for the optimal translation of stress response transcripts that are significantly b...

  3. Biosynthesis and functions of sulfur modifications in tRNA

    Directory of Open Access Journals (Sweden)

    Naoki eShigi

    2014-04-01

    Full Text Available Sulfur is an essential element for a variety of cellular constituents in all living organisms. In tRNA molecules, there are many sulfur-containing nucleosides, such as the derivatives of 2‑thiouridine (s2U, 4-thiouridine (s4U, 2-thiocytidine (s2C, and 2-methylthioadenosine (ms2A. Earlier studies established the functions of these modifications for accurate and efficient translation, including proper recognition of the codons in mRNA or stabilization of tRNA structure. In many cases, the biosynthesis of these sulfur modifications starts with cysteine desulfurases, which catalyze the generation of persulfide (an activated form of sulfur from cysteine. Many sulfur-carrier proteins are responsible for delivering this activated sulfur to each biosynthesis pathway. Finally, specific modification enzymes activate target tRNAs and then incorporate sulfur atoms. Intriguingly, the biosynthesis of 2-thiouridine in all domains of life is functionally and evolutionarily related to the ubiquitin-like post-translational modification system of cellular proteins in eukaryotes. This review summarizes the recent characterization of the biosynthesis of sulfur modifications in tRNA and the novel roles of this modification in cellular functions in various model organisms, with a special emphasis on 2-thiouridine derivatives. Each biosynthesis pathway of sulfur-containing molecules is mutually modulated via sulfur trafficking, and 2-thiouridine and codon usage bias have been proposed to control the translation of specific genes.

  4. Biosynthesis and functions of sulfur modifications in tRNA.

    Science.gov (United States)

    Shigi, Naoki

    2014-01-01

    Sulfur is an essential element for a variety of cellular constituents in all living organisms. In tRNA molecules, there are many sulfur-containing nucleosides, such as the derivatives of 2-thiouridine (s(2)U), 4-thiouridine (s(4)U), 2-thiocytidine (s(2)C), and 2-methylthioadenosine (ms(2)A). Earlier studies established the functions of these modifications for accurate and efficient translation, including proper recognition of the codons in mRNA or stabilization of tRNA structure. In many cases, the biosynthesis of these sulfur modifications starts with cysteine desulfurases, which catalyze the generation of persulfide (an activated form of sulfur) from cysteine. Many sulfur-carrier proteins are responsible for delivering this activated sulfur to each biosynthesis pathway. Finally, specific "modification enzymes" activate target tRNAs and then incorporate sulfur atoms. Intriguingly, the biosynthesis of 2-thiouridine in all domains of life is functionally and evolutionarily related to the ubiquitin-like post-translational modification system of cellular proteins in eukaryotes. This review summarizes the recent characterization of the biosynthesis of sulfur modifications in tRNA and the novel roles of this modification in cellular functions in various model organisms, with a special emphasis on 2-thiouridine derivatives. Each biosynthesis pathway of sulfur-containing molecules is mutually modulated via sulfur trafficking, and 2-thiouridine and codon usage bias have been proposed to control the translation of specific genes. PMID:24765101

  5. The interaction between Aquifex aeolicus leucyl-tRNA synthetase and its cognate tRNALeu%超嗜热菌Aquifex aeolicus亮氨酰-tRNA合成酶和tRNALeu的相互作用

    Institute of Scientific and Technical Information of China (English)

    赵明炜; 王恩多

    2007-01-01

    cognate tRNAs and ensures the accurate translation of the genetic code in the first step of the protein synthesis[1]. On the basis of conserved sequence and characteristic structural motifs, aaRSs can be divided into two classes (class Ⅰ and Ⅱ ) with ten members in each class[2]. Leucyl-tRNA synthetase (LeuRS) belongs to class la aaRSs and the canonical LeuRSs are monomer. Aquifex aeolicus αβ-LeuRS is the only known heterodimeric LeuRS[3]. By fusion and recombination of the genes encoding the α and β subunits from A. aeolicus αβ-LeuRS and the equivalent amino-and carboxy-terminal parts of E. coli LeuRS (identified as α' and β'), five monomeric and five heterodimeric LeuRS mutants were obtained. Seven of these were successfully overexpressed in vivo and purified, while three dimeric mutants with the β' part of E. coli LeuRS were not successfully expressed.The seven purified mutants catalyzed amino acid activation, although several exhibited reduced aminoacylation properties. Removal of the last 36 residues of the α subunit of the A. aeolicus enzyme was determined to be deleterious for tRNA charging. The subunit exchange showed that the cross-speciesspecific recognition of A. aeolicus tRNALeu occurs at the α subunit. None of the mixed E. coli-A.aeolicus enzymes were as thermostable as the native αβ-LeuRS. However, the fusion of the α and βpeptides from A. aeolicus as a single chain analogous to canonical LeuRS resulted in a product more resistant to heat denaturation than the original enzyme. The editing reactions catalyzed by aaRSs are critical for the faithful protein synthesis by correcting errors. We reported that only the isolated editing domain (CP1 domain) of αβ-LeuRS catalyzes the hydrolytic editing of both mischarged tRNALeu and minihelixLeu . Within the domain, we identified a crucial 20-amino-acid motif to the editing of CP1 of αβ-LeuRS that confers the editing capacity to the inactive isolated CP1 domain of E. coli LeuRS. However,the motif

  6. Thiolation Controls Cytoplasmic tRNA Stability and Acts as a Negative Determinant for tRNA Editing in Mitochondria

    Czech Academy of Sciences Publication Activity Database

    Wohlgamuth-Benedum, J. M.; RUBIO, M. A. T.; Paris, Zdeněk; Long, Shaojun; Poliak, Pavel; Lukeš, Julius; Alfonzo, J. D.

    2009-01-01

    Roč. 284, č. 36 (2009), s. 23947-23953. ISSN 0021-9258 R&D Projects: GA ČR GA204/06/1558; GA MŠk LC07032; GA MŠk 2B06129 Institutional research plan: CEZ:AV0Z60220518 Keywords : T. brucei * tRNA * 2-thiolation * Fe-S cluster * editing Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.328, year: 2009

  7. Crystallization and preliminary X-ray crystallographic analysis of bacterial tRNA(Sec) in complex with seryl-tRNA synthetase.

    Science.gov (United States)

    Itoh, Yuzuru; Sekine, Shun Ichi; Yokoyama, Shigeyuki

    2012-06-01

    Selenocysteine (Sec) is translationally incorporated into proteins in response to the UGA codon. The tRNA specific to Sec (tRNA(Sec)) is first ligated with serine by seryl-tRNA synthetase (SerRS). To elucidate the tertiary structure of bacterial tRNA(Sec) and its specific interaction with SerRS, the bacterial tRNA(Sec) from Aquifex aeolicus was crystallized as the heterologous complex with the archaeal SerRS from Methanopyrus kandleri. Although X-ray diffraction by crystals of tRNA(Sec) in complex with wild-type SerRS was rather poor (to 5.7 Å resolution), the resolution was improved by introducing point mutations targeting the crystal-packing interface. Heavy-atom labelling also contributed to resolution improvement. A 3.2 Å resolution diffraction data set for phase determination was obtained from a K(2)Pt(CN)(4)-soaked crystal. PMID:22684069

  8. Biallelic Mutations of Methionyl-tRNA Synthetase Cause a Specific Type of Pulmonary Alveolar Proteinosis Prevalent on Réunion Island.

    Science.gov (United States)

    Hadchouel, Alice; Wieland, Thomas; Griese, Matthias; Baruffini, Enrico; Lorenz-Depiereux, Bettina; Enaud, Laurent; Graf, Elisabeth; Dubus, Jean Christophe; Halioui-Louhaichi, Sonia; Coulomb, Aurore; Delacourt, Christophe; Eckstein, Gertrud; Zarbock, Ralf; Schwarzmayr, Thomas; Cartault, François; Meitinger, Thomas; Lodi, Tiziana; de Blic, Jacques; Strom, Tim M

    2015-05-01

    Methionyl-tRNA synthetase (MARS) catalyzes the ligation of methionine to tRNA and is critical for protein biosynthesis. We identified biallelic missense mutations in MARS in a specific form of pediatric pulmonary alveolar proteinosis (PAP), a severe lung disorder that is prevalent on the island of Réunion and the molecular basis of which is unresolved. Mutations were found in 26 individuals from Réunion and nearby islands and in two families from other countries. Functional consequences of the mutated alleles were assessed by growth of wild-type and mutant strains and methionine-incorporation assays in yeast. Enzyme activity was attenuated in a liquid medium without methionine but could be restored by methionine supplementation. In summary, identification of a founder mutation in MARS led to the molecular definition of a specific type of PAP and will enable carrier screening in the affected community and possibly open new treatment opportunities. PMID:25913036

  9. Interaction of tRNA with MEK2 in pancreatic cancer cells

    Science.gov (United States)

    Wang, Xiaoyun; Chow, Christina R.; Ebine, Kazumi; Lee, Jiyoung; Rosner, Marsha R.; Pan, Tao; Munshi, Hidayatullah G.

    2016-01-01

    Although the translational function of tRNA has long been established, extra translational functions of tRNA are still being discovered. We previously developed a computational method to systematically predict new tRNA-protein complexes and experimentally validated six candidate proteins, including the mitogen-activated protein kinase kinase 2 (MEK2), that interact with tRNA in HEK293T cells. However, consequences of the interaction between tRNA and these proteins remain to be elucidated. Here we tested the consequence of the interaction between tRNA and MEK2 in pancreatic cancer cell lines. We also generated disease and drug resistance-derived MEK2 mutants (Q60P, P128Q, S154F, E207K) to evaluate the function of the tRNA-MEK2 interaction. Our results demonstrate that tRNA interacts with the wild-type and mutant MEK2 in pancreatic cancer cells; furthermore, the MEK2 inhibitor U0126 significantly reduces the tRNA-MEK2 interaction. In addition, tRNA affects the catalytic activity of the wild type and mutant MEK2 proteins in different ways. Overall, our findings demonstrate the interaction of tRNA with MEK2 in pancreatic cancer cells and suggest that tRNA may impact MEK2 activity in cancer cells. PMID:27301426

  10. Analysis of the complement and molecular evolution of tRNA genes in cow

    Directory of Open Access Journals (Sweden)

    Barris Wesley C

    2009-04-01

    Full Text Available Abstract Background Detailed information regarding the number and organization of transfer RNA (tRNA genes at the genome level is becoming readily available with the increase of DNA sequencing of whole genomes. However the identification of functional tRNA genes is challenging for species that have large numbers of repetitive elements containing tRNA derived sequences, such as Bos taurus. Reliable identification and annotation of entire sets of tRNA genes allows the evolution of tRNA genes to be understood on a genomic scale. Results In this study, we explored the B. taurus genome using bioinformatics and comparative genomics approaches to catalogue and analyze cow tRNA genes. The initial analysis of the cow genome using tRNAscan-SE identified 31,868 putative tRNA genes and 189,183 pseudogenes, where 28,830 of the 31,868 predicted tRNA genes were classified as repetitive elements by the RepeatMasker program. We then used comparative genomics to further discriminate between functional tRNA genes and tRNA-derived sequences for the remaining set of 3,038 putative tRNA genes. For our analysis, we used the human, chimpanzee, mouse, rat, horse, dog, chicken and fugu genomes to predict that the number of active tRNA genes in cow lies in the vicinity of 439. Of this set, 150 tRNA genes were 100% identical in their sequences across all nine vertebrate genomes studied. Using clustering analyses, we identified a new tRNA-GlyCCC subfamily present in all analyzed mammalian genomes. We suggest that this subfamily originated from an ancestral tRNA-GlyGCC gene via a point mutation prior to the radiation of the mammalian lineages. Lastly, in a separate analysis we created phylogenetic profiles for each putative cow tRNA gene using a representative set of genomes to gain an overview of common evolutionary histories of tRNA genes. Conclusion The use of a combination of bioinformatics and comparative genomics approaches has allowed the confident identification of a

  11. Silencing Near tRNA Genes Requires Nucleolar Localization*S

    OpenAIRE

    Wang, Li; Haeusler, Rebecca A.; Good, Paul D.; Thompson, Martin; Nagar, Sapna; Engelke, David R.

    2005-01-01

    Transcription by RNA polymerase II is antagonized by the presence of a nearby tRNA gene in Saccharomyces cerevisiae. To test hypotheses concerning the mechanism of this tRNA gene-mediated (tgm) silencing, the effects of specific gene deletions were determined. The results show that the mechanism of silencing near tRNA genes is fundamentally different from other forms of transcriptional silencing in yeast. Rather, tgm silencing is dependent on the ability to cluster the dispersed tRNA genes in...

  12. Mod5 protein binds to tRNA gene complexes and affects local transcriptional silencing

    OpenAIRE

    Pratt-Hyatt, Matthew; Pai, Dave A.; Haeusler, Rebecca A.; Wozniak, Glenn G.; Good, Paul D.; Miller, Erin L.; McLeod, Ian X.; Yates, John R.; Hopper, Anita K.; Engelke, David R.

    2013-01-01

    This study provides new insight into the requirements for observed silencing of RNA polymerase II transcription near tRNA genes. Mod5 is a conserved tRNA modification enzyme found in both the nucleus and cytoplasm, although it only modifies tRNAs in the cytoplasm. Mod5 is required for silencing near tRNA genes, and it is bound to both nuclear tRNA gene complexes and nuclear pre-tRNA transcripts. Possible mechanisms for this form of RNA-mediated transcriptional silencing are discussed.

  13. Structures of Trypanosoma brucei methionyl-tRNA synthetase with urea-based inhibitors provide guidance for drug design against sleeping sickness.

    Directory of Open Access Journals (Sweden)

    Cho Yeow Koh

    2014-04-01

    Full Text Available Methionyl-tRNA synthetase of Trypanosoma brucei (TbMetRS is an important target in the development of new antitrypanosomal drugs. The enzyme is essential, highly flexible and displaying a large degree of changes in protein domains and binding pockets in the presence of substrate, product and inhibitors. Targeting this protein will benefit from a profound understanding of how its structure adapts to ligand binding. A series of urea-based inhibitors (UBIs has been developed with IC50 values as low as 19 nM against the enzyme. The UBIs were shown to be orally available and permeable through the blood-brain barrier, and are therefore candidates for development of drugs for the treatment of late stage human African trypanosomiasis. Here, we expand the structural diversity of inhibitors from the previously reported collection and tested for their inhibitory effect on TbMetRS and on the growth of T. brucei cells. The binding modes and binding pockets of 14 UBIs are revealed by determination of their crystal structures in complex with TbMetRS at resolutions between 2.2 Å to 2.9 Å. The structures show binding of the UBIs through conformational selection, including occupancy of the enlarged methionine pocket and the auxiliary pocket. General principles underlying the affinity of UBIs for TbMetRS are derived from these structures, in particular the optimum way to fill the two binding pockets. The conserved auxiliary pocket might play a role in binding tRNA. In addition, a crystal structure of a ternary TbMetRS•inhibitor•AMPPCP complex indicates that the UBIs are not competing with ATP for binding, instead are interacting with ATP through hydrogen bond. This suggests a possibility that a general 'ATP-engaging' binding mode can be utilized for the design and development of inhibitors targeting tRNA synthetases of other disease-causing pathogen.

  14. Aminoacyl-tRNA-charged eukaryotic elongation factor 1A is a bona fide substrate for Legionelle pneumophila effector glucosyltransferases

    DEFF Research Database (Denmark)

    Tzivelekidis, Tina; Jank, Thomas; Pohl, Corinna;

    2011-01-01

    Legionella pneumophila, which is the causative organism of Legionnaires disease, translocates numerous effector proteins into the host cell cytosol by a type IV secretion system during infection. Among the most potent effector proteins of Legionella are glucosyltransferases (Lgt’s), which selecti...... the ternary complex, consisting of eEF1A, aminoacyl-tRNA and GTP, is the bona fide substrate for Lgt’s....

  15. Substrate tRNA Recognition Mechanism of a Multisite-specific tRNA Methyltransferase, Aquifex aeolicus Trm1, Based on the X-ray Crystal Structure*

    OpenAIRE

    Awai, Takako; Ochi, Anna; Ihsanawati,; Sengoku, Toru; Hirata, Akira; Bessho, Yoshitaka; Yokoyama, Shigeyuki; Hori, Hiroyuki

    2011-01-01

    Archaeal and eukaryotic tRNA (N2,N2-guanine)-dimethyltransferase (Trm1) produces N2,N2-dimethylguanine at position 26 in tRNA. In contrast, Trm1 from Aquifex aeolicus, a hyper-thermophilic eubacterium, modifies G27 as well as G26. Here, a gel mobility shift assay revealed that the T-arm in tRNA is the binding site of A. aeolicus Trm1. To address the multisite specificity, we performed an x-ray crystal structure study. The overall structure of A. aeolicus Trm1 is similar to that of archaeal Tr...

  16. Genetics Home Reference: carbamoyl phosphate synthetase I deficiency

    Science.gov (United States)

    Skip to main content Your Guide to Understanding Genetic Conditions Enable Javascript for addthis links to activate. ... Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Home Health Conditions carbamoyl phosphate synthetase I deficiency ...

  17. Loss of a Conserved tRNA Anticodon Modification Perturbs Plant Immunity.

    Science.gov (United States)

    Ramírez, Vicente; Gonzalez, Beatriz; López, Ana; Castelló, María José; Gil, María José; Etherington, Graham J; Zheng, Bo; Chen, Peng; Vera, Pablo

    2015-10-01

    tRNA is the most highly modified class of RNA species, and modifications are found in tRNAs from all organisms that have been examined. Despite their vastly different chemical structures and their presence in different tRNAs, occurring in different locations in tRNA, the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s). Recent discoveries have revealed unprecedented complexity in the modification patterns of tRNA, their regulation and function, suggesting that each modified nucleoside in tRNA may have its own specific function. However, in plants, our knowledge on the role of individual tRNA modifications and how they are regulated is very limited. In a genetic screen designed to identify factors regulating disease resistance and activation of defenses in Arabidopsis, we identified SUPPRESSOR OF CSB3 9 (SCS9). Our results reveal SCS9 encodes a tRNA methyltransferase that mediates the 2´-O-ribose methylation of selected tRNA species in the anticodon loop. These SCS9-mediated tRNA modifications enhance during the course of infection with the bacterial pathogen Pseudomonas syringae DC3000, and lack of such tRNA modification, as observed in scs9 mutants, severely compromise plant immunity against the same pathogen without affecting the salicylic acid (SA) signaling pathway which regulates plant immune responses. Our results support a model that gives importance to the control of certain tRNA modifications for mounting an effective immune response in Arabidopsis, and therefore expands the repertoire of molecular components essential for an efficient disease resistance response. PMID:26492405

  18. Loss of a Conserved tRNA Anticodon Modification Perturbs Plant Immunity.

    Directory of Open Access Journals (Sweden)

    Vicente Ramírez

    2015-10-01

    Full Text Available tRNA is the most highly modified class of RNA species, and modifications are found in tRNAs from all organisms that have been examined. Despite their vastly different chemical structures and their presence in different tRNAs, occurring in different locations in tRNA, the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s. Recent discoveries have revealed unprecedented complexity in the modification patterns of tRNA, their regulation and function, suggesting that each modified nucleoside in tRNA may have its own specific function. However, in plants, our knowledge on the role of individual tRNA modifications and how they are regulated is very limited. In a genetic screen designed to identify factors regulating disease resistance and activation of defenses in Arabidopsis, we identified SUPPRESSOR OF CSB3 9 (SCS9. Our results reveal SCS9 encodes a tRNA methyltransferase that mediates the 2´-O-ribose methylation of selected tRNA species in the anticodon loop. These SCS9-mediated tRNA modifications enhance during the course of infection with the bacterial pathogen Pseudomonas syringae DC3000, and lack of such tRNA modification, as observed in scs9 mutants, severely compromise plant immunity against the same pathogen without affecting the salicylic acid (SA signaling pathway which regulates plant immune responses. Our results support a model that gives importance to the control of certain tRNA modifications for mounting an effective immune response in Arabidopsis, and therefore expands the repertoire of molecular components essential for an efficient disease resistance response.

  19. Organisation and sequence determination of glutamine-dependent carbamoyl phosphate synthetase II in Toxoplasma gondii.

    Science.gov (United States)

    Fox, Barbara A; Bzik, David J

    2003-01-01

    Carbamoyl phosphate synthetase II encodes the first enzymic step of de novo pyrimidine biosynthesis. Carbamoyl phosphate synthetase II is essential for Toxoplasma gondii replication and virulence. In this study, we characterised the primary structure of a 28kb gene encoding Toxoplasma gondii carbamoyl phosphate synthetase II. The carbamoyl phosphate synthetase II gene was interrupted by 36 introns. The predicted protein encoded by the 37 carbamoyl phosphate synthetase II exons was a 1,687 amino acid polypeptide with an N-terminal glutamine amidotransferase domain fused with C-terminal carbamoyl phosphate synthetase domains. This bifunctional organisation of carbamoyl phosphate synthetase II is unique, so far, to protozoan parasites from the phylum Apicomplexa (Plasmodium, Babesia, Toxoplasma) or zoomastigina (Trypanosoma, Leishmania). Apicomplexan parasites possessed the largest carbamoyl phosphate synthetase II enzymes due to insertions in the glutamine amidotransferase and carbamoyl phosphate synthetase domains that were not present in the corresponding gene segments from bacteria, plants, fungi and mammals. The C-terminal allosteric regulatory domain, the carbamoyl phosphate synthetase linker domain and the oligomerisation domain were also distinct from the corresponding domains in other species. The novel C-terminal regulatory domain may explain the lack of activation of Toxoplasma gondii carbamoyl phosphate synthetase II by the allosteric effector 5-phosphoribosyl 1-pyrophosphate. Toxoplasma gondii growth in vitro was markedly inhibited by the glutamine antagonist acivicin, an inhibitor of glutamine amidotransferase activity typically associated with carbamoyl phosphate synthetase II, guanosine monophosphate synthetase, or CTP synthetase. PMID:12547350

  20. tRNA - RMG | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available [ Credits ] BLAST Search Image Search Home About Archive Update History Contact us ...genomes of other plants. Data file File name: rmg_trna.zip File URL: ftp://ftp.biosciencedbc.jp/archive/rmg/...cription Download License Update History of This Database Site Policy | Contact Us tRNA - RMG | LSDB Archive ...

  1. An NMR Approach to tRNA Tertiary Structure in Solution

    NARCIS (Netherlands)

    Robillard, G.T.; Tarr, C.E.; Vosman, F.; Sussman, J.L.

    1977-01-01

    Atomic coordinates of E. Coli tRNA1Val have been generated from the X-ray crystal structure of Yeast tRNAPhe by base substitution followed by idealization. The NMR spectrum of E. Coli tRNA1Val was then calculated using these coordinates and ring current calculations. The similarity between the calcu

  2. Recognition of aminoacyl-tRNA: a common molecular mechanism revealed by cryo-EM.

    Science.gov (United States)

    Li, Wen; Agirrezabala, Xabier; Lei, Jianlin; Bouakaz, Lamine; Brunelle, Julie L; Ortiz-Meoz, Rodrigo F; Green, Rachel; Sanyal, Suparna; Ehrenberg, Måns; Frank, Joachim

    2008-12-17

    The accuracy of ribosomal translation is achieved by an initial selection and a proofreading step, mediated by EF-Tu, which forms a ternary complex with aminoacyl(aa)-tRNA. To study the binding modes of different aa-tRNAs, we compared cryo-EM maps of the kirromycin-stalled ribosome bound with ternary complexes containing Phe-tRNA(Phe), Trp-tRNA(Trp), or Leu-tRNA(LeuI). The three maps suggest a common binding manner of cognate aa-tRNAs in their specific binding with both the ribosome and EF-Tu. All three aa-tRNAs have the same 'loaded spring' conformation with a kink and twist between the D-stem and anticodon stem. The three complexes are similarly integrated in an interaction network, extending from the anticodon loop through h44 and protein S12 to the EF-Tu-binding CCA end of aa-tRNA, proposed to signal cognate codon-anticodon interaction to the GTPase centre and tune the accuracy of aa-tRNA selection. PMID:19020518

  3. Aminoacyl-anthraquinone conjugates as telomerase inhibitors: synthesis, biophysical and biological evaluation.

    Science.gov (United States)

    Zagotto, Giuseppe; Sissi, Claudia; Lucatello, Lorena; Pivetta, Claudia; Cadamuro, Sergio A; Fox, Keith R; Neidle, Stephen; Palumbo, Manlio

    2008-09-25

    The telomerase-telomere complex is a prospective anticancer target. To inhibit enzyme activity by induction of G-quadruplex in human telomeres, we have synthesized a small library of 2,6- and 2,7-amino-acyl/ peptidyl anthraquinones with diverse connecting linkers, charge, lipophilicity and bulk. The test compounds modulated G-quadruplex stability to different extents and showed clear preference for quadruplex over duplex DNA. Telomerase inhibition correlated with G-quadruplex stabilization. A SAR analysis showed that type of linkage between the linker and the anthraquinone, together with the position of the side chains and the nature of the amino acid components play a major role both in stabilizing G-quadruplex and producing telomerase inhibition. Short-term cytotoxic activity was poor. However, after prolonged exposure to effective G-quadruplex binders, cells became senescent. These results are of help in the rational design of more efficient G-quadruplex stabilizers, possibly endowed with cancer cell-selective antiproliferative effects. PMID:18754611

  4. tRNA gene diversity in the three domains of life

    Directory of Open Access Journals (Sweden)

    Kosuke eFujishima

    2014-05-01

    Full Text Available Transfer RNA (tRNA is widely known for its key role in decoding mRNA into protein. Despite their necessity and relatively short nucleotide sequences, a large diversity of gene structures and RNA secondary structures of pre-tRNAs and mature tRNAs have recently been discovered in the three domains of life. Growing evidences of disrupted tRNA genes in the genomes of Archaea reveals unique gene structures such as, intron-containing tRNA, split tRNA, and permuted tRNA. Coding sequence for these tRNAs are either separated with introns, fragmented, or permuted at the genome level. Although evolutionary scenario behind the tRNA gene disruption is still unclear, diversity of tRNA structure seems to be co-evolved with their processing enzyme, so-called RNA splicing endonuclease. Metazoan mitochondrial tRNAs (mtRNAs are known for their unique lack of either one or two arms from the typical tRNA cloverleaf structure, while still maintaining functionality. Recently identified nematode-specific V-arm containing tRNAs (nev-tRNAs possess long variable arms that are specific to eukaryotic class II tRNASer and tRNALeu but also decode class I tRNA codons. Moreover, many tRNA-like sequences have been found in the genomes of different organisms and viruses. Thus this review is aimed to cover the latest knowledge on tRNA gene diversity and further recapitulate the evolutionary and biological aspects that caused such uniqueness.

  5. The Effect of N-terminal Changes on Arginyl-tRNA Synthetase from Escherichia coli%N端的改变对大肠杆菌精氨酰-tRNA合成酶的影响

    Institute of Scientific and Technical Information of China (English)

    刘文; 刘默芳; 夏宪; 王恩多; 王应睐

    2002-01-01

    得到了缺失Asn2的大肠杆菌(E.coli)精氨酰-tRNA合成酶(ArgRS)的变种和在其N端添加酵母ArgRS的N端23个氨基酸残基的嵌合变种. 它们的基因在大肠杆菌中表达时, 可能由于蛋白质误折叠, 大部分产生了包涵体. 与天然酶相比, 缺失变种保留了全部的氨基酸活化活力, 但氨基酰化活力下降了26%; 嵌合变种的以上两种活力下降了90%以上, 不能氨基酰化酵母tRNAArg. 缺失Asn2和Ile3的变种在E.coli中虽被表达, 但不稳定. 与天然酶相比, 嵌合变种的荧光光谱的最大发射波长向长波移动, 强度减小. 表明变种酶的构象和天然酶不同, 色氨酸更暴露. 用远紫外CD光谱预测变种酶的二级结构表明, 嵌合酶的α螺旋更少, β折叠更多, 无规卷曲稍多. E.coli ArgRS的N端结构域对活力和正确折叠是重要的.%An Asn2 deleted mutant of Escherichia coli arginyl-tRNA synthetase deleted Asn2 and a chimera mutant, in which the N-terminal 23 amino acid residues of yeast arginyl-tRNA synthetase were appended to the N-terminus of Escherichia coli synthetase, were synthesized and studied. The expression of the deletion and chimera mutants in Escherichia coli formed inclusion bodies, presumably due to improper folding of the proteins. Relative to the native enzyme, the deletion mutant showed full amino acid activation activity and a 26% reduction in aminoacylation activity, while the chimera mutant lost 93% and 96% activities in amino acid activation and aminoacylation, respectively, and did not aminoacylate yeast tRNAArg at all. The mutant deleted Asn2 and Ile3 was able to be expressed in Escherichia coli but not stable to be purified. The emission maximum wavelength in the fluorescence spectra of the chimera mutants shifted to longer one and the corresponding intensities decreased, when compared with those of the native enzyme. The data show that the conformation of the mutants are different and the tryptophan residues in

  6. SCREENING OF ANTIMICROBIAL ACTIVITY AND GENES CODING POLYKETIDE SYNTHETASE AND NONRIBOSOMAL PEPTIDE SYNTHETASE OF ACTINOMYCETE ISOLATES

    Directory of Open Access Journals (Sweden)

    Silvia Kovácsová

    2013-12-01

    Full Text Available The aim of this study was to observe antimicrobial activity using agar plate diffusion method and screening genes coding polyketide synthetase (PKS-I and nonribosomal peptide synthetase (NRPS from actinomycetes. A total of 105 actinomycete strains were isolated from arable soil. Antimicrobial activity was demonstrated at 54 strains against at least 1 of total 12 indicator organisms. Antifungal properties were recorded more often than antibacterial properties. The presence of PKS-I and NRPS genes were founded at 61 of total 105 strains. The number of strains with mentioned biosynthetic enzyme gene fragments matching the anticipated length were 19 (18% and 50 (47% respectively. Overall, five actinomycete strains carried all the biosynthetical genes, yet no antimicrobial activity was found against any of tested pathogens. On the other hand, twenty-one strains showed antimicrobial activity even though we were not able to amplify any of the PKS or NRPS genes from them. Combination of the two methods showed broad-spectrum antimicrobial activity of actinomycetes isolated from arable soil, which indicate that actinomycetes are valuable reservoirs of novel bioactive compounds.

  7. Profiling and tandem mass spectrometry analysis of aminoacylated phospholipids in Bacillus subtilis  [version 2; referees: 1 approved, 2 approved with reservations

    Directory of Open Access Journals (Sweden)

    Metin Atila

    2016-04-01

    Full Text Available Cationic modulation of the dominantly negative electrostatic structure of phospholipids plays an important role in bacterial response to changes in the environment. In addition to zwitterionic phosphatidylethanolamine, Gram-positive bacteria are also abundant in positively charged lysyl-phosphatidylglycerol. Increased amounts of both types of lipids render Gram-positive bacterial cells more resistant to cationic antibiotic peptides such as defensins.  Lysyl and alanyl-phosphatidylglycerol as well as alanyl-cardiolipin have also been studied by mass spectroscopy. Phospholipids modified by other amino acids have been discovered by chemical analysis of the lipid lysate but have yet to be studied by mass spectroscopy. We exploited the high sensitivity of modern mass spectroscopy in searching for substructures in complex mixtures to establish a sensitive and thorough screen for aminoacylated phospholipids. The search for deprotonated aminoacyl anions in lipid extracted from Bacillus subtilis strain 168 yielded strong evidence as well as relative abundance of aminoacyl-phosphatidylglycerols, which serves as a crude measure of the specificity of aminoacyl-phosphatidylglycerol synthase MprF. No aminoacyl-cardiolipin was found. More importantly, the second most abundant species in this category is D-alanyl-phosphatidylglycerol, suggesting a possible role in the D-alanylation pathway of wall- and lipo-teichoic acids.

  8. Profiling and tandem mass spectrometry analysis of aminoacylated phospholipids in Bacillus subtilis  [version 1; referees: 1 approved, 2 approved with reservations

    Directory of Open Access Journals (Sweden)

    Metin Atila

    2016-01-01

    Full Text Available Cationic modulation of the dominantly negative electrostatic structure of phospholipids plays an important role in bacterial response to changes in the environment. In addition to zwitterionic phosphatidylethanolamine, Gram-positive bacteria are also abundant in positively charged lysyl-phosphatidylglycerol. Increased amounts of both types of lipids render Gram-positive bacterial cells more resistant to cationic antibiotic peptides such as defensins.  Lysyl and alanyl-phosphatidylglycerol as well as alanyl-cardiolipin have also been studied by mass spectroscopy. Phospholipids modified by other amino acids have been discovered by chemical analysis of the lipid lysate but have yet to be studied by mass spectroscopy. We exploited the high sensitivity of modern mass spectroscopy in searching for substructures in complex mixtures to establish a sensitive and thorough screen for aminoacylated phospholipids. The search for deprotonated aminoacyl anions in lipid extracted from Bacillus subtilis strain 168 yielded strong evidence as well as relative abundance of aminoacyl-phosphatidylglycerols, which serves as a crude measure of the specificity of aminoacyl-phosphatidylglycerol synthase MprF. No aminoacyl-cardiolipin was found. More importantly, the second most abundant species in this category is D-alanyl-phosphatidylglycerol, suggesting a possible role in the D-alanylation pathway of wall- and lipo-teichoic acids.

  9. Analogies between the tRNA methylating enzymes and tRNA's in embryonic and tumor tissues

    Energy Technology Data Exchange (ETDEWEB)

    Borek, E.

    1975-01-01

    Progress is reported in the following areas of research, role of tRNA in protein synthesis and as a carrier of amino acids; histidine pathway in Salmonella typhimurium; role of tRNA in regulation of translation; ribosomal binding reactions; role of tRNA in hemoglobin synthesis; population of tRNA's in mutant of Drosophila; methylation of tRNA and DNA by dimethylnitrosamine; purification of DNA methylase from HeLa cell nuclei; effects of age on levels of excretion of tRNA breakdown products in cancer patients; and tyrosyl tRNA's in embryonic and adult liver and in hepatomas. (HLW)

  10. Study of the Binding Energies between Unnatural Amino Acids and Engineered Orthogonal Tyrosyl-tRNA Synthetases

    Science.gov (United States)

    Ren, Wei; Truong, Tan M.; Ai, Hui-Wang

    2015-07-01

    We utilized several computational approaches to evaluate the binding energies of tyrosine (Tyr) and several unnatural Tyr analogs, to several orthogonal aaRSes derived from Methanocaldococcus jannaschii and Escherichia coli tyrosyl-tRNA synthetases. The present study reveals the following: (1) AutoDock Vina and ROSETTA were able to distinguish binding energy differences for individual pairs of favorable and unfavorable aaRS-amino acid complexes, but were unable to cluster together all experimentally verified favorable complexes from unfavorable aaRS-Tyr complexes; (2) MD-MM/PBSA provided the best prediction accuracy in terms of clustering favorable and unfavorable enzyme-substrate complexes, but also required the highest computational cost; and (3) MM/PBSA based on single energy-minimized structures has a significantly lower computational cost compared to MD-MM/PBSA, but still produced sufficiently accurate predictions to cluster aaRS-amino acid interactions. Although amino acid-aaRS binding is just the first step in a complex series of processes to acylate a tRNA with its corresponding amino acid, the difference in binding energy, as shown by MD-MM/PBSA, is important for a mutant orthogonal aaRS to distinguish between a favorable unnatural amino acid (unAA) substrate from unfavorable natural amino acid substrates. Our computational study should assist further designing and engineering of orthogonal aaRSes for the genetic encoding of novel unAAs.

  11. Pseudouridine-deficient transfer RNAs from Escherichia coli B and their use as substrates for pseudouridine synthetase.

    Science.gov (United States)

    Kwong, L K; Moore, V G; Kaiser, I I

    1977-09-25

    Transfer RNAs isolated from Escherichia coli B grown in the presence of 2-thiouracil are deficient in pseudouridine. Much of this deficiency is from the T psi C region, which has only about 50% of its normal pseudouridine content. The other modified nucleoside from this region, ribothymidine, is reduced by only about 10%. Studies showed that 2-thiouracil is incoproated into the RNA of E. coli during growth in the presence of the analog. This incorporation appears to result from the replacement of uracil, occur in a random manner, and involve all RNA species. The extent of incorporation varies from 1 to 3 mol %, depending upon the preparation and RNA species examined. Electrophoresis on polyacrylamide gels and chromatography on Sephadex G-75 and reverse phase (Systen 5) columns of normal and 2-thiouracil-containing tRNAs revealed no profile differences. No accumulation of any precursor tRNA in the thiopyrimidine-treated cells is found. A partial recovery of the pseudouridine content of 2-thiouracil-containing tRNAs can be achieved in vivo by removal of the 2-thiouracil from the culture media. These transfer RNAs have also been used as substrates to study the properties of a partially purified preparation of pseudouridine synthetase II invitro and should be useful as substrates in the further purification of this enzyme. PMID:330528

  12. A Conserved Proline Triplet in Val-tRNA Synthetase and the Origin of Elongation Factor P

    Directory of Open Access Journals (Sweden)

    Agata L. Starosta

    2014-10-01

    Full Text Available Bacterial ribosomes stall on polyproline stretches and require the elongation factor P (EF-P to relieve the arrest. Yet it remains unclear why evolution has favored the development of EF-P rather than selecting against the occurrence of polyproline stretches in proteins. We have discovered that only a single polyproline stretch is invariant across all domains of life, namely a proline triplet in ValS, the tRNA synthetase, that charges tRNAVal with valine. Here, we show that expression of ValS in vivo and in vitro requires EF-P and demonstrate that the proline triplet located in the active site of ValS is important for efficient charging of tRNAVal with valine and preventing formation of mischarged Thr-tRNAVal as well as efficient growth of E. coli in vivo. We suggest that the critical role of the proline triplet for ValS activity may explain why bacterial cells coevolved the EF-P rescue system.

  13. Evolutionary divergence of chloroplast FAD synthetase proteins

    Directory of Open Access Journals (Sweden)

    Arilla-Luna Sonia

    2010-10-01

    Full Text Available Abstract Background Flavin adenine dinucleotide synthetases (FADSs - a group of bifunctional enzymes that carry out the dual functions of riboflavin phosphorylation to produce flavin mononucleotide (FMN and its subsequent adenylation to generate FAD in most prokaryotes - were studied in plants in terms of sequence, structure and evolutionary history. Results Using a variety of bioinformatics methods we have found that FADS enzymes localized to the chloroplasts, which we term as plant-like FADS proteins, are distributed across a variety of green plant lineages and constitute a divergent protein family clearly of cyanobacterial origin. The C-terminal module of these enzymes does not contain the typical riboflavin kinase active site sequence, while the N-terminal module is broadly conserved. These results agree with a previous work reported by Sandoval et al. in 2008. Furthermore, our observations and preliminary experimental results indicate that the C-terminus of plant-like FADS proteins may contain a catalytic activity, but different to that of their prokaryotic counterparts. In fact, homology models predict that plant-specific conserved residues constitute a distinct active site in the C-terminus. Conclusions A structure-based sequence alignment and an in-depth evolutionary survey of FADS proteins, thought to be crucial in plant metabolism, are reported, which will be essential for the correct annotation of plant genomes and further structural and functional studies. This work is a contribution to our understanding of the evolutionary history of plant-like FADS enzymes, which constitute a new family of FADS proteins whose C-terminal module might be involved in a distinct catalytic activity.

  14. The glutamine synthetase gene family in Populus

    Directory of Open Access Journals (Sweden)

    Cánovas Francisco M

    2011-08-01

    Full Text Available Abstract Background Glutamine synthetase (GS; EC: 6.3.1.2, L-glutamate: ammonia ligase ADP-forming is a key enzyme in ammonium assimilation and metabolism of higher plants. The current work was undertaken to develop a more comprehensive understanding of molecular and biochemical features of GS gene family in poplar, and to characterize the developmental regulation of GS expression in various tissues and at various times during the poplar perennial growth. Results The GS gene family consists of 8 different genes exhibiting all structural and regulatory elements consistent with their roles as functional genes. Our results indicate that the family members are organized in 4 groups of duplicated genes, 3 of which code for cytosolic GS isoforms (GS1 and 1 which codes for the choroplastic GS isoform (GS2. Our analysis shows that Populus trichocarpa is the first plant species in which it was observed the complete GS family duplicated. Detailed expression analyses have revealed specific spatial and seasonal patterns of GS expression in poplar. These data provide insights into the metabolic function of GS isoforms in poplar and pave the way for future functional studies. Conclusions Our data suggest that GS duplicates could have been retained in order to increase the amount of enzyme in a particular cell type. This possibility could contribute to the homeostasis of nitrogen metabolism in functions associated to changes in glutamine-derived metabolic products. The presence of duplicated GS genes in poplar could also contribute to diversification of the enzymatic properties for a particular GS isoform through the assembly of GS polypeptides into homo oligomeric and/or hetero oligomeric holoenzymes in specific cell types.

  15. Changes in the activity levels of glutamine synthetase, glutaminase and glycogen synthetase in rats subjected to hypoxic stress

    Science.gov (United States)

    Vats, P.; Mukherjee, A. K.; Kumria, M. M. L.; Singh, S. N.; Patil, S. K. B.; Rangnathan, S.; Sridharan, K.

    Exposure to high altitude causes loss of body mass and alterations in metabolic processes, especially carbohydrate and protein metabolism. The present study was conducted to elucidate the role of glutamine synthetase, glutaminase and glycogen synthetase under conditions of chronic intermittent hypoxia. Four groups, each consisting of 12 male albino rats (Wistar strain), were exposed to a simulated altitude of 7620 m in a hypobaric chamber for 6 h per day for 1, 7, 14 and 21 days, respectively. Blood haemoglobin, blood glucose, protein levels in the liver, muscle and plasma, glycogen content, and glutaminase, glutamine synthetase and glycogen synthetase activities in liver and muscle were determined in all groups of exposed and in a group of unexposed animals. Food intake and changes in body mass were also monitored. There was a significant reduction in body mass (28-30%) in hypoxia-exposed groups as compared to controls, with a corresponding decrease in food intake. There was rise in blood haemoglobin and plasma protein in response to acclimatisation. Over a three-fold increase in liver glycogen content was observed following 1 day of hypoxic exposure (4.76+/-0.78 mg.g-1 wet tissue in normal unexposed rats; 15.82+/-2.30 mg.g-1 wet tissue in rats exposed to hypoxia for 1 day). This returned to normal in later stages of exposure. However, there was no change in glycogen synthetase activity except for a decrease in the 21-days hypoxia-exposed group. There was a slight increase in muscle glycogen content in the 1-day exposed group which declined significantly by 56.5, 50.6 and 42% following 7, 14, and 21 days of exposure, respectively. Muscle glycogen synthetase activity was also decreased following 21 days of exposure. There was an increase in glutaminase activity in the liver and muscle in the 7-, 14- and 21-day exposed groups. Glutamine synthetase activity was higher in the liver in 7- and 14-day exposed groups; this returned to normal following 21 days of exposure

  16. Examining the Gm18 and m1G Modification Positions in tRNA Sequences

    Science.gov (United States)

    Subramanian, Mayavan; Srinivasan, Thangavelu

    2014-01-01

    The tRNA structure contains conserved modifications that are responsible for its stability and are involved in the initiation and accuracy of the translation process. tRNA modification enzymes are prevalent in bacteria, archaea, and eukaryotes. tRNA Gm18 methyltransferase (TrmH) and tRNA m1G37 methyltransferase (TrmD) are prevalent and essential enzymes in bacterial populations. TrmH involves itself in methylation process at the 2'-OH group of ribose at the 18th position of guanosine (G) in tRNAs. TrmD methylates the G residue next to the anticodon in selected tRNA subsets. Initially, m1G37 modification was reported to take place on three conserved tRNA subsets (tRNAArg, tRNALeu, tRNAPro); later on, few archaea and eukaryotes organisms revealed that other tRNAs also have the m1G37 modification. The present study reveals Gm18, m1G37 modification, and positions of m1G that take place next to the anticodon in tRNA sequences. We selected extremophile organisms and attempted to retrieve the m1G and Gm18 modification bases in tRNA sequences. Results showed that the Gm18 modification G residue occurs in all tRNA subsets except three tRNAs (tRNAMet, tRNAPro, tRNAVal). Whereas the m1G37 modification base G is formed only on tRNAArg, tRNALeu, tRNAPro, and tRNAHis, the rest of the tRNAs contain adenine (A) next to the anticodon. Thus, we hypothesize that Gm18 modification and m1G modification occur irrespective of a G residue in tRNAs. PMID:25031570

  17. 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.

  18. A fungal anticodon nuclease ribotoxin exploits a secondary cleavage site to evade tRNA repair.

    Science.gov (United States)

    Meineke, Birthe; Kast, Alene; Schwer, Beate; Meinhardt, Friedhelm; Shuman, Stewart; Klassen, Roland

    2012-09-01

    PaOrf2 and γ-toxin subunits of Pichia acaciae toxin (PaT) and Kluyveromyces lactis zymocin are tRNA anticodon nucleases. These secreted ribotoxins are assimilated by Saccharomyces cerevisiae, wherein they arrest growth by depleting specific tRNAs. Toxicity can be recapitulated by induced intracellular expression of PaOrf2 or γ-toxin in S. cerevisiae. Mutational analysis of γ-toxin has identified amino acids required for ribotoxicity in vivo and RNA transesterification in vitro. Here, we report that PaOrf2 residues Glu9 and His287 (putative counterparts of γ-toxin Glu9 and His209) are essential for toxicity. Our results suggest a similar basis for RNA transesterification by PaOrf2 and γ-toxin, despite their dissimilar primary structures and distinctive tRNA target specificities. PaOrf2 makes two sequential incisions in tRNA, the first of which occurs 3' from the mcm(5)s(2)U wobble nucleoside and depends on mcm(5). A second incision two nucleotides upstream results in the net excision of a di-nucleotide. Expression of phage and plant tRNA repair systems can relieve PaOrf2 toxicity when tRNA cleavage is restricted to the secondary site in elp3 cells that lack the mcm(5) wobble U modification. Whereas the endogenous yeast tRNA ligase Trl1 can heal tRNA halves produced by PaOrf2 cleavage in elp3 cells, its RNA sealing activity is inadequate to complete the repair. Compatible sealing activity can be provided in trans by plant tRNA ligase. The damage-rescuing ability of tRNA repair systems is lost when PaOrf2 can break tRNA at both sites. These results highlight the logic of a two-incision mechanism of tRNA anticodon damage that evades productive repair by tRNA ligases. PMID:22836353

  19. Mutations affecting excision of the intron from a eukaryotic dimeric tRNA precursor.

    OpenAIRE

    Willis, I; Hottinger, H; Pearson, D.; Chisholm, V; Leupold, U; Söll, D

    1984-01-01

    The nucleotide sequences of a Schizosaccharomyces pombe opal suppressor serine tRNA gene (sup9-e) and of 12 in vivo-generated mutant genes, which have lost the ability to suppress UGA mutations, have been determined. Analysis of the expression of these genes in Saccharomyces cerevisiae in vitro and in vivo systems has revealed defects in tRNA gene transcription and precursor tRNA processing. Single base changes in the D-loop, the intron and the extra arm affect the efficiency of splicing of t...

  20. Isolation and nucleotide sequence of a mouse histidine tRNA gene.

    OpenAIRE

    Han, J. H.; Harding, J D

    1982-01-01

    We have sequenced a 1307 base pair mouse genomic DNA fragment which contains a histidine tRNA gene. The sequence of the putative mouse histidine tRNA differs from the published sequence of sheep liver histidine tRNA by a single base change in the D-loop. It does not contain an unpaired 5' terminal G residue, as reported for Drosophila and sheep histidine tRNAs. The gene does not contain introns. The 3' flanking region contains a typical RNA polymerase III termination site of 6 consecutive T r...

  1. A one-step method for in vitro production of tRNA transcripts

    OpenAIRE

    Korenčić, Dragana; Söll, Dieter; Ambrogelly, Alexandre

    2002-01-01

    Sequencing of a large number of microbial genomes has led to the discovery of new enzymes involved in tRNA biosynthesis and tRNA function. Preparation of a great variety of RNA molecules is, therefore, of major interest for biochemical characterization of these proteins. We describe a fast, cost-effective and efficient method for in vitro production of tRNA transcripts. T7 RNA polymerase requires a double-stranded DNA promoter in order to initiate transcription; however, elongation does not r...

  2. Recurrent Isolated Neonatal Hemolytic Anemia: Think About Glutathione Synthetase Deficiency.

    Science.gov (United States)

    Signolet, Isabelle; Chenouard, Rachel; Oca, Florine; Barth, Magalie; Reynier, Pascal; Denis, Marie-Christine; Simard, Gilles

    2016-09-01

    Hemolytic anemia (HA) of the newborn should be considered in cases of rapidly developing, severe, or persistent hyperbilirubinemia. Several causes of corpuscular hemolysis have been described, among which red blood cell enzyme defects are of particular concern. We report a rare case of red blood cell enzyme defect in a male infant, who presented during his first months of life with recurrent and isolated neonatal hemolysis. All main causes were ruled out. At 6.5 months of age, the patient presented with gastroenteritis requiring hospitalization; fortuitously, urine organic acid chromatography revealed a large peak of 5-oxoproline. Before the association between HA and 5-oxoprolinuria was noted, glutathione synthetase deficiency was suspected and confirmed by a low glutathione synthetase concentration and a collapse of glutathione synthetase activity in erythrocytes. Moreover, molecular diagnosis revealed 2 mutations in the glutathione synthetase gene: a previously reported missense mutation (c.[656A>G]; p.[Asp219Gly]) and a mutation not yet described in the binding site of the enzyme (c.[902T>C]; p.[Leu301Pro]). However, 15 days later, a control sample revealed no signs of 5-oxoprolinuria and the clinical history discovered administration of acetaminophen in the 48 hours before hospitalization. Thus, in this patient, acetaminophen exposure allowed the diagnosis of a mild form of glutathione synthetase deficiency, characterized by isolated HA. Early diagnosis is important because treatment with bicarbonate, vitamins C and E, and elimination of trigger factors are recommended to improve long-term outcomes. Glutathione synthetase deficiency should be screened for in cases of unexplained newborn HA. PMID:27581854

  3. Novel Insights into Regulation of Asparagine Synthetase in Conifers

    OpenAIRE

    Canales, Javier; Rueda-López, Marina; Craven-Bartle, Blanca; Avila, Concepción; Cánovas, Francisco M.

    2012-01-01

    Asparagine, a key amino acid for nitrogen storage and transport in plants, is synthesized via the ATP-dependent reaction catalyzed by the enzyme asparagine synthetase (AS; EC 6.3.5.4). In this work, we present the molecular analysis of two full-length cDNAs that encode asparagine synthetase in maritime pine (Pinus pinaster Ait.), PpAS1, and PpAS2. Phylogenetic analyses of the deduced amino acid sequences revealed that both genes are class II AS, suggesting an ancient origin of these genes in ...

  4. Lysine tRNA and cell division: a G1 cell cycle mutant is temperature sensitive for the modification of tRNA5Lys to tRNA4Lys.

    OpenAIRE

    Ortwerth, B J; Lin, V K; Lewis, J.; Wang, R. J.

    1984-01-01

    Ts-694 is a temperature sensitive mutant of hamster cells which is blocked in the G1 phase of the cell cycle at the restrictive temperature of 39 degrees. A comparison of the Lys-tRNA isoacceptors by RPC-5 chromatography showed a decrease in tRNA5Lys and an increase in tRNA4Lys at 39 degrees. This was identical to the changes seen in confluent cultures at the permissive temperature of 33 degrees. These Lys-tRNA changes were not seen in ts-694 cells blocked in G1 by isoleucine deficiency, nor ...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-09-06

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

  6. Base-pairing versatility determines wobble sites in tRNA anticodons of vertebrate mitogenomes.

    Directory of Open Access Journals (Sweden)

    Miguel M Fonseca

    Full Text Available BACKGROUND: Vertebrate mitochondrial genomes typically have one transfer RNA (tRNA for each synonymous codon family. This limited anticodon repertoire implies that each tRNA anticodon needs to wobble (establish a non-Watson-Crick base pairing between two nucleotides in RNA molecules to recognize one or more synonymous codons. Different hypotheses have been proposed to explain the factors that determine the nucleotide composition of wobble sites in vertebrate mitochondrial tRNA anticodons. Until now, the two major postulates--the "codon-anticodon adaptation hypothesis" and the "wobble versatility hypothesis"--have not been formally tested in vertebrate mitochondria because both make the same predictions regarding the composition of anticodon wobble sites. The same is true for the more recent "wobble cost hypothesis". PRINCIPAL FINDINGS: In this study we have analyzed the occurrence of synonymous codons and tRNA anticodon wobble sites in 1553 complete vertebrate mitochondrial genomes, focusing on three fish species with mtDNA codon usage bias reversal (L-strand is GT-rich. These mitogenomes constitute an excellent opportunity to study the evolution of the wobble nucleotide composition of tRNA anticodons because due to the reversal the predictions for the anticodon wobble sites differ between the existing hypotheses. We observed that none of the wobble sites of tRNA anticodons in these unusual mitochondrial genomes coevolved to match the new overall codon usage bias, suggesting that nucleotides at the wobble sites of tRNA anticodons in vertebrate mitochondrial genomes are determined by wobble versatility. CONCLUSIONS/SIGNIFICANCE: Our results suggest that, at wobble sites of tRNA anticodons in vertebrate mitogenomes, selection favors the most versatile nucleotide in terms of wobble base-pairing stability and that wobble site composition is not influenced by codon usage. These results are in agreement with the "wobble versatility hypothesis".

  7. Competing pathways control host resistance to virus via tRNA modification and programmed ribosomal frameshifting

    OpenAIRE

    Maynard, Nathaniel D.; Macklin, Derek N.; Kirkegaard, Karla; Covert, Markus W

    2012-01-01

    Viral infection depends on a complex interplay between host and viral factors. Here, we link host susceptibility to viral infection to a network encompassing sulfur metabolism, tRNA modification, competitive binding, and programmed ribosomal frameshifting (PRF). We first demonstrate that the iron-sulfur cluster biosynthesis pathway in Escherichia coli exerts a protective effect during lambda phage infection, while a tRNA thiolation pathway enhances viral infection. We show that tRNALys uridin...

  8. Increased hepatic glycogen synthetase and decreased phosphorylase in trained rats

    DEFF Research Database (Denmark)

    Galbo, H; Saugmann, P; Richter, Erik

    1979-01-01

    Rats were either physically trained by a 12 wk swimming program or were freely eating or weight matched, sedentary controls. Trained rats had a higher relative liver weight and total hepatic glycogen synthetase (EC 2.4.1.11) activity and a lower phosphorylase (EC 2.4.1.1) activity than the other...

  9. Prostaglandin synthetase inhibitor in an infant with congenital chloride diarrhoea.

    OpenAIRE

    Minford, A M; Barr, D G

    1980-01-01

    Hyper-reninaemia, hypokaluria, and hypokalaemia in an infant with congenital chloride diarrhoea improved during treatment with a prostaglandin synthetase inhibitor, ketoprofen. There was evidence of increased activity of therenin-aldosterone system when ketoprofen was stopped. It is suggested that prostaglandins may be involved in stimulating the renin-aldosterone system in congenital chloride diarrhoea.

  10. Binding of Divalent Magnesium by Escherichia coli Phosphoribosyl Diphosphate Synthetase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne

    1997-01-01

    The mechanism of binding of the substrates MgATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-d-ribosyl a-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, a,ß-methylene ATP and (+)-1-a,2-a...

  11. Binding of divalent magnesium by Escherichia coli phosphoribosyl diphosphate synthetase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne

    1997-01-01

    The mechanism of binding of the substrates Mg x ATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-D-ribosyl (alpha-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, alpha,beta-methylene ATP ...

  12. The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling

    Energy Technology Data Exchange (ETDEWEB)

    Bernard, S.M.; Habash, D.Z.

    2009-07-02

    Glutamine synthetase assimilates ammonium into amino acids, thus it is a key enzyme for nitrogen metabolism. The cytosolic isoenzymes of glutamine synthetase assimilate ammonium derived from primary nitrogen uptake and from various internal nitrogen recycling pathways. In this way, cytosolic glutamine synthetase is crucial for the remobilization of protein-derived nitrogen. Cytosolic glutamine synthetase is encoded by a small family of genes that are well conserved across plant species. Members of the cytosolic glutamine synthetase gene family are regulated in response to plant nitrogen status, as well as to environmental cues, such as nitrogen availability and biotic/abiotic stresses. The complex regulation of cytosolic glutamine synthetase at the transcriptional to post-translational levels is key to the establishment of a specific physiological role for each isoenzyme. The diverse physiological roles of cytosolic glutamine synthetase isoenzymes are important in relation to current agricultural and ecological issues.

  13. Site-directed mutagenesis of Arg58 and Asp86 of elongation factor Tu from Escherichia coli: effects on the GTPase reaction and aminoacyl-tRNA binding

    DEFF Research Database (Denmark)

    Knudsen, Charlotte Rohde; Clark, Brian F. C.

    1996-01-01

    Elongation factor Tu from Escherichia coli was mutated separately at positions Asp86 and Arg58, in order to shed light both on the GTPase mechanism of elongation factor Tu and on the binding of aminoacyl-tRNA. In addition, the binding of guanine nucleotides was investigated by determination of th...

  14. tRNA binding properties of eukaryotic translation initiation factor 2 from Encephalitozoon cuniculi.

    Science.gov (United States)

    Naveau, Marie; Lazennec-Schurdevin, Christine; Panvert, Michel; Mechulam, Yves; Schmitt, Emmanuelle

    2010-10-12

    A critical consequence of the initiation of translation is the setting of the reading frame for mRNA decoding. In eukaryotic and archaeal cells, heterotrimeric initiation factor e/aIF2, in its GTP form, specifically binds Met-tRNA(i)(Met) throughout the translation initiation process. After start codon recognition, the factor, in its GDP-bound form, loses affinity for Met-tRNA(i)(Met) and eventually dissociates from the initiation complex. The role of each aIF2 subunit in tRNA binding has been extensively studied in archaeal systems. The isolated archaeal γ subunit is able to bind tRNA, but the α subunit is required for strong binding. Until now, difficulties during purification have hampered the study of the role of each of the three subunits of eukaryotic eIF2 in specific binding of the initiator tRNA. Here, we have produced the three subunits of eIF2 from Encephalitozoon cuniculi, isolated or assembled into heterodimers or into the full heterotrimer. Using assays following protection of Met-tRNA(i)(Met) against deacylation, we show that the eukaryotic γ subunit is able to bind by itself the initiator tRNA. However, the two peripheral α and β subunits are required for strong binding and contribute equally to tRNA binding affinity. The core domains of α and β probably act indirectly by stabilizing the tRNA binding site on the γ subunit. These results, together with those previously obtained with archaeal aIF2 and yeast eIF2, show species-specific distributions of the roles of the peripheral subunits of e/aIF2 in tRNA binding. PMID:20822097

  15. tRNA sequence data, annotation data and curation data - tRNADB-CE | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available [ Credits ] BLAST Search Image Search Home About Archive Update History Contact us tRNADB-CE tRNA sequence... data, annotation data and curation data Data detail Data name tRNA sequence data, an... first intron 1st Intron end position End position of first intron Seq tRNA sequence Upstream seq. tRNA gene upstream sequence...-leaf secondary structures of tRNA gene Downstream seq. tRNA gene downstream sequence (10 bps) 1st Intron seq. First intron sequence...nd position of second intron 2st Intron seq. Second intron sequence Decision from

  16. Kinetic proofreading at single molecular level: aminoacylation of tRNA(Ile and the role of water as an editor.

    Directory of Open Access Journals (Sweden)

    Mantu Santra

    Full Text Available Proofreading/editing in protein synthesis is essential for accurate translation of information from the genetic code. In this article we present a theoretical investigation of efficiency of a kinetic proofreading mechanism that employs hydrolysis of the wrong substrate as the discriminatory step in enzyme catalytic reactions. We consider aminoacylation of tRNA(Ile which is a crucial step in protein synthesis and for which experimental results are now available. We present an augmented kinetic scheme and then employ methods of stochastic simulation algorithm to obtain time dependent concentrations of different substances involved in the reaction and their rates of formation. We obtain the rates of product formation and ATP hydrolysis for both correct and wrong substrates (isoleucine and valine in our case, respectively, in single molecular enzyme as well as ensemble enzyme kinetics. The present theoretical scheme correctly reproduces (i the amplitude of the discrimination factor in the overall rates between isoleucine and valine which is obtained as (1.8×10(2.(4.33×10(2 = 7.8×10(4, (ii the rates of ATP hydrolysis for both Ile and Val at different substrate concentrations in the aminoacylation of tRNA(Ile. The present study shows a non-michaelis type dependence of rate of reaction on tRNA(Ile concentration in case of valine. The overall editing in steady state is found to be independent of amino acid concentration. Interestingly, the computed ATP hydrolysis rate for valine at high substrate concentration is same as the rate of formation of Ile-tRNA(Ile whereas at intermediate substrate concentration the ATP hydrolysis rate is relatively low. We find that the presence of additional editing domain in class I editing enzyme makes the kinetic proofreading more efficient through enhanced hydrolysis of wrong product at the editing CP1 domain.

  17. Glutamine synthetase gene evolution: A good molecular clock

    International Nuclear Information System (INIS)

    Glutamine synthetase gene evolution in various animals, plants, and bacteria was evaluated by a general stationary Markov model. The evolutionary process proved to be unexpectedly regular even for a time span as long as that between the divergence of prokaryotes from eukaryotes. This enabled us to draw phylogenetic trees for species whose phylogeny cannot be easily reconstructed from the fossil record. The calculation of the times of divergence of the various organelle-specific enzymes led us to hypothesize that the pea and bean chloroplast genes for these enzymes originated from the duplication of nuclear genes as a result of the different metabolic needs of the various species. The data indicate that the duplication of plastid glutamine synthetase genes occurred long after the endosymbiotic events that produced the organelles themselves

  18. tRNA binding, positioning, and modification by the pseudouridine synthase Pus10.

    Science.gov (United States)

    Kamalampeta, Rajashekhar; Keffer-Wilkes, Laura C; Kothe, Ute

    2013-10-23

    Pus10 is the most recently identified pseudouridine synthase found in archaea and higher eukaryotes. It modifies uridine 55 in the TΨC arm of tRNAs. Here, we report the first quantitative biochemical analysis of tRNA binding and pseudouridine formation by Pyrococcus furiosus Pus10. The affinity of Pus10 for both substrate and product tRNA is high (Kd of 30nM), and product formation occurs with a Km of 400nM and a kcat of 0.9s(-1). Site-directed mutagenesis was used to demonstrate that the thumb loop in the catalytic domain is important for efficient catalysis; we propose that the thumb loop positions the tRNA within the active site. Furthermore, a new catalytic arginine residue was identified (arginine 208), which is likely responsible for triggering flipping of the target uridine into the active site of Pus10. Lastly, our data support the proposal that the THUMP-containing domain, found in the N-terminus of Pus10, contributes to binding of tRNA. Together, our findings are consistent with the hypothesis that tRNA binding by Pus10 occurs through an induced-fit mechanism, which is a prerequisite for efficient pseudouridine formation. PMID:23743107

  19. Locating the binding sites of antioxidants resveratrol, genistein and curcumin with tRNA.

    Science.gov (United States)

    N'soukpoé-Kossi, C N; Bourassa, P; Mandeville, J S; Bekale, L; Bariyanga, J; Tajmir-Riahi, H A

    2015-09-01

    We located the binding sites of antioxidants resveratrol, genistein and curcumin on tRNA in aqueous solution at physiological conditions using constant tRNA concentration and various polyphenol contents. FTIR, UV-visible, CD spectroscopic methods and molecular modeling were used to determine polyphenol binding sites, the binding constant and the effects of polyphenol complexation on tRNA conformation and particle formation. Structural analysis showed that polyphenols bind tRNA via G-C and A-U base pairs through hydrophilic, hydrophobic and H-bonding contacts with overall binding constants of K(res-tRNA)=8.95(±0.80)×10(3) M(-1), K(gen-tRNA)=3.07(±0.5)×10(3) M(-1) and K(cur-tRNA)=1.55(±0.3)×10(4) M(-1). Molecular modeling showed the participation of several nucleobases in polyphenol-tRNA adduct formation with free binding energy of -4.43 for resveratrol, -4.26 kcal/mol for genistein and -4.84 kcal/mol for curcumin, indicating that the interaction process is spontaneous at room temperature. While tRNA remains in A-family structure, major biopolymer aggregation and particle formation occurred at high polyphenol contents. PMID:26093317

  20. The glutamine synthetase gene family in Populus

    OpenAIRE

    Cánovas Francisco M; Kirby Edward G; Avila Concepción; Canales Javier; García-Gutiérrez Angel; Castro-Rodríguez Vanessa

    2011-01-01

    Abstract Background Glutamine synthetase (GS; EC: 6.3.1.2, L-glutamate: ammonia ligase ADP-forming) is a key enzyme in ammonium assimilation and metabolism of higher plants. The current work was undertaken to develop a more comprehensive understanding of molecular and biochemical features of GS gene family in poplar, and to characterize the developmental regulation of GS expression in various tissues and at various times during the poplar perennial growth. Results The GS gene family consists ...

  1. Co-purification of dihydrofolate synthetase and N10formyltetrahydropteroyldiglutamate synthetase from E. coli

    International Nuclear Information System (INIS)

    The enzymatic activities which add a single glutamate to dihydropteroate (H2Pte) and to N10formyltetrahydropteroylglutamate (N10formylH4PteGlu) remained at a constant ratio when various purification techniques were used, including ammonium sulfate fractionation, isoelectric focusing, polyacrylamide gel electrophoresis, and column chromatography on Sephadex G-100, DEAE-Sephadex, CM-Sephadex, ADP-Sepharose, N10formylfolate-agarose or matrix Gel Red-A. The best combination of these methods yielded 900-fold purified enzyme. However, the kinetic properties were dependent upon the substrate used. H2Pte was a noncompetitive inhibitor (Kii . 1.1 microM) of the utilization of N10formylH4PteGlu, but no inhibition was detected in the reciprocal experiment. Aminopterin was a competitive inhibitor (Kis . 370 microM) of the reaction with N10formylH4PteGlu but was not inhibitory with H2Pte as substrate. In the dihydrofolate synthetase reaction, in Tris-HCl, pH 8.9 with 50 mM KCl, the apparent Km values for glutamate and MgATP were 3.5 mM and 8.1 microM respectively. With N10formylH4PteGlu as substrate, these Km values were 1.2 mM and 80 microM. Since the two activities were not separated by protein purification procedures but exhibited different kinetic properties (including lack of reciprocal inhibition), the data suggest these reactions are catalyzed on independent sites of a common protein

  2. Selective inhibition of type 2 fatty acid synthetase by the antibiotic thiolactomycin

    Energy Technology Data Exchange (ETDEWEB)

    Nishida, Ikuo; Kawaguchi, Akihiko; Yamada, Mitsuhiro (Tokyo Univ. (Japan). Faculty of Science)

    1984-03-01

    The antibiotic thiolactomycin inhibits the fatty acid synthesis from both (1-/sup 14/C)-acetate and (2/sup 14/C) malonyl-CoA of spinach leaves, developing castor bean endosperms and avocado mesocarp. On the other hand, fatty acid synthetases of Brevibacterium ammoniagenes and Corynebacterium glutamicum are much less sensitive to this antibiotic. As Hayashi et al. have indicated in their paper that thiolactomycin inhibits fatty acid synthetase of Escherichia coli but has little effect on the synthetases of yeast and rat liver, thiolactomycin is suggested to be a selective inhibitor of type 2 fatty acid synthetases.

  3. Selective inhibition of type 2 fatty acid synthetase by the antibiotic thiolactomycin

    International Nuclear Information System (INIS)

    The antibiotic thiolactomycin inhibits the fatty acid synthesis from both [1-14C]-acetate and [214C] malonyl-CoA of spinach leaves, developing castor bean endosperms and avocado mesocarp. On the other hand, fatty acid synthetases of Brevibacterium ammoniagenes and Corynebacterium glutamicum are much less sensitive to this antibiotic. As Hayashi et al. have indicated in their paper that thiolactomycin inhibits fatty acid synthetase of Escherichia coli but has little effect on the synthetases of yeast and rat liver, thiolactomycin is suggested to be a selective inhibitor of type 2 fatty acid synthetases. (author)

  4. From Prebiotics to Probiotics: The Evolution and Functions of tRNA Modifications.

    Science.gov (United States)

    McKenney, Katherine M; Alfonzo, Juan D

    2016-01-01

    All nucleic acids in cells are subject to post-transcriptional chemical modifications. These are catalyzed by a myriad of enzymes with exquisite specificity and that utilize an often-exotic array of chemical substrates. In no molecule are modifications more prevalent than in transfer RNAs. In the present document, we will attempt to take a chemical rollercoaster ride from prebiotic times to the present, with nucleoside modifications as key players and tRNA as the centerpiece that drove the evolution of biological systems to where we are today. These ideas will be put forth while touching on several examples of tRNA modification enzymes and their modus operandi in cells. In passing, we submit that the choice of tRNA is not a whimsical one but rather highlights its critical function as an essential invention for the evolution of protein enzymes. PMID:26985907

  5. A voltage-gated pore for translocation of tRNA

    Energy Technology Data Exchange (ETDEWEB)

    Koley, Sandip; Adhya, Samit, E-mail: nilugrandson@gmail.com

    2013-09-13

    Highlights: •A tRNA translocating complex was assembled from purified proteins. •The complex translocates tRNA at a membrane potential of ∼60 mV. •Translocation requires Cys and His residues in the Fe–S center of RIC6 subunit. -- Abstract: Very little is known about how nucleic acids are translocated across membranes. The multi-subunit RNA Import Complex (RIC) from mitochondria of the kinetoplastid protozoon Leishmania tropica induces translocation of tRNAs across artificial or natural membranes, but the nature of the translocation pore remains unknown. We show that subunits RIC6 and RIC9 assemble on the membrane in presence of subunit RIC4A to form complex R3. Atomic Force Microscopy of R3 revealed particles with an asymmetric surface groove of ∼20 nm rim diameter and ∼1 nm depth. R3 induced translocation of tRNA into liposomes when the pH of the medium was lowered to ∼6 in the absence of ATP. R3-mediated tRNA translocation could also be induced at neutral pH by a K{sup +} diffusion potential with an optimum of 60–70 mV. Point mutations in the Cys{sub 2}–His{sub 2} Fe-binding motif of RIC6, which is homologous to the respiratory Complex III Fe–S protein, abrogated import induced by low pH but not by K{sup +} diffusion potential. These results indicate that the R3 complex forms a pore that is gated by a proton-generated membrane potential and that the Fe–S binding region of RIC6 has a role in proton translocation. The tRNA import complex of L. tropica thus contains a novel macromolecular channel distinct from the mitochondrial protein import pore that is apparently involved in tRNA import in some species.

  6. Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA

    OpenAIRE

    Barhoom, Sima; Kaur, Jaskiran; Cooperman, Barry S.; Smorodinsky, Nechama I.; Smilansky, Zeev; Ehrlich, Marcelo; Elroy-Stein, Orna

    2011-01-01

    We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution through quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). The localization of fl-tRNA to active translation sites was confirmed through its co-localization with cellular factors and its dynamic alterations upon inhibitio...

  7. Hyperaccurate and error-prone ribosomes exploit distinct mechanisms during tRNA selection

    OpenAIRE

    Zaher, Hani S.; Green, Rachel

    2010-01-01

    Escherichia coli strains displaying hyper-accurate (restrictive) and ribosomal ambiguity (ram) phenotypes have long been associated with alterations in rpsL and rpsD/rpsE, respectively. Crystallographic evidence shows the ribosomal proteins S12 and S4/S5 (corresponding to these genes) to be located in separate regions of the small ribosomal subunit that are important for domain-closure thought to take place during tRNA selection. Mechanistically, the process of tRNA selection is separated int...

  8. Protozoan ALKBH8 Oxygenases Display both DNA Repair and tRNA Modification Activities

    DEFF Research Database (Denmark)

    Zdżalik, Daria; Vågbø, Cathrine B; Kirpekar, Finn;

    2014-01-01

    1-8 and FTO. Mammalian and plant ALKBH8 are tRNA hydroxylases targeting 5-methoxycarbonylmethyl-modified uridine (mcm5U) at the wobble position of tRNAGly(UCC). In contrast, the genomes of some bacteria encode a protein with strong sequence homology to ALKBH8, and robust DNA repair activity was......, interestingly, two protozoan ALKBH8s also catalyzed wobble uridine modification of tRNA, thus displaying a dual in vitro activity. Also, we found the modification status of tRNAGly(UCC) to be unaltered in an ALKBH8 deficient mutant of Agrobacterium tumefaciens, indicating that bacterial ALKBH8s have a function...

  9. Anticodon loop mutations perturb reading frame maintenance by the E site tRNA

    OpenAIRE

    Sanders, Christina L.; Lohr, Kristin J.; Gambill, Holly L.; Curran, Ryan B.; Curran, James F.

    2008-01-01

    The ribosomal E site helps hold the reading frame. Certain tRNA mutations affect translation, and anticodon loop mutations can be especially detrimental. We studied the effects of mutations saturating the anticodon loop of the amber suppressor tRNA, Su7, on the ability to help hold the reading frame when in the E site. We also tested three mutations in the anticodon stem, as well as a mutation in the D stem (the “Hirsh” mutation). We used the Escherichia coli RF2 programmed frameshift site to...

  10. Monitoring Protein Synthesis in Living Cells with Fluorescent Labeled tRNA FRET Pairs

    OpenAIRE

    Barhoom, Sima; Farrel, Ian; Dahary, Dvir; Ehrlich, Marcelo; Cooperman, Barry S.; Elroy-Stein, Orna; Smilansky, Zeev

    2013-01-01

    We introduce Protein Synthesis Monitoring (PSM) – a technique to monitor protein synthesis in live cells. In PSM, we transfect cells with tRNA labeled as FRET donors and acceptors. A FRET signal is generated only when a donor- and an acceptor-labeled tRNA come in close contact (< 7nM), as they do on the ribosome during elongation. The intensity of the FRET signal correlates with the number of ribosomes engaged in protein synthesis, providing a real-time, live-cell assay for measuring rates of...

  11. Movement of the 3'-end of tRNA through the peptidyl transferase centre and its inhibition by antibiotics

    DEFF Research Database (Denmark)

    Kirillov, Stanislav; Porse, Bo Torben; Vester, Birthe;

    1997-01-01

    Determining how antibiotics inhibit ribosomal activity requires a detailed understanding of the interactions and relative movement of tRNA, mRNA and the ribosome. Recent models for the formation of hybrid tRNA binding sites during the elongation cycle have provided a basis for re-evaluating earlier...... experimental data and, especially, those relevant to substrate movements through the peptidyl transferase centre. With the exception of deacylated tRNA, which binds at the E-site, ribosomal interactions of the 3'-ends of the tRNA substrates generate only a small part of the total free energy of tRNA......-ribosome binding. Nevertheless, these relatively weak interactions determine the unidirectional movement of tRNAs through the ribosome and, moreover, they appear to be particularly susceptible to perturbation by antibiotics. Here we summarise current ideas relating particularly to the movement of the 3'-ends of tRNA...

  12. eEF1A Mediates the Nuclear Export of SNAG-Containing Proteins via the Exportin5-Aminoacyl-tRNA Complex

    Directory of Open Access Journals (Sweden)

    José Manuel Mingot

    2013-11-01

    Full Text Available Exportin5 mediates the nuclear export of double-stranded RNAs, including pre-microRNAs, adenoviral RNAs, and tRNAs. When tRNAs are aminoacylated, the Exportin5-aminoacyl (aa-tRNA complex recruits and coexports the translation elongation factor eEF1A. Here, we show that eEF1A binds to Snail transcription factors when bound to their main target, the E-cadherin promoter, facilitating their export to the cytoplasm in association with the aa-tRNA-Exportin5 complex. Snail binds to eEF1A through the SNAG domain, a protein nuclear export signal present in several transcription factor families, and this binding is regulated by phosphorylation. Thus, we describe a nuclear role for eEF1A and provide a mechanism for protein nuclear export that attenuates the activity of SNAG-containing transcription factors.

  13. Response of transgenic poplar overexpressing cytosolic glutamine synthetase to phosphinothricin.

    Science.gov (United States)

    Pascual, María Belén; Jing, Zhong Ping; Kirby, Edward G; Cánovas, Francisco M; Gallardo, Fernando

    2008-01-01

    Glutamine synthetase (GS) is the main enzyme involved in ammonia assimilation in plants and is the target of phosphinothricin (PPT), an herbicide commonly used for weed control in agriculture. As a result of the inhibition of GS, PPT also blocks photorespiration, resulting in the depletion of leaf amino acid pools leading to the plant death. Hybrid transgenic poplar (Populus tremula x P. alba INRA clone 7171-B4) overexpressing cytosolic GS is characterized by enhanced vegetative growth [Gallardo, F., Fu, J., Cantón, F.R., García-Gutiérrez, A., Cánovas, F.M., Kirby, E.G., 1999. Expression of a conifer glutamine synthetase gene in transgenic poplar. Planta 210, 19-26; Fu, J., Sampalo, R., Gallardo, F., Cánovas, F.M., Kirby, E.G., 2003. Assembly of a cytosolic pine glutamine synthetase holoenzyme in leaves of transgenic poplar leads to enhanced vegetative growth in young plants. Plant Cell Environ. 26, 411-418; Jing, Z.P., Gallardo, F., Pascual, M.B., Sampalo, R., Romero, J., Torres de Navarra, A., Cánovas, F.M., 2004. Improved growth in a field trial of transgenic hybrid poplar overexpressing glutamine synthetase. New Phytol. 164, 137-145], increased photosynthetic and photorespiratory capacities [El-Khatib, R.T., Hamerlynck, E.P., Gallardo, F., Kirby, E.G., 2004. Transgenic poplar characterized by ectopic expression of a pine cytosolic glutamine synthetase gene exhibits enhanced tolerance to water stress. Tree Physiol. 24, 729-736], enhanced tolerance to water stress (El-Khatib et al., 2004), and enhanced nitrogen use efficiency [Man, H.-M., Boriel, R., El-Khatib, R.T., Kirby, E.G., 2005. Characterization of transgenic poplar with ectopic expression of pine cytosolic glutamine synthetase under conditions of varying nitrogen availability. New Phytol. 167, 31-39]. In vitro plantlets of GS transgenic poplar exhibited enhanced resistance to PPT when compared with non-transgenic controls. After 30 days exposure to PPT at an equivalent dose of 275 g ha(-1), growth

  14. Uroporphyrinogen-I-synthetase activity in red blood cells of lead-exposed workers

    Energy Technology Data Exchange (ETDEWEB)

    El-Waseef, A.

    1982-01-01

    Lead-exposed (n . 26) and control (n . 12) subjects were investigated for their blood lead concentration erythrocyte 5-amino-laevulinic acid dehydratase (5-ALAD) and erythrocyte uroporphyrinogen-I-synthetase (URO-I-S) activity; 5-amino-laevulinic acid (5-ALA) and porphobilinogen (PBG) were used as substrates in the synthetase assay. In the lead workers erythrocyte 5-ALA dehydratase was grossly inhibited but with PBG as substrate the synthetase activity was not significantly different from the control group. With 5-ALA as substrate the synthetase assay showed marked inhibition. Addition of zinc (0.1 mmol/l) and dithiotheritol (0.5 mmol/l) brought the activities of both the dehydratase and synthetase (using 5-ALA as substrate) back into the ranges seen in the control group. With porphobilinogen as substrate higher concentrations of zinc caused inhibition of the synthetase, whilst reduction of added zinc to 0.01 mmol/l resulted in stimulation of the synthetase. A good correlation (r . 0.87) was obtained in synthetase assay when PBG and 5-aminolaevulinate (with added zinc and dithiothreitol) were used as substrates. With these additions 5-ALA may be used as a substrate in the URO-I-S assay in the investigation of latent cases of acute intermittent porphyria.

  15. Tissue- and Time-Specific Expression of Otherwise Identical tRNA Genes.

    Science.gov (United States)

    Sagi, Dror; Rak, Roni; Gingold, Hila; Adir, Idan; Maayan, Gadi; Dahan, Orna; Broday, Limor; Pilpel, Yitzhak; Rechavi, Oded

    2016-08-01

    Codon usage bias affects protein translation because tRNAs that recognize synonymous codons differ in their abundance. Although the current dogma states that tRNA expression is exclusively regulated by intrinsic control elements (A- and B-box sequences), we revealed, using a reporter that monitors the levels of individual tRNA genes in Caenorhabditis elegans, that eight tryptophan tRNA genes, 100% identical in sequence, are expressed in different tissues and change their expression dynamically. Furthermore, the expression levels of the sup-7 tRNA gene at day 6 were found to predict the animal's lifespan. We discovered that the expression of tRNAs that reside within introns of protein-coding genes is affected by the host gene's promoter. Pairing between specific Pol II genes and the tRNAs that are contained in their introns is most likely adaptive, since a genome-wide analysis revealed that the presence of specific intronic tRNAs within specific orthologous genes is conserved across Caenorhabditis species. PMID:27560950

  16. Fluorescence anisotropy: analysis of tRNA binding to the T box riboswitch antiterminator RNA.

    Science.gov (United States)

    Zhou, S; Anupam, R; Hines, J V

    2015-01-01

    Fluorescence anisotropy can be utilized in drug discovery screening assays to identify compounds that disrupt medicinally important RNA-macromolecular complexes. Here we describe the application of this technique to monitor tRNA binding to T box riboswitch antiterminator RNA. PMID:25352143

  17. Limited diagnostic value of enzyme analysis in patients with mitochondrial tRNA mutations

    DEFF Research Database (Denmark)

    Wibrand, Flemming; Jeppesen, Tina Dysgaard; Frederiksen, Anja L;

    2010-01-01

    We evaluated the diagnostic value of respiratory chain (RC) enzyme analysis of muscle in adult patients with mitochondrial myopathy (MM). RC enzyme activity was measured in muscle biopsies from 39 patients who carry either the 3243A>G mutation, other tRNA point mutations, or single, large...

  18. Mitochondrial tRNA cleavage by tRNA-targeting ribonuclease causes mitochondrial dysfunction observed in mitochondrial disease

    Energy Technology Data Exchange (ETDEWEB)

    Ogawa, Tetsuhiro, E-mail: atetsu@mail.ecc.u-tokyo.ac.jp; Shimizu, Ayano; Takahashi, Kazutoshi; Hidaka, Makoto; Masaki, Haruhiko, E-mail: amasaki@mail.ecc.u-tokyo.ac.jp

    2014-08-15

    Highlights: • MTS-tagged ribonuclease was translocated successfully to the mitochondrial matrix. • MTS-tagged ribonuclease cleaved mt tRNA and reduced COX activity. • Easy and reproducible method of inducing mt tRNA dysfunction. - Abstract: Mitochondrial DNA (mtDNA) is a genome possessed by mitochondria. Since reactive oxygen species (ROS) are generated during aerobic respiration in mitochondria, mtDNA is commonly exposed to the risk of DNA damage. Mitochondrial disease is caused by mitochondrial dysfunction, and mutations or deletions on mitochondrial tRNA (mt tRNA) genes are often observed in mtDNA of patients with the disease. Hence, the correlation between mt tRNA activity and mitochondrial dysfunction has been assessed. Then, cybrid cells, which are constructed by the fusion of an enucleated cell harboring altered mtDNA with a ρ{sup 0} cell, have long been used for the analysis due to difficulty in mtDNA manipulation. Here, we propose a new method that involves mt tRNA cleavage by a bacterial tRNA-specific ribonuclease. The ribonuclease tagged with a mitochondrial-targeting sequence (MTS) was successfully translocated to the mitochondrial matrix. Additionally, mt tRNA cleavage, which resulted in the decrease of cytochrome c oxidase (COX) activity, was observed.

  19. Mitochondrial tRNA cleavage by tRNA-targeting ribonuclease causes mitochondrial dysfunction observed in mitochondrial disease

    International Nuclear Information System (INIS)

    Highlights: • MTS-tagged ribonuclease was translocated successfully to the mitochondrial matrix. • MTS-tagged ribonuclease cleaved mt tRNA and reduced COX activity. • Easy and reproducible method of inducing mt tRNA dysfunction. - Abstract: Mitochondrial DNA (mtDNA) is a genome possessed by mitochondria. Since reactive oxygen species (ROS) are generated during aerobic respiration in mitochondria, mtDNA is commonly exposed to the risk of DNA damage. Mitochondrial disease is caused by mitochondrial dysfunction, and mutations or deletions on mitochondrial tRNA (mt tRNA) genes are often observed in mtDNA of patients with the disease. Hence, the correlation between mt tRNA activity and mitochondrial dysfunction has been assessed. Then, cybrid cells, which are constructed by the fusion of an enucleated cell harboring altered mtDNA with a ρ0 cell, have long been used for the analysis due to difficulty in mtDNA manipulation. Here, we propose a new method that involves mt tRNA cleavage by a bacterial tRNA-specific ribonuclease. The ribonuclease tagged with a mitochondrial-targeting sequence (MTS) was successfully translocated to the mitochondrial matrix. Additionally, mt tRNA cleavage, which resulted in the decrease of cytochrome c oxidase (COX) activity, was observed

  20. Monitoring Protein Synthesis in Living Cells with Fluorescent Labeled tRNA FRET Pairs

    Science.gov (United States)

    Barhoom, Sima; Farrel, Ian; Dahary, Dvir; Ehrlich, Marcelo; Cooperman, Barry S.; Elroy-Stein, Orna; Smilansky, Zeev

    2013-01-01

    We introduce Protein Synthesis Monitoring (PSM) – a technique to monitor protein synthesis in live cells. In PSM, we transfect cells with tRNA labeled as FRET donors and acceptors. A FRET signal is generated only when a donor- and an acceptor-labeled tRNA come in close contact (< 7nM), as they do on the ribosome during elongation. The intensity of the FRET signal correlates with the number of ribosomes engaged in protein synthesis, providing a real-time, live-cell assay for measuring rates of protein synthesis. PSM can be used to monitor the rate of either total protein synthesis (overall PSM), using bulk tRNAs, or of the synthesis of a specific protein (specific PSM), using specific pairs of tRNA to mark the protein of interest. PSM has sub-micron spatial and sub-second temporal resolutions. Cells continue to live and grow normally, and the synthesized proteins are unchanged since the labeling is on the tRNA itself and not on the amino acid. We have demonstrated specific PSM for monitoring synthesis of a viral protein during viral infection using Isoleucine tRNA, and for monitoring synthesis of collagen during fibrosis in mouse fibroblasts using tRNA-Gly and tRNA-Pro, as collagen is distinguished by many repeats of Gly-Pro dipeptides. We will discuss these results as well as additional applications of PSM in basic research, drug discovery, cell sorting, neurobiology, cancer, biomanufacturing, viral infections, and various protein-synthesis specific diseases.

  1. Holocarboxylase synthetase deficiency pre and post newborn screening

    Directory of Open Access Journals (Sweden)

    Taraka R. Donti

    2016-06-01

    Full Text Available Holocarboxylase synthetase deficiency is an autosomal recessive disorder of biotin metabolism resulting in multiple carboxylase deficiency. The typical presentation described in the medical literature is of neonatal onset within hours to weeks of birth with emesis, hypotonia, lethargy, seizures, metabolic ketolactic acidosis, hyperammonemia, developmental delay, skin rash and alopecia. The condition is screened for by newborn screening (NBS tandem mass spectroscopy by elevated hydroxypentanoylcarnitine on dried blood spots. Urine organic acid profile may demonstrate elevated lactic, 3-OH isovaleric, 3-OH propionic, 3-MCC, methylcitric acids, and tiglylglycine consistent with loss of function of the above carboxylases. Here we describe a cohort of patients, 2 diagnosed pre-NBS and 3 post-NBS with broad differences in initial presentation and phenotype. In addition, prior to the advent of NBS, there are isolated reports of late-onset holocarboxylase synthetase deficiency in the medical literature, which describe patients diagnosed between 1 and 8 years of life, however to our knowledge there are no reports of late-onset HCLS being missed by NBS. Also we report two cases, each with novel pathogenic variants HCLS, diagnosed at age 3 years and 21 months respectively. The first patient had a normal newborn screen whilst the second had an abnormal newborn screen but was misdiagnosed as 3-methylcrotonylcarboxylase (3-MCC deficiency and subsequently lost to follow-up until they presented again with severe metabolic acidosis.

  2. Holocarboxylase synthetase deficiency pre and post newborn screening.

    Science.gov (United States)

    Donti, Taraka R; Blackburn, Patrick R; Atwal, Paldeep S

    2016-06-01

    Holocarboxylase synthetase deficiency is an autosomal recessive disorder of biotin metabolism resulting in multiple carboxylase deficiency. The typical presentation described in the medical literature is of neonatal onset within hours to weeks of birth with emesis, hypotonia, lethargy, seizures, metabolic ketolactic acidosis, hyperammonemia, developmental delay, skin rash and alopecia. The condition is screened for by newborn screening (NBS) tandem mass spectroscopy by elevated hydroxypentanoylcarnitine on dried blood spots. Urine organic acid profile may demonstrate elevated lactic, 3-OH isovaleric, 3-OH propionic, 3-MCC, methylcitric acids, and tiglylglycine consistent with loss of function of the above carboxylases. Here we describe a cohort of patients, 2 diagnosed pre-NBS and 3 post-NBS with broad differences in initial presentation and phenotype. In addition, prior to the advent of NBS, there are isolated reports of late-onset holocarboxylase synthetase deficiency in the medical literature, which describe patients diagnosed between 1 and 8 years of life, however to our knowledge there are no reports of late-onset HCLS being missed by NBS. Also we report two cases, each with novel pathogenic variants HCLS, diagnosed at age 3 years and 21 months respectively. The first patient had a normal newborn screen whilst the second had an abnormal newborn screen but was misdiagnosed as 3-methylcrotonylcarboxylase (3-MCC) deficiency and subsequently lost to follow-up until they presented again with severe metabolic acidosis. PMID:27114915

  3. Purification and properties of the dihydrofolate synthetase from Serratia indica

    International Nuclear Information System (INIS)

    The dihydrofolate synthetase (EC6.3.2.12) responsible for catalyzing the synthesis of dihydrofolic acid from dihydropteroic acid and L-glutamic acid was purified about 130-fold from extracts of Serratia indica IFO 3759 by ammonium sulfate fractionation, DEAE-Sephadex column chromatography, Sephadex G-200 gel filtration, and DEAE-cellulose column chromatography. The enzyme preparation obtained was shown to be homogeneous by DEAE-cellulose column chromatography and ultracentrifugal analysis. The sedimentation coefficient of this enzyme was 3.9 S, and the molecular weight was determined to be about 47,000 by Sephadex G-100. The optimum pH for the reaction was 9.0. The enzymatic reaction required dihydropteroate, L-glutamate and ATP as substrates, and Mg2+ and K+ as cofactors. γ-L-Glutamyl-L-glutamic acid cannot replace L-glutamic acid as the substrate. Neither pteroic acid nor tetrahydropteroic acid can be used as the substrate. ATP was partially replaced by ITP or GTP. The enzyme reaction was inhibited by the addition of ADP, but not by AMP. One mole of dihydrofolate, 1 mole of ADP and 1 mole of orthophosphate were produced from each 1 mole of dihydropteroic acid, L-glutamic acid, and ATP. These results suggest that the systematic name for the dihydrofolate synthetase is 7,8-dihydropteroate: L-glutamate ligase (ADP). (auth.)

  4. Co-evolution of tRNA 3′ trailer sequences with 3′ processing enzymes in bacteria

    OpenAIRE

    Li, Zhongwei; Gong, Xin; JOSHI, VEDANG H.; LI, MUXIN

    2005-01-01

    Maturation of the tRNA 3′ terminus is a complicated process in bacteria. Usually, it is initiated by an endonucleolytic cleavage carried out by RNase E and Z in different bacteria. In Escherichia coli, RNase E cleaves AU-rich sequences downstream of tRNA, producing processing intermediates with a few extra residues at the 3′ end; these are then removed by exoribonuclease trimming to generate the mature 3′ end. Here we show that essentially all E. coli tRNA precursors contain a potential RNase...

  5. Structural elements and organization of the ancestral translational machinery

    Science.gov (United States)

    Rein, R.; Srinivasan, S.; McDonald, J.; Raghunathan, G.; Shibata, M.

    1987-09-01

    The molecular mechanisms underlying the primitive translational apparatus have been studied in light of present day protein biosynthesis. Using the structural information available from the contemporary system as a key to its function, both the structural necessities for an early adaptor and the multipoint recognition properties of such adaptors have been investigated. This was done by first critically examining the potential feasibility of right- and left-handed hairpin adaptor models. Second, a molecular model of the contemporary transpeptidation complex has been constructed in order to ascertain the structural requirements of the adaptor molecule needed for peptidyl transfer. Third, a model of the tRNATyr-tyrosyl tRNA synthetase complex including the positioning of the disordered region is proposed. This model is used to illustrate those required recognition properties of aminoacyl synthetase which lead to a perspective on the structure of the ancestor synthetase.

  6. Selection of functional tRNA primers and primer binding site sequences from a retroviral combinatorial library: identification of new functional tRNA primers in murine leukemia virus replication

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Duch, M; Pedersen, F S

    2000-01-01

    Retroviral reverse transcription is initiated from a cellular tRNA molecule and all known exogenous isolates of murine leukemia virus utilise a tRNA(Pro)molecule. While several studies suggest flexibility in murine leukemia virus primer utilisation, studies on human immunodeficiency virus and avian...... retro-viruses have revealed evidence of molecular adapt-ation towards the specific tRNA isoacceptor used as replication primer. In this study, murine leukemia virus tRNA utilisation is investigated by in vivo screening of a retroviral vector combinatorial library with randomised primer binding sites....... While most of the selected primer binding sites are complementary to the 3'-end of tRNA((Pro)), we also retrieved PBS sequences matching four other tRNA molecules and demonstrate that Akv murine leukemia virus vectors may efficiently replicate using tRNA(Arg(CCU)), tRNA(Phe(GAA))and a hitherto unknown...

  7. Chemical synthesis of 5-azacytidine nucleotides and preparation of tRNAs containing 5-azacytidine in its 3'-terminus.

    OpenAIRE

    Zielinski, W S; Sprinzl, M.

    1984-01-01

    5-azacytidine-5'-triphosphate prepared from 5-azacytidine by chemical phosphorylation is a substrate for AMP (CMP) tRNA nucleotidyl transferase from yeast. tRNAsPhe from yeast containing 5-azacytidine in their 3'-termini were prepared enzymatically. tRNAPhe-Cpn5CpA and tRNAPhe-n5Cpn5CpA can be aminoacylated by phenylalanyl-tRNA synthetase from yeast and they are active in the poly(U)-dependent synthesis of poly(Phe) on E. coli ribosomes. The decomposition of 5-azacytidine via hydrolysis of th...

  8. Selective posttranslational modification of phage-displayed polypeptides

    Energy Technology Data Exchange (ETDEWEB)

    Tsao, Meng-Lin; Tian, Feng; Schultz, Peter

    2013-11-19

    The invention relates to posttranslational modification of phage-displayed polypeptides. These displayed polypeptides comprise at least one unnatural amino acid, e.g., an aryl-azide amino acid such as p-azido-L-phenylalanine, or an alkynyl-amino acid such as para-propargyloxyphenylalanine, which are incorporated into the phage-displayed fusion polypeptide at a selected position by using an in vivo orthogonal translation system comprising a suitable orthogonal aminoacyl-tRNA synthetase and a suitable orthogonal tRNA species. These unnatural amino acids advantageously provide targets for posttranslational modifications such as azide-alkyne [3+2] cycloaddition reactions and Staudinger modifications.

  9. Encapsulation of glutamine synthetase in mouse erythrocytes: a new procedure for ammonia detoxification.

    Science.gov (United States)

    Kosenko, Elena A; Venediktova, Natalia I; Kudryavtsev, Andrey A; Ataullakhanov, Fazoil I; Kaminsky, Yury G; Felipo, Vicente; Montoliu, Carmina

    2008-12-01

    There are a number of pathological situations in which ammonia levels increase leading to hyperammonemia, which may cause neurological alterations and can lead to coma and death. Currently, there are no efficient treatments allowing rapid and sustained decrease of ammonia levels in these situations. A way to increase ammonia detoxification would be to increase its incorporation in glutamine by glutamine synthetase. The aim of this work was to develop a procedure to encapsulate glutamine synthetase in mouse erythrocytes and to assess whether administration of these erythrocytes containing glutamine synthetase (GS) reduce ammonia levels in hyperammonemic mice. The procedure developed allowed the encapsulation of 3 +/- 0.25 IU of GS / mL of erythrocytes with a 70% cell recovery. Most metabolites, including ATP, remained unaltered in glutamine synthetase-loaded erythrocytes (named ammocytes by us) compared with native erythrocytes. The glutamine synthetase-loaded ammocytes injected in mice survived and retained essentially all of their glutamine synthetase activity for at least 48 h in vivo. Injection of these ammocytes into hyperammonemic mice reduced ammonia levels in the blood by about 50%. The results reported indicate that ammocytes are able to keep their integrity, normal energy metabolism, the inserted glutamine synthetase activity, and can be useful to reduce ammonia levels in hyperammonemic situations. PMID:19088795

  10. Antenatal and postnatal radiologic diagnosis of holocarboxylase synthetase deficiency: a systematic review

    Energy Technology Data Exchange (ETDEWEB)

    Bandaralage, Sahan P.S. [Gold Coast Hospital and Health Service, Southport, Queensland (Australia); Griffith University, School of Medicine, Southport, Queensland (Australia); Farnaghi, Soheil [Caboolture Hospital, Caboolture, Queensland (Australia); Dulhunty, Joel M.; Kothari, Alka [Redcliffe Hospital, Redcliffe, Queensland (Australia); The University of Queensland, School of Medicine, Herston, Queensland (Australia)

    2016-03-15

    Holocarboxylase synthetase deficiency results in impaired activation of enzymes implicated in glucose, fatty acid and amino acid metabolism. Antenatal imaging and postnatal imaging are useful in making the diagnosis. Untreated holocarboxylase synthetase deficiency is fatal, while antenatal and postnatal biotin supplementation is associated with good clinical outcomes. Although biochemical assays are required for definitive diagnosis, certain radiologic features assist in the diagnosis of holocarboxylase synthetase deficiency. To review evidence regarding radiologic diagnostic features of holocarboxylase synthetase deficiency in the antenatal and postnatal period. A systematic review of all published cases of holocarboxylase synthetase deficiency identified by a search of Pubmed, Scopus and Web of Science. A total of 75 patients with holocarboxylase synthetase deficiency were identified from the systematic review, which screened 687 manuscripts. Most patients with imaging (19/22, 86%) had abnormal findings, the most common being subependymal cysts, ventriculomegaly and intraventricular hemorrhage. Although the radiologic features of subependymal cysts, ventriculomegaly, intraventricular hemorrhage and intrauterine growth restriction may be found in the setting of other pathologies, these findings should prompt consideration of holocarboxylase synthetase deficiency in at-risk children. (orig.)

  11. Antenatal and postnatal radiologic diagnosis of holocarboxylase synthetase deficiency: a systematic review

    International Nuclear Information System (INIS)

    Holocarboxylase synthetase deficiency results in impaired activation of enzymes implicated in glucose, fatty acid and amino acid metabolism. Antenatal imaging and postnatal imaging are useful in making the diagnosis. Untreated holocarboxylase synthetase deficiency is fatal, while antenatal and postnatal biotin supplementation is associated with good clinical outcomes. Although biochemical assays are required for definitive diagnosis, certain radiologic features assist in the diagnosis of holocarboxylase synthetase deficiency. To review evidence regarding radiologic diagnostic features of holocarboxylase synthetase deficiency in the antenatal and postnatal period. A systematic review of all published cases of holocarboxylase synthetase deficiency identified by a search of Pubmed, Scopus and Web of Science. A total of 75 patients with holocarboxylase synthetase deficiency were identified from the systematic review, which screened 687 manuscripts. Most patients with imaging (19/22, 86%) had abnormal findings, the most common being subependymal cysts, ventriculomegaly and intraventricular hemorrhage. Although the radiologic features of subependymal cysts, ventriculomegaly, intraventricular hemorrhage and intrauterine growth restriction may be found in the setting of other pathologies, these findings should prompt consideration of holocarboxylase synthetase deficiency in at-risk children. (orig.)

  12. Mode of Action of RNase BN/RNase Z on tRNA Precursors: RNase BN DOES NOT REMOVE THE CCA SEQUENCE FROM tRNA*

    OpenAIRE

    Dutta, Tanmay; Deutscher, Murray P.

    2010-01-01

    RNase BN, the Escherichia coli homolog of RNase Z, was previously shown to act as both a distributive exoribonuclease and an endoribonuclease on model RNA substrates and to be inhibited by the presence of a 3′-terminal CCA sequence. Here, we examined the mode of action of RNase BN on bacteriophage and bacterial tRNA precursors, particularly in light of a recent report suggesting that RNase BN removes CCA sequences (Takaku, H., and Nashimoto, M. (2008) Genes Cells 13, 1087–1097). We show that ...

  13. Thiol synthetases of legumes: Immunogold localization and differential gene regulation by phytohormones

    OpenAIRE

    Clemente, María Rebeca; Bustos-Sanmamed, Pilar; Loscos Aranda, Jorge; James, Euan Kevin; Pérez-Rontomé, Carmen; Navascués Ortega, Joaquín; Gay, Marina; Becana Ausejo, Manuel

    2012-01-01

    In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1–48 h with 50 μM of hormones. Immu...

  14. Treatment of renal colic by prostaglandin synthetase inhibitors and avafortan (analgesic antispasmodic).

    Science.gov (United States)

    el-Sherif, A E; Foda, R; Norlen, L J; Yahia, H

    1990-12-01

    In a study of the pain-relieving effect of 3 drugs commonly used to treat acute renal colic in this hospital, intravenous indomethacin and intramuscular diclofenac (prostaglandin synthetase inhibitors) were compared with intravenous Avafortan (analgesic antispasmodic). As first-line analgesics, prostaglandin synthetase inhibitors, if given intravenously, offer an effective alternative to Avafortan. Of 145 patients studied, 32 required a second injection for complete relief of pain. Administering a second dose of prostaglandin synthetase inhibitors resulted in equally significant pain relief rate even though the route was intramuscular. PMID:2265331

  15. Transfer-messenger RNA and SmpB mediate bacteriostasis in Escherichia coli cells against tRNA cleavage.

    Science.gov (United States)

    Sakai, Fusako; Sugita, Risa; Chang, Jung-Wei; Ogawa, Tetsuhiro; Tsumadori, Natsuko; Takahashi, Kazutoshi; Hidaka, Makoto; Masaki, Haruhiko

    2015-10-01

    RNAs, such as mRNA, rRNA and tRNA, are essential macromolecules for cell survival and maintenance. Any perturbation of these molecules, such as by degradation or mutation, can be toxic to cells and may occasionally induce cell death. Therefore, cells have mechanisms known as quality control systems to eliminate abnormal RNAs. Although tRNA is a stable molecule, the anticodon loop is quite susceptible to tRNA-targeting RNases such as colicin E5 and colicin D. However, the mechanism underlying cellular reaction to tRNA cleavage remains unclear. It had long been believed that tRNA cleavage by colicins E5 and D promptly induces cell death because colony formation of the sensitive cells is severely reduced; this indicates that cells do not resist the tRNA cleavage. Here, we show that Escherichia coli cells enter a bacteriostatic state against the tRNA cleavage of colicins D and E5. The bacteriostasis requires small protein B (SmpB) and transfer-messenger RNA (tmRNA), which are known to mediate trans-translation. Furthermore, another type of colicin, colicin E3 cleaving rRNA, immediately reduces the viability of sensitive cells. Moreover, nascent peptide degradation has an additive effect on bacteriostasis. Considering the recent observation that tRNA cleavage may be used as a means of cell-to-cell communication, tRNA cleavage could be used by bacteria not only to dominate other bacteria living in the same niche, but also to regulate growth of their own or other cells. PMID:26199088

  16. tRNADB-CE: tRNA gene database well-timed in the era of big sequence data

    Directory of Open Access Journals (Sweden)

    Takashi eAbe

    2014-05-01

    Full Text Available The tRNA Gene Data Base Curated by Experts tRNADB-CE (http://trna.ie.niigata-u.ac.jp was constructed by analyzing 1,966 complete and 5,272 draft genomes of prokaryotes, 171 viruses’, 121 chloroplasts’, and 12 eukaryotes’ genomes plus fragment sequences obtained by metagenome studies of environmental samples. 595,115 tRNA genes in total, and thus two times of genes compiled previously, have been registered, for which sequence, clover-leaf structure, and results of sequence-similarity and oligonucleotide-pattern searches can be browsed. To provide collective knowledge with help from experts in tRNA researches, we added a column for enregistering comments to each tRNA. By grouping bacterial tRNAs with an identical sequence, we have found high phylogenetic preservation of tRNA sequences, especially at the phylum level. Since many species-unknown tRNAs from metagenomic sequences have sequences identical to those found in species-known prokaryotes, the identical sequence group can provide phylogenetic markers to investigate the microbial community in an environmental ecosystem. This strategy can be applied to a huge amount of short sequences obtained from next-generation sequencers, as showing that tRNADB-CE is a well-timed database in the era of big sequence data. It is also discussed that BLSOM with oligonucleotide composition is useful for efficient knowledge discovery from big sequence data.

  17. tRNA modifying enzymes, NSUN2 and METTL1, determine sensitivity to 5-fluorouracil in HeLa cells.

    Directory of Open Access Journals (Sweden)

    Mayumi Okamoto

    2014-09-01

    Full Text Available Nonessential tRNA modifications by methyltransferases are evolutionarily conserved and have been reported to stabilize mature tRNA molecules and prevent rapid tRNA decay (RTD. The tRNA modifying enzymes, NSUN2 and METTL1, are mammalian orthologs of yeast Trm4 and Trm8, which are required for protecting tRNA against RTD. A simultaneous overexpression of NSUN2 and METTL1 is widely observed among human cancers suggesting that targeting of both proteins provides a novel powerful strategy for cancer chemotherapy. Here, we show that combined knockdown of NSUN2 and METTL1 in HeLa cells drastically potentiate sensitivity of cells to 5-fluorouracil (5-FU whereas heat stress of cells revealed no effects. Since NSUN2 and METTL1 are phosphorylated by Aurora-B and Akt, respectively, and their tRNA modifying activities are suppressed by phosphorylation, overexpression of constitutively dephosphorylated forms of both methyltransferases is able to suppress 5-FU sensitivity. Thus, NSUN2 and METTL1 are implicated in 5-FU sensitivity in HeLa cells. Interfering with methylation of tRNAs might provide a promising rationale to improve 5-FU chemotherapy of cancer.

  18. RNA fragments mimicking tRNA analogs interact with cytochrome c.

    Science.gov (United States)

    Pawlowska, Roza; Janicka, Magdalena; Jedrzejczyk, Dominika; Chworos, Arkadiusz

    2016-04-01

    In times, when drug seeking assays focus on the natural molecular triggers and their analogs, a deeper insight into molecular mechanisms governing the initial step of intrinsic apoptosis (cytochrome c release) is essential to suppress the immortality of pathologically changed cells. In this study, we examined RNA molecules mimicking mitochondrial tRNAs interacting with cytochrome c and possibly affecting its cellular function. tRNA analogs were designed and synthesized prior to the conformational analysis and gel assays clearly stating the nucleic acid-protein complex formation. The circular dichroism spectroscopic (CD) and microscale thermophoresis examination revealed the structural and conformational differences between four tRNA analogs in their interactions with cytochrome c. Obtained CD spectra and gel studies resulted in the complex ratio estimation and conclusion that not only the complex formation may be preferential towards specific tRNAs present in the cell, but nucleobase modifications are not essential for such interaction. PMID:26892782

  19. Cavitation as a mechanism of substrate discrimination by adenylosuccinate synthetases.

    Science.gov (United States)

    Iancu, Cristina V; Zhou, Yang; Borza, Tudor; Fromm, Herbert J; Honzatko, Richard B

    2006-09-26

    Adenylosuccinate synthetase catalyzes the first committed step in the de novo biosynthesis of AMP, coupling L-aspartate and IMP to form adenylosuccinate. Km values of IMP and 2'-deoxy-IMP are nearly identical with each substrate supporting comparable maximal velocities. Nonetheless, the Km value for L-aspartate and the Ki value for hadacidin (a competitive inhibitor with respect to L-aspartate) are 29-57-fold lower in the presence of IMP than in the presence of 2'-deoxy-IMP. Crystal structures of the synthetase ligated with hadacidin, GDP, and either 6-phosphoryl-IMP or 2'-deoxy-6-phosphoryl-IMP are identical except for the presence of a cavity normally occupied by the 2'-hydroxyl group of IMP. In the presence of 6-phosphoryl-IMP and GDP (hadacidin absent), the L-aspartate pocket can retain its fully ligated conformation, forming hydrogen bonds between the 2'-hydroxyl group of IMP and sequence-invariant residues. In the presence of 2'-deoxy-6-phosphoryl-IMP and GDP, however, the L-aspartate pocket is poorly ordered. The absence of the 2'-hydroxyl group of the deoxyribonucleotide may destabilize binding of the ligand to the L-aspartate pocket by disrupting hydrogen bonds that maintain a favorable protein conformation and by the introduction of a cavity into the fully ligated active site. At an approximate energy cost of 2.2 kcal/mol, the unfavorable thermodynamics of cavity formation may be the major factor in destabilizing ligands at the L-aspartate pocket. PMID:16981730

  20. Transformation of Bacillus Subtilis with cloned thymidylate synthetases

    Energy Technology Data Exchange (ETDEWEB)

    Rubin, Edward M.

    1980-01-01

    Bacillus subtilis carries two genes, thyA and thyB, each encoding different protein products, with thymidylate synthetase (TSase) activity. Either of these genes alone is sufficient for thymidine independence in B. subtilis. In addition there exist two B. subtilis temperate bacteriophages which upon infection of thymine requiring auxotrophs results in conversion of the organism to thymine independence. Chimeric plasmids selected for Thy/sup +/ transforming activity in E. coli were constructed and then used as a source of defined highly enriched DNA with which to transform competent B. subtilis. These plasmids were studied for their: (1) abiility to transform B. subtilis to thymine independence; (2) site of integration within the B. subtilis chromosome upon transformation; (3) phenotype of Thy/sup +/ plasmid generated transformants; and (4) nucleotide sequence homology among the cloned DNA fragments conferring thymine independence. Plasmids containing the two bacteriophage thy genes displayed the phenotype associated with thyA, whereas the plasmids containing the cloned B. subtilis chromosomal genes displayed the phenotype associated with thyB. Utilizing similar technology, the ability of an entirely foreign hybred bacterial plasmiid to transform B. subtilis was examined. In this case the gene from E. coli encoding thymidylate synthetase was cloned in the plasmid pBR322. The resulting chimeric plasmid was effective in transforming both E. coli and B. subtilis to thymine prototrophy. Uncloned linear E. coli chromosomal DNA was unable to transform thymine requiring strains of B. subtilis to thymine independence. Although the Thy/sup +/ transformants of E. coli contained plasmid DNA, the Thy/sup +/ transformants derived from the transformation of B. subtilis did not contain detectable extrachromosomal DNA. Instead the DNA from the chimeric plasmid was integrated into the chromosome of B. subtilis. (ERB)

  1. Mitochondrial tRNA import in Trypanosoma brucei is independent of thiolation and the Rieske protein

    Czech Academy of Sciences Publication Activity Database

    Paris, Zdeněk; RUBIO, M. A. T.; Lukeš, Julius; Alfonzo, J. D.

    2009-01-01

    Roč. 15, č. 7 (2009), s. 1398-1406. ISSN 1355-8382 R&D Projects: GA ČR GA204/06/1558; GA MŠk LC07032; GA MŠk 2B06129 Institutional research plan: CEZ:AV0Z60220518 Keywords : T. brucei * tRNA import * 2-thiolation * RIC * Rieske * Fe-S cluster Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.198, year: 2009

  2. RNA Polymerase III Output Is Functionally Linked to tRNA Dimethyl-G26 Modification.

    Directory of Open Access Journals (Sweden)

    Aneeshkumar G Arimbasseri

    2015-12-01

    Full Text Available Control of the differential abundance or activity of tRNAs can be important determinants of gene regulation. RNA polymerase (RNAP III synthesizes all tRNAs in eukaryotes and it derepression is associated with cancer. Maf1 is a conserved general repressor of RNAP III under the control of the target of rapamycin (TOR that acts to integrate transcriptional output and protein synthetic demand toward metabolic economy. Studies in budding yeast have indicated that the global tRNA gene activation that occurs with derepression of RNAP III via maf1-deletion is accompanied by a paradoxical loss of tRNA-mediated nonsense suppressor activity, manifested as an antisuppression phenotype, by an unknown mechanism. We show that maf1-antisuppression also occurs in the fission yeast S. pombe amidst general activation of RNAP III. We used tRNA-HydroSeq to document that little changes occurred in the relative levels of different tRNAs in maf1Δ cells. By contrast, the efficiency of N2,N2-dimethyl G26 (m(22G26 modification on certain tRNAs was decreased in response to maf1-deletion and associated with antisuppression, and was validated by other methods. Over-expression of Trm1, which produces m(22G26, reversed maf1-antisuppression. A model that emerges is that competition by increased tRNA levels in maf1Δ cells leads to m(22G26 hypomodification due to limiting Trm1, reducing the activity of suppressor-tRNASerUCA and accounting for antisuppression. Consistent with this, we show that RNAP III mutations associated with hypomyelinating leukodystrophy decrease tRNA transcription, increase m(22G26 efficiency and reverse antisuppression. Extending this more broadly, we show that a decrease in tRNA synthesis by treatment with rapamycin leads to increased m(22G26 modification and that this response is conserved among highly divergent yeasts and human cells.

  3. Controlling translation elongation efficiency: tRNA regulation of ribosome flux on the mRNA.

    Science.gov (United States)

    Gorgoni, Barbara; Marshall, Elizabeth; McFarland, Matthew R; Romano, M Carmen; Stansfield, Ian

    2014-02-01

    Gene expression can be regulated by a wide variety of mechanisms. One example concerns the growing body of evidence that the protein-production rate can be regulated at the level of translation elongation by controlling ribosome flux across the mRNA. Variations in the abundance of tRNA molecules cause different rates of translation of their counterpart codons. This, in turn, produces a variable landscape of translational rate across each and every mRNA, with the dynamic formation and deformation of ribosomal queues being regulated by both tRNA availability and the rates of translation initiation and termination. In the present article, a range of examples of tRNA control of gene expression are reviewed, and the use of mathematical modelling to develop a predictive understanding of the consequences of that regulation is discussed and explained. These findings encourage a view that predicting the protein-synthesis rate of each mRNA requires a holistic understanding of how each stage of translation, including elongation, contributes to the overall protein-production rate. PMID:24450645

  4. Global analysis of transcriptionally engaged yeast RNA polymerase III reveals extended tRNA transcripts.

    Science.gov (United States)

    Turowski, Tomasz W; Leśniewska, Ewa; Delan-Forino, Clementine; Sayou, Camille; Boguta, Magdalena; Tollervey, David

    2016-07-01

    RNA polymerase III (RNAPIII) synthesizes a range of highly abundant small stable RNAs, principally pre-tRNAs. Here we report the genome-wide analysis of nascent transcripts attached to RNAPIII under permissive and restrictive growth conditions. This revealed strikingly uneven polymerase distributions across transcription units, generally with a predominant 5' peak. This peak was higher for more heavily transcribed genes, suggesting that initiation site clearance is rate-limiting during RNAPIII transcription. Down-regulation of RNAPIII transcription under stress conditions was found to be uneven; a subset of tRNA genes showed low response to nutrient shift or loss of the major transcription regulator Maf1, suggesting potential "housekeeping" roles. Many tRNA genes were found to generate long, 3'-extended forms due to read-through of the canonical poly(U) terminators. The degree of read-through was anti-correlated with the density of U-residues in the nascent tRNA, and multiple, functional terminators can be located far downstream. The steady-state levels of 3'-extended pre-tRNA transcripts are low, apparently due to targeting by the nuclear surveillance machinery, especially the RNA binding protein Nab2, cofactors for the nuclear exosome, and the 5'-exonuclease Rat1. PMID:27206856

  5. Mitochondrial genomes of praying mantises (Dictyoptera, Mantodea): rearrangement, duplication, and reassignment of tRNA genes.

    Science.gov (United States)

    Ye, Fei; Lan, Xu-E; Zhu, Wen-Bo; You, Ping

    2016-01-01

    Insect mitochondrial genomes (mitogenomes) contain a conserved set of 37 genes for an extensive diversity of lineages. Previously reported dictyopteran mitogenomes share this conserved mitochondrial gene arrangement, although surprisingly little is known about the mitogenome of Mantodea. We sequenced eight mantodean mitogenomes including the first representatives of two families: Hymenopodidae and Liturgusidae. Only two of these genomes retain the typical insect gene arrangement. In three Liturgusidae species, the trnM genes have translocated. Four species of mantis (Creobroter gemmata, Mantis religiosa, Statilia sp., and Theopompa sp.-HN) have multiple identical tandem duplication of trnR, and Statilia sp. additionally includes five extra duplicate trnW. These extra trnR and trnW in Statilia sp. are erratically arranged and form another novel gene order. Interestingly, the extra trnW is converted from trnR by the process of point mutation at anticodon, which is the first case of tRNA reassignment for an insect. Furthermore, no significant differences were observed amongst mantodean mitogenomes with variable copies of tRNA according to comparative analysis of codon usage. Combined with phylogenetic analysis, the characteristics of tRNA only possess limited phylogenetic information in this research. Nevertheless, these features of gene rearrangement, duplication, and reassignment provide valuable information toward understanding mitogenome evolution in insects. PMID:27157299

  6. High cerebral guanidinoacetate and variable creatine concentrations in argininosuccinate synthetase and lyase deficiency : Implications for treatment?

    NARCIS (Netherlands)

    van Spronsen, F. J.; Reijngoud, D. J.; Verhoeven, N. M.; Soorani-Lunsing, R. J.; Jakobs, C.; Sijens, P. E.

    2006-01-01

    Cerebral creatine and guanidinoacetate and blood and urine metabolites were studied in four patients with argininosuccinate synthetase (ASS) or argininosuccinate lyase (ASL) deficiency receiving large doses of arginine. Urine and blood metabolites varied largely. Cerebral guanidinoacetate was increa

  7. Heterogeneity of holocarboxylase synthetase in patients with biotin-responsive multiple carboxylase deficiency.

    OpenAIRE

    Burri, B J; Sweetman, L.; Nyhan, W L

    1985-01-01

    Holocarboxylase synthetase activity has been determined in fibroblasts of seven patients with the neonatal form of biotin-responsive multiple carboxylase deficiency. The normal Km for biotin was 15 +/- 3 nmol/l, while in the patients the values ranged from 48 to 1,062 nmol/l. The mean maximum velocity was 27% of normal. Differences among the values obtained for the Km for biotin and the heat stability of holocarboxylase synthetase suggested that the patients studied represented at least four ...

  8. Oligo-2',5'-adenylate synthetase activity in peripheral blood mononuclear leukocytes in various diseases.

    OpenAIRE

    Fujii, N; Kotake, S.; Hirose, S; Ohno, S; Yasuda, I.; Sagawa, A; Ishikawa, K.; Minagawa, T

    1984-01-01

    Interferon induces oligo-2',5'-adenylate synthetase in cells. In various diseases, interferon was detectable in the circulation or was produced spontaneously from peripheral blood mononuclear leukocytes. The oligo-2',5'-adenylate synthetase activity in peripheral blood mononuclear leukocytes was examined in various diseases, including systemic lupus erythematosus, sarcoidosis, Vogt-Koyanagi-Harada disease, and Behcet's disease. The activity of this enzyme was significantly increased in system...

  9. Superposition of two tRNASer acceptor stem crystal structures: Comparison of structure, ligands and hydration

    International Nuclear Information System (INIS)

    We solved the X-ray structures of two Escherichia coli tRNASer acceptor stem microhelices. As both tRNAs are aminoacylated by the same seryl-tRNA-synthetase, we performed a comparative structure analysis of both duplexes to investigate the helical conformation, the hydration patterns and magnesium binding sites. It is well accepted, that the hydration of RNA plays an important role in RNA-protein interactions and that the extensive solvent content of the minor groove has a special function in RNA. The detailed comparison of both tRNASer microhelices provides insights into the structural arrangement of the isoacceptor tRNA aminoacyl stems with respect to the surrounding water molecules and may eventually help us to understand their biological function at atomic resolution.

  10. The gene encoding human glutathione synthetase (GSS) maps to the long arm of chromosome 20 at band 11.2

    Energy Technology Data Exchange (ETDEWEB)

    Webb, G.C.; Vaska, V.L.; Ford, J.H. [Queen Elizabeth Hospital, Woodville (Australia)] [and others

    1995-12-10

    Two forms of glutathione synthetase deficiency have been described. While one form is mild, causing hemolytic anemia, the other more severe form causes 5-oxoprolinuria with secondary neurological involvement. Despite the existence of two deficiency phenotypes, Southern blots hybridized with a glutathione synthetase cDNA suggest that there is a single glutathione synthetase gene in the human genome. Analysis of somatic cell hybrids showed the human glutathione synthetase gene (GSS) to be located on chromosome 20, and this assignment has been refined to subband 20q11.2 using in situ hybridization. 16 refs., 2 figs.

  11. Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains.

    Science.gov (United States)

    Ishikawa, Fumihiro; Miyamoto, Kengo; Konno, Sho; Kasai, Shota; Kakeya, Hideaki

    2015-12-18

    A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the Escherichia coli expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the kcat/Km value with the noncognate substrate Sal and a corresponding 48-fold decrease in the kcat/Km value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique

  12. A human tRNA methyltransferase 9-like protein prevents tumour growth by regulating LIN9 and HIF1-α.

    Science.gov (United States)

    Begley, Ulrike; Sosa, Maria Soledad; Avivar-Valderas, Alvaro; Patil, Ashish; Endres, Lauren; Estrada, Yeriel; Chan, Clement T Y; Su, Dan; Dedon, Peter C; Aguirre-Ghiso, Julio A; Begley, Thomas

    2013-03-01

    Emerging evidence points to aberrant regulation of translation as a driver of cell transformation in cancer. Given the direct control of translation by tRNA modifications, tRNA modifying enzymes may function as regulators of cancer progression. Here, we show that a tRNA methyltransferase 9-like (hTRM9L/KIAA1456) mRNA is down-regulated in breast, bladder, colorectal, cervix and testicular carcinomas. In the aggressive SW620 and HCT116 colon carcinoma cell lines, hTRM9L is silenced and its re-expression and methyltransferase activity dramatically suppressed tumour growth in vivo. This growth inhibition was linked to decreased proliferation, senescence-like G0/G1-arrest and up-regulation of the RB interacting protein LIN9. Additionally, SW620 cells re-expressing hTRM9L did not respond to hypoxia via HIF1-α-dependent induction of GLUT1. Importantly, hTRM9L-negative tumours were highly sensitive to aminoglycoside antibiotics and this was associated with altered tRNA modification levels compared to antibiotic resistant hTRM9L-expressing SW620 cells. Our study links hTRM9L and tRNA modifications to inhibition of tumour growth via LIN9 and HIF1-α-dependent mechanisms. It also suggests that aminoglycoside antibiotics may be useful to treat hTRM9L-deficient tumours. PMID:23381944

  13. Chitin synthetase in encysting Giardia lamblia and Entamoeba invadens

    Energy Technology Data Exchange (ETDEWEB)

    Das, S.; Gillin, F.D.

    1987-05-01

    Giardia lamblia (Gl) and Entamoeba invadens (Ei) are protozoan parasites with two morphologic stages in their life cycles. Motile trophozoites colonize the intestine of humans and reptiles respectively. Water resistant cysts, which can survive outside the host, transmit infection. In vitro cyst formation of Ei from trophozoites has been reported, and the authors have recently induced in vitro encystation of Gl. Although the cyst walls of both parasites contain chitin, it synthesis by encysting trophozoites has not been reported. The authors now show that encystation conditions greatly increase chitin synthetase (CS) specific activity (incorporation of /sup 3/H GlcNAc from UDP-GlcNAc into TCA-or alcohol-precipitable material). Extracts of encysting Gl incorporated 3.6 nmol/mg protein in 5 hr compared to < 0.005 in controls. Extracts of encysting Fi incorporated 4.8 n mol/mg protein, compared to 1.7 in the control. CS activity of both parasites requires preformed chitin. The Gl enzyme requires a reducing agent, is inhibited by digitonin and the CS inhibitors, polyoxin D and Nikkomycin, but not by tunicamycin. The product is digested by chitinase. Ei enzyme does not require a reducing agent and is stimulated by 1 mg/ml digitonin, but inhibited by higher concentrations. These studies demonstrate CS enzymes which may play important roles in encystation of Gl and Ei.

  14. Isolation and characterization of glutamine synthetase genes in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Chen, Q; Silflow, C D

    1996-11-01

    To elucidate the role of glutamine synthetase (GS) in nitrogen assimilation in the green alga Chlamydomonas reinhardtii we used maize GS1 (the cytosolic form) and GS2 (the chloroplastic form) cDNAs as hybridization probes to isolate C. reinhardtii cDNA clones. The amino acid sequences derived from the C. reinhardtii clones have extensive homology with GS enzymes from higher plants. A putative amino-terminal transit peptide encoded by the GS2 cDNA suggests that the protein localizes to the chloroplast. Genomic DNA blot analysis indicated that GS1 is encoded by a single gene, whereas two genomic fragments hybridized to the GS2 cDNA probe. All GS2 cDNA clones corresponded to only one of the two GS2 genomic sequences. We provide evidence that ammonium, nitrate, and light regulate GS transcript accumulation in green algae. Our results indicate that the level of GS1 transcripts is repressed by ammonium but induced by nitrate. The level of GS2 transcripts is not affected by ammonium or nitrate. Expression of both GS1 and GS2 genes is regulated by light, but perhaps through different mechanisms. Unlike in higher plants, no decreased level of GS2 transcripts was detected when cells were grown under conditions that repress photorespiration. Analysis of GS transcript levels in mutants with defects in the nitrate assimilation pathway show that nitrate assimilation and ammonium assimilation are regulated independently. PMID:8938407

  15. Chitin synthetase in encysting Giardia lamblia and Entamoeba invadens

    International Nuclear Information System (INIS)

    Giardia lamblia (Gl) and Entamoeba invadens (Ei) are protozoan parasites with two morphologic stages in their life cycles. Motile trophozoites colonize the intestine of humans and reptiles respectively. Water resistant cysts, which can survive outside the host, transmit infection. In vitro cyst formation of Ei from trophozoites has been reported, and the authors have recently induced in vitro encystation of Gl. Although the cyst walls of both parasites contain chitin, it synthesis by encysting trophozoites has not been reported. The authors now show that encystation conditions greatly increase chitin synthetase (CS) specific activity (incorporation of 3H GlcNAc from UDP-GlcNAc into TCA-or alcohol-precipitable material). Extracts of encysting Gl incorporated 3.6 nmol/mg protein in 5 hr compared to < 0.005 in controls. Extracts of encysting Fi incorporated 4.8 n mol/mg protein, compared to 1.7 in the control. CS activity of both parasites requires preformed chitin. The Gl enzyme requires a reducing agent, is inhibited by digitonin and the CS inhibitors, polyoxin D and Nikkomycin, but not by tunicamycin. The product is digested by chitinase. Ei enzyme does not require a reducing agent and is stimulated by 1 mg/ml digitonin, but inhibited by higher concentrations. These studies demonstrate CS enzymes which may play important roles in encystation of Gl and Ei

  16. Overexpression of glutamine synthetases confers transgenic rice herbicide resistance

    Institute of Scientific and Technical Information of China (English)

    Sun Hui; Huang Qiman; Su Jin

    2005-01-01

    Glutamine synthetase (GS, E.C.6.3.1.2) is a key enzyme involved in the assimilation of inorganic nitrogen in higher plants and gram-negative microorganisms. GS is the targeting enzyme of a herbicide phosphinothricin (PPT) or Basta. In order to generate PPT-resistant transgenic rice via overexpression of GS, we constructed a plant expression vector p2GS harboring two different isoenzymes GS1 and GS2 cDNAs under the control of constitutive promoters of rice Act1 and maize Ubiquitin(Ubi) genes. The p2GS was introduced into rice genome by Agrobacterium-mediated transformation and confirmed by PCR and Southern blot hybridization. GS-transgene expression was first detected by Northern blot analyses. Results from Basta test indicated that GS-transgenic plants can tolerate as high as 0.3% Basta solution. In addition, our results also demonstrated that GS overexpression conferred transformed rice calli PPT resistance. Thus, GS cassette can serve as a selective marker gene instead of bar cassette for selection of PPT transformants.

  17. Glutamine synthetase and glutamyltransferase in developing chick and rat tissues

    Energy Technology Data Exchange (ETDEWEB)

    Herzfeld, A.; Raper, S.M.

    1975-01-01

    Concomitant determination of ..gamma..-glutamine-hydroxylamine-glutamyltransferase (GT) and ..gamma..-glutamyl hydroxamate synthetase (GS) activities in chick retina, brain and liver between the 13th day of incubation and a day after hatching showed that while both activities increased late in incubation, in neural tissues their rises were not simultaneous throughout development; in liver both activities were already high on the 14th day of incubation and changed in parallel thereafter. GS and GT activities could be evoked prematurely in all three tissues by cortisol, but GT activity showed higher responses to the hormone. GT and GS were separable by differential solubilization in chick liver but not in retina of chick or rat. The wide spread of the ratios of GT : GS activities in homogenates of a number of chick and rat tissues (1 : 1 to 73 : 1) indicates that the GS reaction is not catalyzed by the same protein that catalyzes the GT reactions. Relative amounts of GT(T) (free of GS activity) and of variants of GS (with different competences to catalyze the GT reaction) may govern the changes between the two activities during development and their distribution among different adult tissues in chick and rat.

  18. Nitric oxide synthetase and Helicobacter pylori in patients undergoing appendicectomy.

    LENUS (Irish Health Repository)

    Kell, M R

    2012-02-03

    BACKGROUND: This study was designed to determine whether Helicobacter pylori forms part of the normal microenvironment of the appendix, whether it plays a role in the pathogenesis of acute appendicitis, and whether it is associated with increased expression of inducible nitric oxide synthetase (iNOS) in appendicular macrophages. METHODS: Serology for H. pylori was performed on 51 consecutive patients undergoing emergency appendicectomy. Appendix samples were tested for urease activity, cultured and stained for H. pylori, graded according to the degree of inflammatory infiltrate, and probed immunohistochemically for iNOS expression. RESULTS: The mean age of the patients was 21 (range 7-51) years. Seventeen patients (33 per cent) were seropositive for H. pylori but no evidence of H. pylori was found in any appendix specimen. However, an enhanced inflammatory cell infiltration was observed in seropositive patients (P < 0.04) and the expression of macrophage iNOS in the mucosa of normal and inflamed appendix specimens was increased (P < 0.01). CONCLUSION: H. pylori does not colonize the appendix and is unlikely to be a pathogenic stimulus for appendicitis. Priming effects on mucosal immunology downstream from the foregut may occur after infection with H. pylori.

  19. The T box regulatory element controlling expression of the class I lysyl-tRNA synthetase of Bacillus cereus strain 14579 is functional and can be partially induced by reduced charging of asparaginyl-tRNAAsn

    LENUS (Irish Health Repository)

    Foy, Niall

    2010-07-22

    Abstract Background Lysyl-tRNA synthetase (LysRS) is unique within the aminoacyl-tRNA synthetase family in that both class I (LysRS1) and class II (LysRS2) enzymes exist. LysRS1 enzymes are found in Archaebacteria and some eubacteria while all other organisms have LysRS2 enzymes. All sequenced strains of Bacillus cereus (except AH820) and Bacillus thuringiensis however encode both a class I and a class II LysRS. The lysK gene (encoding LysRS1) of B. cereus strain 14579 has an associated T box element, the first reported instance of potential T box control of LysRS expression. Results A global study of 891 completely sequenced bacterial genomes identified T box elements associated with control of LysRS expression in only four bacterial species: B. cereus, B. thuringiensis, Symbiobacterium thermophilum and Clostridium beijerinckii. Here we investigate the T box element found in the regulatory region of the lysK gene in B. cereus strain 14579. We show that this T box element is functional, responding in a canonical manner to an increased level of uncharged tRNALys but, unusually, also responding to an increased level of uncharged tRNAAsn. We also show that B. subtilis strains with T box regulated expression of the endogenous lysS or the heterologous lysK genes are viable. Conclusions The T box element controlling lysK (encoding LysRS1) expression in B. cereus strain 14579 is functional, but unusually responds to depletion of charged tRNALys and tRNAAsn. This may have the advantage of making LysRS1 expression responsive to a wider range of nutritional stresses. The viability of B. subtilis strains with a single LysRS1 or LysRS2, whose expression is controlled by this T box element, makes the rarity of the occurrence of such control of LysRS expression puzzling.

  20. The T box regulatory element controlling expression of the class I lysyl-tRNA synthetase of Bacillus cereus strain 14579 is functional and can be partially induced by reduced charging of asparaginyl-tRNAAsn

    Directory of Open Access Journals (Sweden)

    Conant Gavin C

    2010-07-01

    Full Text Available Abstract Background Lysyl-tRNA synthetase (LysRS is unique within the aminoacyl-tRNA synthetase family in that both class I (LysRS1 and class II (LysRS2 enzymes exist. LysRS1 enzymes are found in Archaebacteria and some eubacteria while all other organisms have LysRS2 enzymes. All sequenced strains of Bacillus cereus (except AH820 and Bacillus thuringiensis however encode both a class I and a class II LysRS. The lysK gene (encoding LysRS1 of B. cereus strain 14579 has an associated T box element, the first reported instance of potential T box control of LysRS expression. Results A global study of 891 completely sequenced bacterial genomes identified T box elements associated with control of LysRS expression in only four bacterial species: B. cereus, B. thuringiensis, Symbiobacterium thermophilum and Clostridium beijerinckii. Here we investigate the T box element found in the regulatory region of the lysK gene in B. cereus strain 14579. We show that this T box element is functional, responding in a canonical manner to an increased level of uncharged tRNALys but, unusually, also responding to an increased level of uncharged tRNAAsn. We also show that B. subtilis strains with T box regulated expression of the endogenous lysS or the heterologous lysK genes are viable. Conclusions The T box element controlling lysK (encoding LysRS1 expression in B. cereus strain 14579 is functional, but unusually responds to depletion of charged tRNALys and tRNAAsn. This may have the advantage of making LysRS1 expression responsive to a wider range of nutritional stresses. The viability of B. subtilis strains with a single LysRS1 or LysRS2, whose expression is controlled by this T box element, makes the rarity of the occurrence of such control of LysRS expression puzzling.

  1. tRNA properties help shape codon pair preferences in open reading frames

    OpenAIRE

    Buchan, J. Ross; Aucott, Lorna S; Stansfield, Ian

    2006-01-01

    Translation elongation is an accurate and rapid process, dependent upon efficient juxtaposition of tRNAs in the ribosomal A- and P-sites. Here, we sought evidence of A- and P-site tRNA interaction by examining bias in codon pair choice within open reading frames from a range of genomes. Three distinct and marked effects were revealed once codon and dipeptide biases had been subtracted. First, in the majority of genomes, codon pair preference is primarily determined by a tetranucleotide combin...

  2. The role of mitochondrial tRNA variants in female breast cancer.

    Science.gov (United States)

    Meng, Xian-Li; Meng, Hua; Zhang, Wei; Qin, Yu-Hua; Zhao, Ning-Min

    2016-09-01

    Mitochondrial tRNA (Mt-tRNA) variants have been found to be involved in the carcinogenesis of breast cancer. These tRNAs, which played critical roles in mitochondrial protein synthesis, were important regulators in tumorigenesis. Distinguishing the polymorphisms or mutations in mt-tRNA genes was still puzzling for the clinicians and geneticists when confronted with the breast cancer. In this study, we performed a detailed analysis of recently reported mutations in mt-tRNA genes and further discussed the relationship between these variants and breast cancer. PMID:25703847

  3. A fungal anticodon nuclease ribotoxin exploits a secondary cleavage site to evade tRNA repair

    OpenAIRE

    Meineke, Birthe; Kast, Alene; Schwer, Beate; Meinhardt, Friedhelm; Shuman, Stewart; Klassen, Roland

    2012-01-01

    The PaOrf2 and γ-toxin subunits of Pichia acaciae toxin (PaT) and Kluyveromyces lactis zymocin are tRNA anticodon nucleases encoded by cytoplasmic DNA plasmids. Toxicity can be recapitulated conveniently by induced intracellular expression of PaOrf2 or γ-toxin in Saccharomyces cerevisiae. Mutational analysis of γ-toxin has identified amino acids required for ribotoxicity in vivo and RNA transesterification in vitro. Here, the authors report that PaOrf2 residues Glu9 and His287 (putative count...

  4. Selective charging of tRNA isoacceptors induced by amino-acid starvation

    DEFF Research Database (Denmark)

    Dittmar, K. A.; Sørensen, Michael Askvad; Elf, J.;

    2005-01-01

    -acid starvation results in 'selective charging' where the charging levels of some tRNA isoacceptors will be low and those of others will remain high. Here, we developed a microarray for the analysis of charged fractions of tRNAs and measured charging for all Escherichia coli tRNAs before and during leucine......, threonine or arginine starvation. Before starvation, most tRNAs were fully charged. During starvation, the isoacceptors in the leucine, threonine or arginine families showed selective charging when cells were starved for their cognate amino acid, directly confirming the theoretical prediction. Codons read...

  5. Aquifex aeolicus tRNA (N2,N2-Guanine)-dimethyltransferase (Trm1) Catalyzes Transfer of Methyl Groups Not Only to Guanine 26 but Also to Guanine 27 in tRNA*

    OpenAIRE

    Awai, Takako; Kimura, Satoshi; Tomikawa, Chie; Ochi, Anna; Ihsanawati,; Bessho, Yoshitaka; Yokoyama, Shigeyuki; Ohno, Satoshi; Nishikawa, Kazuya; Yokogawa, Takashi; Suzuki, Tsutomu; Hori, Hiroyuki

    2009-01-01

    Transfer RNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes N2,N2-dimethylguanine formation at position 26 (m22G26) in tRNA. In the reaction, N2-guanine at position 26 (m2G26) is generated as an intermediate. The trm1 genes are found only in archaea and eukaryotes, although it has been reported that Aquifex aeolicus, a hyper-thermophilic eubacterium, has a putative trm1 gene. To confirm whether A. aeolicus Trm1 has tRNA methyltransferase activity, we purified recombinant Trm1 protein. I...

  6. Anticodon Modifications in the tRNA Set of LUCA and the Fundamental Regularity in the Standard Genetic Code

    Science.gov (United States)

    van der Gulik, Peter T. S.; Hoff, Wouter D.

    2016-01-01

    Based on (i) an analysis of the regularities in the standard genetic code and (ii) comparative genomics of the anticodon modification machinery in the three branches of life, we derive the tRNA set and its anticodon modifications as it was present in LUCA. Previously we proposed that an early ancestor of LUCA contained a set of 23 tRNAs with unmodified anticodons that was capable of translating all 20 amino acids while reading 55 of the 61 sense codons of the standard genetic code (SGC). Here we use biochemical and genomic evidence to derive that LUCA contained a set of 44 or 45 tRNAs containing 2 or 3 modifications while reading 59 or 60 of the 61 sense codons. Subsequent tRNA modifications occurred independently in the Bacteria and Eucarya, while the Archaea have remained quite close to the tRNA set as it was present in LUCA. PMID:27454314

  7. The ribosome triggers the stringent response by RelA via a highly distorted tRNA.

    Science.gov (United States)

    Agirrezabala, Xabier; Fernández, Israel S; Kelley, Ann C; Cartón, David Gil; Ramakrishnan, Venki; Valle, Mikel

    2013-09-01

    The bacterial stringent response links nutrient starvation with the transcriptional control of genes. This process is initiated by the stringent factor RelA, which senses the presence of deacylated tRNA in the ribosome as a symptom of amino-acid starvation to synthesize the alarmone (p)ppGpp. Here we report a cryo-EM study of RelA bound to ribosomes bearing cognate, deacylated tRNA in the A-site. The data show that RelA on the ribosome stabilizes an unusual distorted form of the tRNA, with the acceptor arm making contact with RelA and far from its normal location in the peptidyl transferase centre. PMID:23877429

  8. La adaptación a la deficiencia de zinc en cianobacterias. Papel de treonil-trna sintetasas duplicadas

    OpenAIRE

    Rubio Gómez, Miguel Ángel

    2016-01-01

    Falta palabras claves Las aminoacil tRNA sintetasas (aaRSs) son las enzimas que catalizan la carga del aminoácido en el tRNA y son las responsables de mantener la fidelidad en la traducción del código genético. Las aaRSs son componentes esenciales de la síntesis proteica y son ubicuas en todos los dominios de la vida (Ibba y Sol, 2000; Perona y Hadd, 2012). La cianobacteria filamentosa Anabaena sp.PCC 7120 contiene dos genes de treonil tRNA sintetasa, alr0335(thrS1) y all4723 (thrS2), ...

  9. Interdomain and Intermodule Organization in Epimerization Domain Containing Nonribosomal Peptide Synthetases.

    Science.gov (United States)

    Chen, Wei-Hung; Li, Kunhua; Guntaka, Naga Sandhya; Bruner, Steven D

    2016-08-19

    Nonribosomal peptide synthetases are large, complex multidomain enzymes responsible for the biosynthesis of a wide range of peptidic natural products. Inherent to synthetase chemistry is the thioester templated mechanism that relies on protein/protein interactions and interdomain dynamics. Several questions related to structure and mechanism remain to be addressed, including the incorporation of accessory domains and intermodule interactions. The inclusion of nonproteinogenic d-amino acids into peptide frameworks is a common and important modification for bioactive nonribosomal peptides. Epimerization domains, embedded in nonribosomal peptide synthetases assembly lines, catalyze the l- to d-amino acid conversion. Here we report the structure of the epimerization domain/peptidyl carrier protein didomain construct from the first module of the cyclic peptide antibiotic gramicidin synthetase. Both holo (phosphopantethiene post-translationally modified) and apo structures were determined, each representing catalytically relevant conformations of the two domains. The structures provide insight into domain-domain recognition, substrate delivery during the assembly line process, in addition to the structural organization of homologous condensation domains, canonical players in all synthetase modules. PMID:27294598

  10. Characterization of arachidonate 5-lipoxygenase and leukotriene A4 synthetase from RBL-1 cells

    International Nuclear Information System (INIS)

    5-lipoxygenase (LO) and leukotriene (LT) A4 synthetase from RBL-1 high speed (105,000 x g for 60 min) supernatants were partially purified by protein-high performance liquid chromatography (HPLC) and characterized in detail. The partially purified preparation contained only 5-LO and LTA4 synthetase and was isolated from 12-LO, peroxidase and LTA4 hydrolase activities. Reaction products were separated by reversed phase HPLC and quantitated by absorption spectrophotometry and radiochemical detection. The enzyme preparation rapidly converted [14C]arachidonate to [14C]5-hydroperoxyeicosatetraenoic acid (HPETE) and [14C]5,12-dihydroperoxyeicosatetraenoic acids (diHETEs). The 5,12-diHETEs were primarily non-enzymatic breakdown products of LTA4 (e.g., 6-trans-LTB4 and 6-trans-12-epi-LTB4). Both the 5-LO and LTA4 synthetase activities were Ca2+- and ATP-dependent. For both enzyme activities, the CA2+ stimulation required the presence of ATP. The fatty acid hydroperoxides, 5-,12-, and 15-HPETE, both stimulated ([ 3 μM]) 5-LO and LTA4 synthetase activities. The rapid isolation and subsequent characterization of 5-LO and LTA4 synthetase provide the bases for the further understanding of the role of the LO pathway in biological processes

  11. Crosslinking of tRNA containing a long extra arm to elongation factor Tu by trans-diamminedichloroplatinum(II)

    DEFF Research Database (Denmark)

    Rasmussen, Nils-Jørgen; Wikman, Friedrik; Clark, Brian F. C.

    1990-01-01

    A tRNA containing a long extra arm, namely E. coli tRNA1Leu has been crosslinked to elongation factor Tu, with the crosslinking reagent trans-diamminedichloroplatinum(II). The nucleotide involved in the crosslinking was identified to be a guanosine in the variable region at position 47F or 47G....

  12. Crystallization and preliminary X-ray diffraction analysis of an Escherichia coli tRNAGly acceptor-stem microhelix

    International Nuclear Information System (INIS)

    In order to investigate the identity elements of the E. coli tRNAGly/GlyRS class II system, a tRNAGly acceptor-stem microhelix was crystallized and a data set was collected to 2.0 Å resolution using synchrotron radiation. The tRNAGly and glycyl-tRNA synthetase (GlyRS) system is an evolutionary special case within the class II aminoacyl-tRNA synthetases because two divergent types of GlyRS exist: an archaebacterial/human type and an eubacterial type. The tRNA identity elements which determine the correct aminoacylation process are located in the aminoacyl domain of tRNAGly. To obtain further insight concerning structural investigation of the identity elements, the Escherichia coli seven-base-pair tRNAGly acceptor-stem helix was crystallized. Data were collected to 2.0 Å resolution using synchrotron radiation. Crystals belong to space group P3121 or P3221, with unit-cell parameters a = b = 35.35, c = 130.82 Å, α = β = 90, γ = 120° and two molecules in the asymmetric unit

  13. PLMItRNA, a database for mitochondrial tRNA genes and tRNAs in photosynthetic eukaryotes

    Science.gov (United States)

    Damiano, Fabrizio; Gallerani, Raffaele; Liuni, Sabino; Licciulli, Flavio; Ceci, Luigi R.

    2001-01-01

    The PLMItRNA database for mitochondrial tRNA molecules and genes in Viridiplantae (green plants) [Volpetti,V., Gallerani,R., DeBenedetto,C., Liuni,S., Licciulli,F. and Ceci,L.R. (2000) Nucleic Acids Res., 28, 159–162] has been enlarged to include algae. The database now contains 436 genes and 16 tRNA entries relative to 25 higher plants, eight green algae, four red algae (Rhodophytae) and two Stramenopiles. The PLMItRNA database is accessible via the WWW at http://bio-www.ba.cnr.it:8000/PLMItRNA. PMID:11125079

  14. Small tandemly repeated DNA sequences of higher plants likely originate from a tRNA gene ancestor.

    OpenAIRE

    Benslimane, A A; Dron, M; Hartmann, C; Rode, A.

    1986-01-01

    Several monomers (177 bp) of a tandemly arranged repetitive nuclear DNA sequence of Brassica oleracea have been cloned and sequenced. They share up to 95% homology between one another and up to 80% with other satellite DNA sequences of Cruciferae, suggesting a common ancestor. Both strands of these monomers show more than 50% homology with many tRNA genes; the best homologies have been obtained with Lys and His yeast mitochondrial tRNA genes (respectively 64% and 60%). These results suggest t...

  15. Duplication and Remolding of tRNA Genes in the Mitochondrial Genome of Reduvius tenebrosus (Hemiptera: Reduviidae)

    OpenAIRE

    Pei Jiang; Hu Li; Fan Song; Yao Cai; Jianyun Wang; Jinpeng Liu; Wanzhi Cai

    2016-01-01

    Most assassin bugs are predators that act as important natural enemies of insect pests. Mitochondrial (mt) genomes of these insects are double-strand circular DNAs that encode 37 genes. In the present study, we explore the duplication and rearrangement of tRNA genes in the mt genome of Reduvius tenebrosus, the first mt genome from the subfamily Reduviinae. The gene order rearranges from CR (control region)-trnI-trnQ-trnM-ND2 to CR-trnQ-trnI2-trnI1-trnM-ND2. We identified 23 tRNA genes, includ...

  16. Escherichia coli B lacks one of the two initiator tRNA species present in E. coli K-12.

    OpenAIRE

    Mandal, N; RajBhandary, U L

    1992-01-01

    We show that the metY locus which specifies tRNA(2fMet) in Escherichia coli K-12 specifies tRNA(1fMet) in E. coli B. This conclusion is based on results of Southern blot analysis of E. coli B and K-12 DNAs and on polymerase chain reaction amplification, cloning, and sequencing of an approximately 200-bp region of DNA corresponding to the metY loci of E. coli B and E. coli K-12. We also show that the metY locus of E. coli B is transcriptionally active. E. coli strains transformed with the mult...

  17. The Pai-associated leuX specific tRNA5(Leu) affects type 1fimbriation in pathogenic Escherichia coli by control of FimB recombinase expression

    DEFF Research Database (Denmark)

    Ritter, A.; Gally, D.; Olsen, Peter Bjarke;

    1997-01-01

    The uropathogenic Escherichia coli strain 536 (06:K15:H31) carries two large chromosomalpathogenicity islands (Pais). Both Pais are flanked by tRNA genes. Spontaneous deletion of Pai IIresults in truncation of the leuX tRNA5Leu gene. This tRNA is required for the expression of type 1fimbriae (Fim...

  18. Coevolution Theory of the Genetic Code at Age Forty: Pathway to Translation and Synthetic Life

    Directory of Open Access Journals (Sweden)

    J. Tze-Fei Wong

    2016-03-01

    Full Text Available The origins of the components of genetic coding are examined in the present study. Genetic information arose from replicator induction by metabolite in accordance with the metabolic expansion law. Messenger RNA and transfer RNA stemmed from a template for binding the aminoacyl-RNA synthetase ribozymes employed to synthesize peptide prosthetic groups on RNAs in the Peptidated RNA World. Coevolution of the genetic code with amino acid biosynthesis generated tRNA paralogs that identify a last universal common ancestor (LUCA of extant life close to Methanopyrus, which in turn points to archaeal tRNA introns as the most primitive introns and the anticodon usage of Methanopyrus as an ancient mode of wobble. The prediction of the coevolution theory of the genetic code that the code should be a mutable code has led to the isolation of optional and mandatory synthetic life forms with altered protein alphabets.

  19. Genetic association analysis of 13 nuclear-encoded mitochondrial candidate genes with type II diabetes mellitus: The DAMAGE study

    DEFF Research Database (Denmark)

    Reiling, Erwin; van Vliet-Ostaptchouk, Jana V; van 't Riet, Esther;

    2009-01-01

    Mitochondria play an important role in many processes, like glucose metabolism, fatty acid oxidation and ATP synthesis. In this study, we aimed to identify association of common polymorphisms in nuclear-encoded genes involved in mitochondrial protein synthesis and biogenesis with type II diabetes...... for correct mitochondrial protein synthesis and biogenesis: aminoacyl tRNA synthetases, translation initiation factors, tRNA modifying enzymes and mitochondrial DNA transcription and replication. SNPs showing evidence for association with T2DM were measured in second stage genotyping (n=10164......-wide association studies, this SNP was also not associated with T2DM (P=0.72). In conclusion, we did not find evidence for association of common variants in 13 nuclear-encoded mitochondrial proteins with T2DM.European Journal of Human Genetics advance online publication, 11 February 2009; doi:10.1038/ejhg.2009.4....

  20. Trm9-Catalyzed tRNA Modifications Regulate Global Protein Expression by Codon-Biased Translation.

    Directory of Open Access Journals (Sweden)

    Wenjun Deng

    2015-12-01

    Full Text Available Post-transcriptional modifications of transfer RNAs (tRNAs have long been recognized to play crucial roles in regulating the rate and fidelity of translation. However, the extent to which they determine global protein production remains poorly understood. Here we use quantitative proteomics to show a direct link between wobble uridine 5-methoxycarbonylmethyl (mcm5 and 5-methoxy-carbonyl-methyl-2-thio (mcm5s2 modifications catalyzed by tRNA methyltransferase 9 (Trm9 in tRNAArg(UCU and tRNAGlu(UUC and selective translation of proteins from genes enriched with their cognate codons. Controlling for bias in protein expression and alternations in mRNA expression, we find that loss of Trm9 selectively impairs expression of proteins from genes enriched with AGA and GAA codons under both normal and stress conditions. Moreover, we show that AGA and GAA codons occur with high frequency in clusters along the transcripts, which may play a role in modulating translation. Consistent with these results, proteins subject to enhanced ribosome pausing in yeast lacking mcm5U and mcm5s2U are more likely to be down-regulated and contain a larger number of AGA/GAA clusters. Together, these results suggest that Trm9-catalyzed tRNA modifications play a significant role in regulating protein expression within the cell.

  1. Competing pathways control host resistance to virus via tRNA modification and programmed ribosomal frameshifting.

    Science.gov (United States)

    Maynard, Nathaniel D; Macklin, Derek N; Kirkegaard, Karla; Covert, Markus W

    2012-01-01

    Viral infection depends on a complex interplay between host and viral factors. Here, we link host susceptibility to viral infection to a network encompassing sulfur metabolism, tRNA modification, competitive binding, and programmed ribosomal frameshifting (PRF). We first demonstrate that the iron-sulfur cluster biosynthesis pathway in Escherichia coli exerts a protective effect during lambda phage infection, while a tRNA thiolation pathway enhances viral infection. We show that tRNA(Lys) uridine 34 modification inhibits PRF to influence the ratio of lambda phage proteins gpG and gpGT. Computational modeling and experiments suggest that the role of the iron-sulfur cluster biosynthesis pathway in infection is indirect, via competitive binding of the shared sulfur donor IscS. Based on the universality of many key components of this network, in both the host and the virus, we anticipate that these findings may have broad relevance to understanding other infections, including viral infection of humans. PMID:22294093

  2. Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA.

    Science.gov (United States)

    Barhoom, Sima; Kaur, Jaskiran; Cooperman, Barry S; Smorodinsky, Nechama I; Smilansky, Zeev; Ehrlich, Marcelo; Elroy-Stein, Orna

    2011-10-01

    We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution through quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). The localization of fl-tRNA to active translation sites was confirmed through its co-localization with cellular factors and its dynamic alterations upon inhibition of protein synthesis. Moreover, fluorescence resonance energy transfer (FRET) signals, generated when fl-tRNAs, separately labeled as a FRET pair occupy adjacent sites on the ribosome, quantitatively reflect levels of protein synthesis in defined cellular regions. In addition, FRET signals enable detection of intra-populational variability in protein synthesis activity. We demonstrate that FtTM allows quantitative comparison of protein synthesis between different cell types, monitoring effects of antibiotics and stress agents, and characterization of changes in spatial compartmentalization of protein synthesis upon viral infection. PMID:21795382

  3. Nonribosomal Peptide Synthetase Genes pesL and pes1 Are Essential for Fumigaclavine C Production in Aspergillus fumigatus

    DEFF Research Database (Denmark)

    O'Hanlon, Karen A.; Gallagher, Lorna; Schrettl, Markus;

    2012-01-01

    The identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen, Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end produc...

  4. Continuous recording of long-chain acyl-coenzyme A synthetase activity using fluorescently labeled bovine serum albumin

    DEFF Research Database (Denmark)

    Demant, Erland J.F.; Nystrøm, Birthe T.

    2001-01-01

    acyl-Coenzyme A, synthetase, activity assay, fluorescence recording, fatty acid probe, serum albumin, hydroxycoumarin, detergent, micelles, Pseudomonas fragi, rat liver microsomes......acyl-Coenzyme A, synthetase, activity assay, fluorescence recording, fatty acid probe, serum albumin, hydroxycoumarin, detergent, micelles, Pseudomonas fragi, rat liver microsomes...

  5. Undetected antisense tRNAs in mitochondrial genomes?

    Directory of Open Access Journals (Sweden)

    Seligmann Hervé

    2010-06-01

    Full Text Available Abstract Background The hypothesis that both mitochondrial (mt complementary DNA strands of tRNA genes code for tRNAs (sense-antisense coding is explored. This could explain why mt tRNA mutations are 6.5 times more frequently pathogenic than in other mt sequences. Antisense tRNA expression is plausible because tRNA punctuation signals mt sense RNA maturation: both sense and antisense tRNAs form secondary structures potentially signalling processing. Sense RNA maturation processes by default 11 antisense tRNAs neighbouring sense genes. If antisense tRNAs are expressed, processed antisense tRNAs should have adapted more for translational activity than unprocessed ones. Four tRNA properties are examined: antisense tRNA 5' and 3' end processing by sense RNA maturation and its accuracy, cloverleaf stability and misacylation potential. Results Processed antisense tRNAs align better with standard tRNA sequences with the same cognate than unprocessed antisense tRNAs, suggesting less misacylations. Misacylation increases with cloverleaf fragility and processing inaccuracy. Cloverleaf fragility, misacylation and processing accuracy of antisense tRNAs decrease with genome-wide usage of their predicted cognate amino acid. Conclusions These properties correlate as if they adaptively coevolved for translational activity by some antisense tRNAs, and to avoid such activity by other antisense tRNAs. Analyses also suggest previously unsuspected particularities of aminoacylation specificity in mt tRNAs: combinations of competition between tRNAs on tRNA synthetases with competition between tRNA synthetases on tRNAs determine specificities of tRNA amino acylations. The latter analyses show that alignment methods used to detect tRNA cognates yield relatively robust results, even when they apparently fail to detect the tRNA's cognate amino acid and indicate high misacylation potential. Reviewers This article was reviewed by Dr Juergen Brosius, Dr Anthony M Poole and

  6. Various mechanisms in cyclopeptide production from precursors synthesized independently of non-ribosomal peptide synthetases

    Institute of Scientific and Technical Information of China (English)

    Wenyan Xu; Liling Li; Liangcheng Du; Ninghua Tan

    2011-01-01

    An increasing number of cyclopeptides have been discovered as products of ribosomal synthetic pathway.The biosynthetic study of these cyclopeptides has revealed interesting new mechanisms for cyclization.This review highlighted the recent discoveries in cyclization mechanisms for cyclopeptides synthesized independently of non-ribosomal peptide synthetases,including endopeptidase-catalyzed cyclization,intein-mediated cyclization,and peptide synthetase-catalyzed cyclization.This information may help to design hybrid ribosomal and non-ribosomal biosynthetic systems to produce novel cyclopeptides with various bioactivities.

  7. A cDNA clone from Zea mays endosperm sucrose synthetase mRNA.

    OpenAIRE

    Geiser, M.; Döring, H P; Wöstemeyer, J; Behrens, U.; Tillmann, E; Starlinger, P.

    1980-01-01

    A cDNA clone for maize endosperm sucrose synthetase of 62o nucleotide pairs length was obtained by cloning double stranded DNA obtained from the total maize endosperm poly(A) RNA in pBR322, and identifying the appropriate clone by hybrid-promoter translation. In Southern blotting to genomic BamHI-digested DNA, a single band only of approximately 20 Kb lights up, indicating that the sucrose synthetase gene is unique, or that closely linked copies are located on this DNA fragment.

  8. Archease from Pyrococcus abyssi improves substrate specificity and solubility of a tRNA m5C methyltransferase

    DEFF Research Database (Denmark)

    Auxilien, Sylvie; El Khadali, Fatima; Rasmussen, Anette;

    2007-01-01

    reading frame (PAB1947), which is shown here to encode a tRNA m(5)C methyltransferase. In vitro, the purified recombinant methyltransferase catalyzes m(5)C formation at several cytosines within tRNAs with preference for C49. The specificity of the methyltransferase is increased by the archease...

  9. A story with a good ending: tRNA 3'-end maturation by CCA-adding enzymes.

    Science.gov (United States)

    Xiong, Yong; Steitz, Thomas A

    2006-02-01

    CCA-adding enzymes (tRNA nucleotidyltransferases) are responsible for the maturation or repair of the functional 3' end of tRNAs. These enzymes are remarkable because they polymerize the essential nucleotides CCA onto the 3' terminus of tRNA precursors without using a nucleic acid template. Recent crystal structures, plus three decades of enzymology, have revealed the elegant mechanisms by which CCA-adding enzymes achieve their substrate specificity in a nucleic acid template independent fashion. The class I CCA-adding enzyme employs both an arginine sidechain and backbone phosphates of the bound tRNA to recognize incoming nucleotides. It switches from C to A addition through changes in the size and shape of the nucleotide-binding pocket, which is progressively altered by the elongating 3' terminus of the tRNA. By contrast, the class II CCA-adding enzyme uses only amino acid sidechains, which form a protein template for incoming nucleotide selection. PMID:16364630

  10. Electrophoretic mobility shift assays: analysis of tRNA binding to the T box riboswitch antiterminator RNA.

    Science.gov (United States)

    Anupam, R; Zhou, S; Hines, J V

    2015-01-01

    Changes in electrophoretic mobility upon complex formation with RNA can be used to probe structure-function relationships that are critical for complex formation. Here, we describe the application of this technique to monitor tRNA binding to the T box riboswitch antiterminator RNA. PMID:25352142

  11. Several RNase T2 enzymes function in induced tRNA and rRNA turnover in the ciliate Tetrahymena

    DEFF Research Database (Denmark)

    Andersen, Kasper Langebjerg; Collins, Kathleen

    2012-01-01

    RNase T2 enzymes are produced by a wide range of organisms and have been implicated to function in diverse cellular processes, including stress-induced anticodon loop cleavage of mature tRNAs to generate tRNA halves. Here we describe a family of eight RNase T2 genes (RNT2A-RNT2H) in the ciliate...

  12. The ribosome triggers the stringent response by RelA via a highly distorted tRNA

    OpenAIRE

    Agirrezabala, Xabier; Fernández, Israel S.; Kelley, Ann C.; Cartón, David Gil; Ramakrishnan, Venki; Valle, Mikel

    2013-01-01

    The bacterial stringent response is initiated by RelA and links nutrient starvation with the transcriptional control of genes. Cryo-EM now shows that RelA on the ribosome stabilizes an unusual distorted form of cognate, deacylated tRNA.

  13. Measurement of Acceptor-TΨC Helix Length of tRNA for Terminal A76-Addition by A-Adding Enzyme.

    Science.gov (United States)

    Yamashita, Seisuke; Martinez, Anna; Tomita, Kozo

    2015-05-01

    The 3'-terminal CCA (C74C75A76-3') of tRNA is required for protein synthesis. In Aquifex aeolicus, the CCA-3' is synthesized by CC-adding and A-adding enzymes, although in most organisms, CCA is synthesized by a single CCA-adding enzyme. The mechanisms by which the A-adding enzyme adds only A76, but not C74C75, onto tRNA remained elusive. The complex structures of the enzyme with various tRNAs revealed the presence of a single tRNA binding site on the enzyme, with the enzyme measuring the acceptor-TΨC helix length of tRNA. The 3'-C75 of tRNA lacking A76 can reach the active site and the size and shape of the nucleotide binding pocket at the insertion stage are suitable for ATP. The 3'-C74 of tRNA lacking C75A76 cannot reach the active site, although CTP or ATP can bind the active pocket. Thus, the A-adding enzyme adds only A76, but not C74C75, onto tRNA. PMID:25914059

  14. Ribosome•RelA structures reveal the mechanism of stringent response activation.

    Science.gov (United States)

    Loveland, Anna B; Bah, Eugene; Madireddy, Rohini; Zhang, Ying; Brilot, Axel F; Grigorieff, Nikolaus; Korostelev, Andrei A

    2016-01-01

    Stringent response is a conserved bacterial stress response underlying virulence and antibiotic resistance. RelA/SpoT-homolog proteins synthesize transcriptional modulators (p)ppGpp, allowing bacteria to adapt to stress. RelA is activated during amino-acid starvation, when cognate deacyl-tRNA binds to the ribosomal A (aminoacyl-tRNA) site. We report four cryo-EM structures of E. coli RelA bound to the 70S ribosome, in the absence and presence of deacyl-tRNA accommodating in the 30S A site. The boomerang-shaped RelA with a wingspan of more than 100 Å wraps around the A/R (30S A-site/RelA-bound) tRNA. The CCA end of the A/R tRNA pins the central TGS domain against the 30S subunit, presenting the (p)ppGpp-synthetase domain near the 30S spur. The ribosome and A/R tRNA are captured in three conformations, revealing hitherto elusive states of tRNA engagement with the ribosomal decoding center. Decoding-center rearrangements are coupled with the step-wise 30S-subunit 'closure', providing insights into the dynamics of high-fidelity tRNA decoding. PMID:27434674

  15. RNA molecular turn-over in Tobacco cell cultures. II. Intramolecular and intermolecular differential turn-over of tRNA

    International Nuclear Information System (INIS)

    Tobacco cells, exponentially growing in a shaken liquid medium were labeled by addition of [32P]-phosphate during 30 minutes or 4 hours. The tRNA of these cells were extracted and fractionated by reversed phase chromatography (RPC5). The different fractions had the same specific radioactivity after a 4 hours labeling period. When the 32P pulse was shortened to 30 minutes the specific radioactivity of the tail fractions eluted from the RPC5 column chromatography was about two fold that of the head fractions. The fractions eluted from the RPC5 column all belonged to the tRNA population. Their electrophoretic behavior on polyacrylamide gels supplemented with formamide was that of tRNA; the enzymatic acylation of the total tRNA extract and of each fraction were satisfactory; moreover the more heavily labeled fractions were more acylated. Only mature tRNA are responsible for the observed differences between the specific radioactivities of the various tRNA fractions. These differences could not be assigned to the nucleotides of the vCvCvA-OH oligonucleotides 3'-end but were assigned to the transcribed part of the tRNA molecules. These results may be understood according to the hypotheses that either the precursor nucleotide pools of the tRNA incorporated [32P]-phosphate at different rates in different cell compartments, or that some tRNA species, tightly bound to the RPC5 solid phase of the columns, are more rapidly turned over. These explanations do not exclude each other

  16. Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2

    DEFF Research Database (Denmark)

    Schwer, Bjoern; Bunkenborg, Jakob; Verdin, Regis O; Andersen, Jens S; Verdin, Eric

    2006-01-01

    We report that human acetyl-CoA synthetase 2 (AceCS2) is a mitochondrial matrix protein. AceCS2 is reversibly acetylated at Lys-642 in the active site of the enzyme. The mitochondrial sirtuin SIRT3 interacts with AceCS2 and deacetylates Lys-642 both in vitro and in vivo. Deacetylation of AceCS2 b...

  17. 2'-phosphodiesterase and 2',5'-oligoadenylate synthetase activities in the lowest metazoans, sponge [porifera

    DEFF Research Database (Denmark)

    Saby, Emilie; Poulsen, Jesper Buchhave; Justesen, Just;

    2009-01-01

    Sponges [porifera], the most ancient metazoans, contain modules related to the vertebrate immune system, including the 2′,5′-oligoadenylate synthetase (OAS). The components of the antiviral 2′,5′-oligoadenylate (2–5A) system (OAS, 2′-Phosphodiesterase (2′-PDE) and RNAse L) of vertebrates have not...

  18. The PRPP Synthetase Spectrum: What Does it Demonstrate About Nucleotide Syndromes?

    NARCIS (Netherlands)

    Duley, J.A.; Christodoulou, J.; Brouwer, A.P.M. de

    2011-01-01

    Defects in X-linked phosphoribosylpyrophosphate synthetase 1 (PRPS1) manifest as follows: (1) PRS-I enzyme "superactivity" (gain-of-function mutations affecting allosteric regions); (2) PRS-I overexpression (which may be linked to miRNA mutation); (3) severe PRS-I deficiency/Arts syndrome (missense

  19. Changes in Activities of Glutamine Synthetase during Grain Filling and Their Relation to Rice Quality

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Four japonica rice varieties differed in cooking and eating qualities were used in a pot experiment to study the relationship between the activities of glutamine synthetase during grain filling and rice quality. The activities of glutamine synthetase gradually increased and then declined as a single peak curve in the course of grain filling. The 15th day after heading was a turning point, before which the enzymatic activities in the inferior rice varieties with high protein content were higher than those in the superior rice varietie with low protein content, and after which it was converse. The activity of glutamine synthetase in grain was correlated with the taste meter value, peak viscosity and breakdown negatively at the early stage of grain filling whereas positively at the middle and late stages. Moreover, it was correlated with the protein content of rice grain and setback positively at the early stage and negatively at the middle and late stages. The correlation degree varied with the course of grain filling. From 15 days to 20 days after heading was a critical stage, in which the direction of correlation between the activity of glutamine synthetase and taste meter value and RVA properties of rice changed.

  20. Regulation of Amidase Formation in Mutants from Pseudomonas aeruginosa PAO Lacking Glutamine Synthetase Activity

    NARCIS (Netherlands)

    Janssen, Dick B.; Herst, Patricia M.; Joosten, Han M.L.J.; Drift, Chris van der

    1982-01-01

    The formation of amidase was studied in mutants from Pseudomonas aeruginosa PAO lacking glutamine synthetase activity. It appeared that catabolite repression of amidase synthesis by succinate was partially relieved when cellular growth was limited by glutamine. Under these conditions, a correlation

  1. Characterization of a Salmonella typhimurium mutant defective in phosphoribosylpyrophosphate synthetase

    DEFF Research Database (Denmark)

    Jochimsen, Bjarne U.; Hove-Jensen, Bjarne; Garber, Bruce B.;

    1985-01-01

    This study describes the isolation and characterization of a mutant (strain GP122) of Salmonella typhimurium with a partial deficiency of phosphoribosylpyrophosphate (PRPP) synthetase activity. This strain was isolated in a purE deoD gpt purine auxotroph by a procedure designed to select guanosin...

  2. Purification and properties of phosphoribosyl-diphosphate synthetase from Bacillus subtilis

    DEFF Research Database (Denmark)

    Arnvig, Kirsten; Hove-Jensen, Bjarne; Switzer, Robert L.

    1990-01-01

    Phosphoribosyl-diphosphate (PPRibP) synthetase from Bacillus subtiliis has been purified to near homogeneity from an Escherichia coli Δprs strain bearing the cloned B. subtilis prs gene, encoding PPRibP synthentase, on a plasmid. The Mr of the subunit (34,000) and its amino-terminal amino acid...

  3. Acyl-CoA synthetase 1 deficiency alters cardiolipin species and impairs mitochondrial function

    DEFF Research Database (Denmark)

    Grevengoed, Trisha J; Martin, Sarah A; Katunga, Lalage; Cooper, Daniel E; Anderson, Ethan J; Murphy, Robert C; Coleman, Rosalind A

    2015-01-01

    Long-chain acyl-CoA synthetase 1 (ACSL1) contributes more than 90% of total cardiac ACSL activity, but its role in phospholipid synthesis has not been determined. Mice with an inducible knockout of ACSL1 (Acsl1(T-/-)) have impaired cardiac fatty acid oxidation and rely on glucose for ATP producti...

  4. Tandem heterocyclization domains in a nonribosomal peptide synthetase essential for siderophore biosynthesis in Vibrio anguillarum

    NARCIS (Netherlands)

    Di Lorenzo, M.; Stork, M.; Naka, H.; Tolmasky, M.E.; Crosa, J.H.

    2008-01-01

    Anguibactin, the siderophore produced by Vibrio anguillarum 775, is synthesized via a nonribosomal peptide synthetase (NRPS) mechanism. Most of the genes required for anguibactin biosynthesis are harbored by the pJM1 plasmid. Complete sequencing of this plasmid identified an orf encoding a 108 kDa p

  5. Phosphoribosyl pyrophosphate synthetase activity affects growth and riboflavin production in Ashbya gossypii

    Directory of Open Access Journals (Sweden)

    Revuelta José L

    2008-09-01

    Full Text Available Abstract Background Phosphoribosyl pyrophosphate (PRPP is a central compound for cellular metabolism and may be considered as a link between carbon and nitrogen metabolism. PRPP is directly involved in the de novo and salvage biosynthesis of GTP, which is the immediate precursor of riboflavin. The industrial production of this vitamin using the fungus Ashbya gossypii is an important biotechnological process that is strongly influenced by substrate availability. Results Here we describe the characterization and manipulation of two genes of A. gossypii encoding PRPP synthetase (AGR371C and AGL080C. We show that the AGR371C and AGL080C gene products participate in PRPP synthesis and exhibit inhibition by ADP. We also observed a major contribution of AGL080C to total PRPP synthetase activity, which was confirmed by an evident growth defect of the Δagl080c strain. Moreover, we report the overexpression of wild-type and mutant deregulated isoforms of Agr371cp and Agl080cp that significantly enhanced the production of riboflavin in the engineered A. gossypii strains. Conclusion It is shown that alterations in PRPP synthetase activity have pleiotropic effects on the fungal growth pattern and that an increase in PRPP synthetase enzymatic activity can be used to enhance riboflavin production in A. gossypii.

  6. Absence of acetohydroxy acid synthetase in a clinical isolate of Neisseria gonorrhoeae requiring isoleucine and valine.

    OpenAIRE

    Lerner, S A; Friedman, E L; Dudek, E J; Kominski, G; Bohnhoff, M.; Morello, J A

    1980-01-01

    A clinical isolate of Neisseria gonorrhoeae with an unusual growth requirement for isoleucine and valine lacked the activity of acetohydroxy acid synthetase, one of the enzymes required for the biosynthesis of these amino acids. A spontaneous mutant which no longer required isoleucine and valine had acquired this enzymatic activity.

  7. Structures of two distinct conformations of holo-non-ribosomal peptide synthetases.

    Science.gov (United States)

    Drake, Eric J; Miller, Bradley R; Shi, Ce; Tarrasch, Jeffrey T; Sundlov, Jesse A; Allen, C Leigh; Skiniotis, Georgios; Aldrich, Courtney C; Gulick, Andrew M

    2016-01-14

    Many important natural products are produced by multidomain non-ribosomal peptide synthetases (NRPSs). During synthesis, intermediates are covalently bound to integrated carrier domains and transported to neighbouring catalytic domains in an assembly line fashion. Understanding the structural basis for catalysis with non-ribosomal peptide synthetases will facilitate bioengineering to create novel products. Here we describe the structures of two different holo-non-ribosomal peptide synthetase modules, each revealing a distinct step in the catalytic cycle. One structure depicts the carrier domain cofactor bound to the peptide bond-forming condensation domain, whereas a second structure captures the installation of the amino acid onto the cofactor within the adenylation domain. These structures demonstrate that a conformational change within the adenylation domain guides transfer of intermediates between domains. Furthermore, one structure shows that the condensation and adenylation domains simultaneously adopt their catalytic conformations, increasing the overall efficiency in a revised structural cycle. These structures and the single-particle electron microscopy analysis demonstrate a highly dynamic domain architecture and provide the foundation for understanding the structural mechanisms that could enable engineering of novel non-ribosomal peptide synthetases. PMID:26762461

  8. Association of IDDM and attenuated response of 2',5'-oligoadenylate synthetase to yellow fever vaccine

    DEFF Research Database (Denmark)

    Bonnevie-Nielsen, V; Larsen, M L; Frifelt, J J;

    1989-01-01

    Basal and yellow fever vaccination-induced 2',5'-oligoadenylate synthetase (2',5'A) activity was determined in blood mononuclear cells (peripheral blood lymphocytes [PBLs]) from insulin-dependent diabetes mellitus (IDDM) and matched control subjects. The live attenuated yellow fever vaccine repre...

  9. tRNA Core Hypothesis for the Transition from the RNA World to the Ribonucleoprotein World

    Directory of Open Access Journals (Sweden)

    Savio T. de Farias

    2016-03-01

    Full Text Available Herein we present the tRNA core hypothesis, which emphasizes the central role of tRNAs molecules in the origin and evolution of fundamental biological processes. tRNAs gave origin to the first genes (mRNA and the peptidyl transferase center (rRNA, proto-tRNAs were at the core of a proto-translation system, and the anticodon and operational codes then arose in tRNAs molecules. Metabolic pathways emerged from evolutionary pressures of the decoding systems. The transitions from the RNA world to the ribonucleoprotein world to modern biological systems were driven by three kinds of tRNAs transitions, to wit, tRNAs leading to both mRNA and rRNA.

  10. The nucleotide sequence of histidine tRNA gamma of Drosophila melanogaster.

    OpenAIRE

    Altwegg, M.; Kubli, E

    1980-01-01

    The nucleotide sequence of D. melanogaster histidine tRNA gamma was determined to be: pG-G-C-C-G-U-G-A-U-C-G-U-C-psi-A-G-D-G-G-D-D-A-G-G-A-C-C-C-C-A-C-G-psi-U-G-U-G- m1G-C-C-G-U-G-G-U-A-A-C-C-m5C-A-G-G-U-psi-C-G-m1A-A-U-C-C-U-G-G-U-C-A-C-G-G-m5C -A-C-C-AOH. An additional unpaired G is found at the 5' end, and the T in the TpsiC loop is replaced by a U.

  11. Crystal structure of tRNA m1A58 methyltransferase TrmI from Aquifex aeolicus in complex with S-adenosyl-l-methionine

    OpenAIRE

    Kuratani, Mitsuo; Yanagisawa, Tatsuo; Ishii, Ryohei; Matsuno, Michiyo; Si, Shu-Yi; Katsura, Kazushige; Ushikoshi-Nakayama, Ryoko; Shibata, Rie; Shirouzu, Mikako; Bessho, Yoshitaka; Yokoyama, Shigeyuki

    2014-01-01

    The N 1-methyladenosine residue at position 58 of tRNA is found in the three domains of life, and contributes to the stability of the three-dimensional L-shaped tRNA structure. In thermophilic bacteria, this modification is important for thermal adaptation, and is catalyzed by the tRNA m1A58 methyltransferase TrmI, using S-adenosyl-l-methionine (AdoMet) as the methyl donor. We present the 2.2 Å crystal structure of TrmI from the extremely thermophilic bacterium Aquifex aeolicus, in complex wi...

  12. Regulation of the intersubunit ammonia tunnel in Mycobacterium tuberculosis glutamine-dependent NAD[superscript +] synthetase

    Energy Technology Data Exchange (ETDEWEB)

    Chuenchor, Watchalee; Doukov, Tzanko I.; Resto, Melissa; Chang, Andrew; Gerratana, Barbara (SSRL); (Maryland)

    2012-08-31

    Glutamine-dependent NAD{sup +} synthetase is an essential enzyme and a validated drug target in Mycobacterium tuberculosis (mtuNadE). It catalyses the ATP-dependent formation of NAD{sup +} from NaAD{sup +} (nicotinic acid-adenine dinucleotide) at the synthetase active site and glutamine hydrolysis at the glutaminase active site. An ammonia tunnel 40 {angstrom} (1 {angstrom} = 0.1 nm) long allows transfer of ammonia from one active site to the other. The enzyme displays stringent kinetic synergism; however, its regulatory mechanism is unclear. In the present paper, we report the structures of the inactive glutaminase C176A variant in an apo form and in three synthetase-ligand complexes with substrates (NaAD{sup +}/ATP), substrate analogue {l_brace}NaAD{sup +}/AMP-CPP (adenosine 5'-[{alpha},{beta}-methylene]triphosphate){r_brace} and intermediate analogues (NaAD{sup +}/AMP/PPi), as well as the structure of wild-type mtuNadE in a product complex (NAD{sup +}/AMP/PPi/glutamate). This series of structures provides snapshots of the ammonia tunnel during the catalytic cycle supported also by kinetics and mutagenesis studies. Three major constriction sites are observed in the tunnel: (i) at the entrance near the glutaminase active site; (ii) in the middle of the tunnel; and (iii) at the end near the synthetase active site. Variation in the number and radius of the tunnel constrictions is apparent in the crystal structures and is related to ligand binding at the synthetase domain. These results provide new insight into the regulation of ammonia transport in the intermolecular tunnel of mtuNadE.

  13. Activation of 2'-5' oligoadenylate synthetase by single-stranded and double-stranded RNA aptamers

    DEFF Research Database (Denmark)

    Hartmann, R; Norby, P L; Martensen, P M;

    1998-01-01

    . Competition experiments indicate that the binding site for the small RNAs on the 2'-5' oligoadenylate synthetase molecule at least partially overlaps that for the synthetic double-stranded RNA, poly(I).poly(C). Several of the RNAs function as potent activators of 2'-5' oligoadenylate synthetase in vitro......, although there is no correlation between binding affinity and ability to activate. The RNA aptamers having the strongest activation potential appear to have few base-paired regions. This suggests that 2'-5' oligoadenylate synthetase, which has previously been believed to be activated only by double-stranded...

  14. Biophysical insights into the intercalative interaction of surfactant cobalt(III) complexes of certain diimine ligands bound to yeast tRNA: Effects of hydrophobicity

    Science.gov (United States)

    Nagaraj, Karuppiah; Sakthinathan, Subramanian; Arunachalam, Sankaralingam

    2015-08-01

    The interaction of two surfactant cobalt(III) complexes, cis-[Co(ip)2(DA)2](ClO4)3 1 and cis-[Co(dpq)2(DA)2](ClO4)3 2 where ip = imidazo[4,5-f][1,10]phenanthroline and dpq = dipyrido[3,2-d:2‧-3‧-f]quinoxaline with yeast tRNA have been explored by using electronic absorption, competitive binding, electrochemical studies and viscosity measurements. The results suggest that these complexes can bind to tRNA by intercalation. The presence of hydrophobic diimine ligand and the long aliphatic double chains of these complexes facilitate its intercalative interaction with the hydrophobic interior of the tRNA. The extent of tRNA binding of complex 2 has greater affinity than that of complex containing imidazo[4,5-f][1,10]phenanthroline ligands.

  15. PLMItRNA, a database for tRNAs and tRNA genes in plant mitochondria: enlargement and updating

    Science.gov (United States)

    Volpetti, Vito; Gallerani, Raffaele; De Benedetto, Caterina; Liuni, Sabino; Licciulli, Flavio; Ceci, Luigi R.

    2000-01-01

    The current version of PLMItRNA has been realized to constitute a database for tRNA molecules and genes identified in the mitochondria of all green plants (Viridiplantae). It is the enlargement of a previous database originally restricted to seed plants [Ceci,L.R., Volpicella,M., Liuni,S., Volpetti,V., Licciulli,F. and Gallerani,R. (1999) Nucleic Acids Res., 27, 156–157]. PLMItRNA reports information and multialignments on 254 genes and 16 tRNA molecules detected in 25 higher plants (one bryophyta and 24 vascular plants) and seven green algae. PLMItRNA is accessible via the WWW at http://bio-WWW.ba.cnr.it:8000/srs6/ PMID:10592210

  16. Determination of the Specificity Landscape for Ribonuclease P Processing of Precursor tRNA 5' Leader Sequences.

    Science.gov (United States)

    Niland, Courtney N; Zhao, Jing; Lin, Hsuan-Chun; Anderson, David R; Jankowsky, Eckhard; Harris, Michael E

    2016-08-19

    Maturation of tRNA depends on a single endonuclease, ribonuclease P (RNase P), to remove highly variable 5' leader sequences from precursor tRNA transcripts. Here, we use high-throughput enzymology to report multiple-turnover and single-turnover kinetics for Escherichia coli RNase P processing of all possible 5' leader sequences, including nucleotides contacting both the RNA and protein subunits of RNase P. The results reveal that the identity of N(-2) and N(-3) relative to the cleavage site at N(1) primarily control alternative substrate selection and act at the level of association not the cleavage step. As a consequence, the specificity for N(-1), which contacts the active site and contributes to catalysis, is suppressed. This study demonstrates high-throughput RNA enzymology as a means to globally determine RNA specificity landscapes and reveals the mechanism of substrate discrimination by a widespread and essential RNA-processing enzyme. PMID:27336323

  17. Nonribosomal Peptide Synthetase Genes pesL and pes1 Are Essential for Fumigaclavine C Production in Aspergillus fumigatus

    OpenAIRE

    O'Hanlon, Karen A.; Gallagher, Lorna; Schrettl, Markus; Jöchl, Christoph; Kavanagh, Kevin; Larsen, Thomas O.; Doyle, Sean

    2012-01-01

    The identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen, Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end product of the complex ergot alkaloid (EA) pathway in A. fumigatus. Deletion of either pesL (ΔpesL) or pes1 (Δpes1) resulted in complete loss of fumigaclavine C biosynthesis, relatively increased production...

  18. Reduced density of glutamine synthetase immunoreactive astrocytes in different cortical areas in major depression but not in bipolar I disorder

    Directory of Open Access Journals (Sweden)

    Hans-Gert eBernstein

    2015-08-01

    Full Text Available There is increasing evidence for disturbances within the glutamate system in patients with affective disorders, which involve disruptions of the glutamate-glutamine- cycle. The mainly astroglia-located enzyme glutamine synthetase catalyzes the ATP-dependent condensation of ammonia and glutamate to form glutamine, thus playing a central role in glutamate and glutamine homoeostasis. However, glutamine synthetase is also expressed in numerous oligodendrocytes, another class of glial cells implicated in mood disorder pathology. To learn more about the role of glia-associated glutamine synthetase in mental illnesses, we decided to find out if numerical densities of glial cells immunostained for the enzyme protein differ between subjects with major depressive disorder, bipolar disorder and psychically healthy control cases. Counting of glutamine synthetase expressing astrocytes and oligodendrocytes in eight cortical and two subcortical brain regions of subjects with mood disorder (N=14, bipolar disorder (N=15 and controls (N=16 revealed that in major depression the densities of astrocytes were significantly reduced in some cortical but not subcortical gray matter areas, whereas no changes were found for oligodendrocytes. In bipolar disorder no alterations of glutamine synthetase-immunoreactive glia were found. From our findings we conclude that (1 glutamine synthetase expressing astrocytes are prominently involved in glutamate-related disturbances in major depression, but not in bipolar disorder and (2 glutamine synthetase expressing oligodendrocytes, though being present in significant numbers in prefrontal cortical areas, play a minor (if any role in mood disorder pathology. The latter assumption is supported by findings of others showing that - at least in the mouse brain cortex - glutamine synthetase immunoreactive oligodendroglial cells are unable to contribute to the glutamate-glutamine cycle due to the complete lack of amino acid transporters

  19. Properties of asparagine synthetase in asparagine-independent variants of Jensen rat sarcoma cells induced by 5-azacytidine.

    OpenAIRE

    Sugiyama, R H; Arfin, S M; Harris, M.

    1983-01-01

    Jensen rat sarcoma cells in culture require L-asparagine for growth and lack detectable levels of asparagine synthetase. Cultures exposed for 24 h to graded concentrations of 5-azacytidine give rise to asparagine-independent variants in high frequency. These prototrophs are stable phenotypically whether maintained in the presence or absence of L-asparagine. Asparagine synthetase activity in several variant clones was uniform in thermolability and several kinetic parameters, as well as in immu...

  20. An entropy based analysis of the relationship between the DOW JONES Index and the TRNA Sentiment series

    OpenAIRE

    Allen, David; McAleer, Michael; Singh, Abhay

    2016-01-01

    textabstractThis paper features an analysis of the relationship between the DOW JONES Industrial Average Index (DJIA) and a sentiment news series using daily data obtained from the Thomson Reuters News Analytics (TRNA)1 provided by SIRCA (The Securities Industry Research Centre of the Asia Pacic). The recent growth in the availability of on-line financial news sources such as internet news and social media sources provides instantaneous access to financial news. Various commercial agencies ha...

  1. eth-1, the Neurospora crassa locus encoding S-adenosylmethionine synthetase: Molecular cloning, sequence analysis and in vivo overexpression

    Energy Technology Data Exchange (ETDEWEB)

    Mautino, M.R.; Barra, J.L.; Rosa, A.L. [Universidad Nacional de Cordoba (Argentina)

    1996-03-01

    Intense biochemical and genetic research on the eth-1 mutant of Neurospora crassa suggested that this locus might encode S-adenosylmethionine synthetase (S-Adomet synthetase). We have used protoplast transformation and phenotypic rescue of a thermosensitive phenotype associated with the eth-1 mutation to clone the locus. Nucleotide sequence analysis demonstrated that it encodes S-Adomet synthetase. Homology analyses of prokaryotic, fungal and higher eukaryotic S-Adomet synthetase polypeptide sequences show a remarkable evolutionary conservation of the enzyme. N. crassa strains carrying S-Adomet synthetase coding sequences fused to a strong heterologous promoter were constructed to assess the phenotypic consequences of in vivo S-Adomet synthetase overexpression. Studies of growth rates and microscopic examination of vegetative development revealed that normal growth and morphogenesis take place in N. crassa even at abnormally high levels of cellular S-Adomet. The degree of cytosine methylation of a naturally methylated genomic region was dependent on the cellular levels of S-Adomet. We conclude that variation in S-Adomet levels in N. crassa cells, which in addition to the status of genomic DNA methylation could modify the flux of other S-Adomet-dependent metabolic pathways, does not affect growth rate or morphogenesis. 90 refs., 5 figs., 1 tab.

  2. The tRNA 30-end Processing Enzyme tRNase Z2 Contributes to Chloroplast Biogenesis in Rice

    Institute of Scientific and Technical Information of China (English)

    Tuan Long; Dong Guo; Dong He; Wenjie Shen; Xianghua Li

    2013-01-01

    tRNase Z (TRZ) is a ubiquitous endonuclease that removes the 30-trailer from precursor tRNAs during maturation. In yeast and animals, TRZ regulates the cell cycle via its (t)RNA processing activity;however, its physiological function in higher plants has not been well characterized. This study describes the identification of a rice (Oryza sativa) TRZ2 mutant; plants homozygous for the osatrz2 mutation were albinos with deficient chlorophyll content. A microscopic analysis of the mutant plants revealed that the transition of proplastids to chloroplasts was arrested at an early stage, and the number and size of the plastids in callus cells was substantially decreased. A genetic complementation test and an RNA interference analysis confirmed that disruption of OsaTRZ2 was responsible for the mutant phenotype. OsaTRZ2 is expressed in all rice tissues, but is preferentially expressed in leaves, sheathes, and calli. OsaTRZ2 was subcellularly localized in chloroplasts, and displayed tRNA 30-end processing activity in both in vitro and in vivo assays. In the osatrz2 mutants, transcription of plastid-encoded and nucleus-encoded RNA polymerases was severely reduced and moderately increased, respectively. These results suggest that the tRNA 30 processing activity of OsaTRZ2 contributes to chloroplast biogenesis.

  3. Effects of aeration on formation and localization of the acetyl coenzyme A synthetases of Saccharomyces cerevisiae

    Science.gov (United States)

    Klein, H. P.; Jahnke, L.

    1979-01-01

    Previous studies on the yeast Saccharomyces cerevisiae have shown that two different forms of the enzyme acetyl coenzyme A synthetase (ACS) are present, depending on the conditions under which the cells are grown. The paper evaluates the usefulness of a method designed to assay both synthetases simultaneously in yeast homogenates. The data presented confirm the possibility of simultaneous detection and estimation of the amount of both ACSs of S. cerevisiae in crude homogenates of this strain, making possible the study of physiological factors involved in the formation of these isoenzymes. One important factor for specifying which of the two enzymes is found in these yeast cells is the presence or absence of oxygen in their environment. Aeration not only affects the ratio of the two ACSs but also appears to affect the cellular distribution of these enzymes. Most of the data presented suggest the possibility that the nonaerobic ACS may serve as a precursor to the aerobic form.

  4. Molecular analysis of intragenic recombination at the tryptophan synthetase locus in Neurospora crassa

    Indian Academy of Sciences (India)

    A. Wiest; D. Barchers; M. Eaton; R. Henderson; R. Schnittker; K. Mccluskey

    2013-12-01

    Fifteen different classically generated and mapped mutations at the tryptophan synthetase locus in Neurospora crassa have been characterized to the level of the primary sequence of the gene. This sequence analysis has demonstrated that intragenic recombination is accurate to order mutations within one open reading frame. While classic genetic analysis correctly ordered the mutations, the position of mutations characterized by gene sequence analysis was more accurate. A leaky mutation was found to have a wild-type primary sequence. The presence of unique polymorphisms in the primary sequence of the trp-3 gene from strain 861 confirms that it has a unique history relative to the other strains studied. Most strains that were previously shown to be immunologically nonreactive with antibody preparations raised against tryptophan synthetase protein were shown to have nonsense mutations. This work defines 14 alleles of the N. crassa trp-3 gene.

  5. Structure of an unusual S-adenosylmethionine synthetase from Campylobacter jejuni.

    Science.gov (United States)

    Zano, Stephen P; Pavlovsky, Alexander G; Viola, Ronald E

    2014-02-01

    S-Adenosylmethionine (AdoMet) participates in a wide range of methylation and other group-transfer reactions and also serves as the precursor for two groups of quorum-sensing molecules that function as regulators of the production of virulence factors in Gram-negative bacteria. The synthesis of AdoMet is catalyzed by AdoMet synthetases (MATs), a ubiquitous family of enzymes found in species ranging from microorganisms to mammals. The AdoMet synthetase from the bacterium Campylobacter jejuni (cjMAT) is an outlier among this homologous enzyme family, with lower sequence identity, numerous insertions and substitutions, and higher catalytic activity compared with other bacterial MATs. Alterations in the structure of this enzyme provide an explanation for its unusual dimeric quaternary structure relative to the other MATs. Taken together with several active-site substitutions, this new structure provides insights into its improved kinetic properties with alternative substrates. PMID:24531478

  6. Targeted Disruption of Nonribosomal Peptide Synthetase pes3 Augments the Virulence of Aspergillus fumigatus

    OpenAIRE

    O'Hanlon, Karen; Cairns, Timothy; Stack, Deirdre; Schrettl, Markus; Bignell, Elaine; Kavanagh, Kevin; Miggin, Sinead; O'Keeffe, Grainne; Larsen, Thomas; Doyle, Sean

    2011-01-01

    Nonribosomal peptide synthesis (NRPS) is a documented virulence factor for the opportunistic pathogen Aspergillus fumigatus and other fungi. Secreted or intracellularly located NRP products include the toxic molecule gliotoxin and the iron-chelating siderophores triacetylfusarinine C and ferricrocin. No structural or immunologically relevant NRP products have been identified in the organism. We investigated the function of the largest gene in A. fumigatus, which encodes the NRP synthetase Pes...

  7. Diversity of Nonribosomal Peptide Synthetase Genes in the Microbial Metagenomes of Marine Sponges

    OpenAIRE

    Ute Hentschel; Sheila Marie Pimentel-Elardo; Sebastian Proksch; Lubomir Grozdanov

    2012-01-01

    Genomic mining revealed one major nonribosomal peptide synthetase (NRPS) phylogenetic cluster in 12 marine sponge species, one ascidian, an actinobacterial isolate and seawater. Phylogenetic analysis predicts its taxonomic affiliation to the actinomycetes and hydroxy-phenyl-glycine as a likely substrate. Additionally, a phylogenetically distinct NRPS gene cluster was discovered in the microbial metagenome of the sponge Aplysina aerophoba, which shows ...

  8. Bimodular Peptide Synthetase SidE Produces Fumarylalanine in the Human Pathogen Aspergillus fumigatus

    OpenAIRE

    Steinchen, Wieland; Lackner, Gerald; Yasmin, Sabiha; Schrettl, Markus; Dahse, Hans-Martin; Haas, Hubertus; Hoffmeister, Dirk

    2013-01-01

    The filamentous mold Aspergillus fumigatus causes invasive aspergillosis, a potentially life-threatening infectious disease, in humans. The sidE gene encodes a bimodular peptide synthetase and was shown previously to be strongly upregulated during initiation of murine lung infection. In this study, we characterized the two adenylation domains of SidE with the ATP-[32P]pyrophosphate exchange assay in vitro, which identified fumarate and l-alanine, respectively, as the preferred substrates. Usi...

  9. Genetic identification of essential indels and domains in carbamoyl phosphate synthetase II of Toxoplasma gondii

    OpenAIRE

    Fox, Barbara A.; Ristuccia, Jessica G.; Bzik, David J.

    2008-01-01

    New treatments need to be developed for the significant human diseases of toxoplasmosis and malaria to circumvent problems with current treatments and drug resistance. Apicomplexan parasites causing these lethal diseases are deficient in pyrimidine salvage suggesting that selective inhibition of de novo pyrimidine biosynthesis can lead to a severe loss of UMP and dTMP pools thereby inhibiting parasite RNA and DNA synthesis. Disruption of Toxoplasma gondii carbamoyl phosphate synthetase II (CP...

  10. Effect of detergents on membrane-associated glucan synthetase from Paracoccidioides brasiliensis.

    OpenAIRE

    San-Blas, G; San-Blas, F

    1982-01-01

    Yeast and mycelial particulate preparations of Paracoccidioides brasiliensis were subjected to the action of several detergents in an attempt to solubilize the glucan synthetase present in these preparations. This was achieved more successfully in the yeast membranes than in the mycelial ones. The enzymatic activity was greatly stimulated in the insoluble fractions upon treatment with some of the detergents used. The results suggest that the yeast and mycelial phases of P. brasiliensis may di...

  11. Glutamine synthetase immunor present in oligodendroglia of regions of the central nervous system

    Science.gov (United States)

    D'Amelio, Fernando; Eng, Lawrence F.; Gibbs, Michael A.

    1990-01-01

    Glutamine synthetase immunoreactive oligodendrocytes were identified in the cerebral cortex, cerebellum, brain stem, and spinal cord. They were mostly confined to the gray matter, particularly close to neurons and processes. The white matter showed few immunoreactive oligodendroglia. It was suggested that some type of oligodendrocytes, specially those in perineuronal location, might fulfill a functional role more akin to astrocytes than to the normally myelinating oligodendroglia.

  12. Virtual Screening to Identify Lead Inhibitors for Bacterial NAD Synthetase (NADs)

    OpenAIRE

    Moro, Whitney Beysselance; Yang, Zhengrong; Kane, Tasha; Brouillette, Christie G; Brouillette, Wayne J.

    2009-01-01

    Virtual screening was employed to identify new drug-like inhibitors of NAD synthetase (NADs) as antibacterial agents. Four databases of commercially available compounds were docked against three subsites of the NADs active site using FlexX in conjunction with CScore. Over 200 commercial compounds were purchased and evaluated in enzyme inhibition and antibacterial assays. 18 compounds inhibited NADs at or below 100 μM (7.6% hit rate), and two were selected for future SAR studies.

  13. Virtual Screening to Identify Lead Inhibitors for Bacterial NAD Synthetase (NADs)

    Science.gov (United States)

    Moro, Whitney Beysselance; Yang, Zhengrong; Kane, Tasha; Brouillette, Christie G.; Brouillette, Wayne J.

    2009-01-01

    Virtual screening was employed to identify new drug-like inhibitors of NAD synthetase (NADs) as antibacterial agents. Four databases of commercially available compounds were docked against three subsites of the NADs active site using FlexX in conjunction with CScore. Over 200 commercial compounds were purchased and evaluated in enzyme inhibition and antibacterial assays. 18 compounds inhibited NADs at or below 100 μM (7.6% hit rate), and two were selected for future SAR studies. PMID:19249205

  14. Holocarboxylase synthetase regulates expression of biotin transporters by chromatin remodeling events at the SMVT locus⋆

    OpenAIRE

    Gralla, Michael; Camporeale, Gabriela; Zempleni, Janos

    2007-01-01

    The sodium-dependent multivitamin transporter (SMVT) is essential for mediating and regulating biotin entry into mammalian cells. In cells, biotin is covalently linked to histones in a reaction catalyzed by holocarboxylase synthetase (HCS); biotinylation of lysine 12-biotinylated histone H4 (K12Bio H4) causes gene silencing. Here, we propose a novel role for HCS in sensing and regulating levels of biotin in eukaryotic cells. We hypothesized that nuclear translocation of HCS increases in respo...

  15. Coexpression of glutamine synthetase and carbamoylphosphate synthase I genes in pancreatic hepatocytes of rat.

    OpenAIRE

    Yeldandi, A. V.; X. D. Tan; Dwivedi, R S; Subbarao, V; Smith, D. D.; Scarpelli, D. G.; Rao, M S; Reddy, J K

    1990-01-01

    In the mammalian liver the distribution of ammonia-detoxifying enzymes, glutamine synthetase (GS) and carbamoylphosphate synthase I (ammonia) (CPS-I), is mutually exclusive in that these enzymes are expressed in two distinct populations of hepatocytes that are zonally demarcated in the liver acinus. In the present study we examined the distribution of GS and CPS-I in pancreatic hepatocytes to ascertain if the expression of these two genes in these hepatocytes is also mutually exclusive. Multi...

  16. Nucleotide synthetase ribozymes may have emerged first in the RNA world

    OpenAIRE

    Ma, Wentao; Yu, Chunwu; Zhang, Wentao; Hu, Jiming

    2007-01-01

    Though the “RNA world” hypothesis has gained a central role in ideas concerning the origin of life, the scenario concerning its emergence remains uncertain. It has been speculated that the first scene may have been the emergence of a template-dependent RNA synthetase ribozyme, which catalyzed its own replication: thus, “RNA replicase.” However, the speculation remains uncertain, primarily because of the large sequence length requirement of such a replicase and the lack of a convincing mechani...

  17. Comparative inhibition of bacterial and microsomal 3-ketodihydrosphingosine synthetases by L-cycloserine and other inhibitors.

    OpenAIRE

    Sundaram, K S; Lev, M

    1984-01-01

    Eleven compounds were examined for their capacity to inhibit the first enzyme of the sphingolipid pathway, 3-ketodihydrosphingosine synthetase. Of these, L-cycloserine was the most potent, affecting both bacterial and brain microsomal enzymes to a significant degree at 0.04 mM. D- and L-cycloserine irreversibly inactivated the enzyme, indicating a suicide substrate mode of action. L-Cycloserine was a more potent inhibitor of the growth of Bacteroides levii than was D-cycloserine, indicating t...

  18. On the active site thiol of gamma-glutamylcysteine synthetase: relationships to catalysis, inhibition, and regulation.

    OpenAIRE

    C. S. Huang; Moore, W. R.; Meister, A.

    1988-01-01

    gamma-Glutamylcysteine synthetase (glutamate-cysteine ligase; EC 6.3.2.2) was isolated from an Escherichia coli strain enriched in the gene for this enzyme by recombinant DNA techniques. The purified enzyme has a specific activity of 1860 units/mg and a molecular weight of 56,000. Comparison of the E. coli enzyme with the well-characterized rat kidney enzyme showed that these enzymes have similar catalytic properties (apparent Km values, substrate specificities, turnover numbers). Both enzyme...

  19. Glutamine Synthetase in Legumes: Recent Advances in Enzyme Structure and Functional Genomics

    OpenAIRE

    Marco Betti; Margarita García-Calderón; Pérez-Delgado, Carmen M.; Alfredo Credali; Guillermo Estivill; Francisco Galván; Vega, José M.; Márquez, Antonio J.

    2012-01-01

    Glutamine synthetase (GS) is the key enzyme involved in the assimilation of ammonia derived either from nitrate reduction, N2 fixation, photorespiration or asparagine breakdown. A small gene family is encoding for different cytosolic (GS1) or plastidic (GS2) isoforms in legumes. We summarize here the recent advances carried out concerning the quaternary structure of GS, as well as the functional relationship existing between GS2 and processes such as nodulation, photore...

  20. Reassimilation of Photorespiratory Ammonium in Lotus japonicus Plants Deficient in Plastidic Glutamine Synthetase.

    Directory of Open Access Journals (Sweden)

    Carmen M Pérez-Delgado

    Full Text Available It is well established that the plastidic isoform of glutamine synthetase (GS2 is the enzyme in charge of photorespiratory ammonium reassimilation in plants. The metabolic events associated to photorespiratory NH4(+ accumulation were analyzed in a Lotus japonicus photorespiratory mutant lacking GS2. The mutant plants accumulated high levels of NH4(+ when photorespiration was active, followed by a sudden drop in the levels of this compound. In this paper it was examined the possible existence of enzymatic pathways alternative to GS2 that could account for this decline in the photorespiratory ammonium. Induction of genes encoding for cytosolic glutamine synthetase (GS1, glutamate dehydrogenase (GDH and asparagine synthetase (ASN was observed in the mutant in correspondence with the diminishment of NH4(+. Measurements of gene expression, polypeptide levels, enzyme activity and metabolite levels were carried out in leaf samples from WT and mutant plants after different periods of time under active photorespiratory conditions. In the case of asparagine synthetase it was not possible to determine enzyme activity and polypeptide content; however, an increased asparagine content in parallel with the induction of ASN gene expression was detected in the mutant plants. This increase in asparagine levels took place concomitantly with an increase in glutamine due to the induction of cytosolic GS1 in the mutant, thus revealing a major role of cytosolic GS1 in the reassimilation and detoxification of photorespiratory NH4(+ when the plastidic GS2 isoform is lacking. Moreover, a diminishment in glutamate levels was observed, that may be explained by the induction of NAD(H-dependent GDH activity.

  1. Effect of post-silking drought on nitrogen partitioning and gene expression patterns of glutamine synthetase and asparagine synthetase in two maize (Zea mays L.) varieties.

    Science.gov (United States)

    Li, Yajun; Wang, Meiling; Zhang, Fengxia; Xu, Yadong; Chen, Xiaohong; Qin, Xiaoliang; Wen, Xiaoxia

    2016-05-01

    Glutamine synthetase (GS) and asparagine synthetase (AS) are proposed to have important function in plant nitrogen (N) remobilization, but their roles under drought stress are not well defined. In this study, the expression dynamics of GS and AS genes were analyzed in two maize varieties (ZD958 and NH101) in relation to post-silking drought stress induced nitrogen partitioning. ZD958 was a 'stay-green' variety with 5% nitrogen harvest index (NHI) lower than NH101. From silking to maturity, the amount of nitrogen remobilized from ear-leaves in ZD958 was evidently lower than NH101, and post-silking drought stress increased the nitrogen remobilization for both varieties. In ear-leaves, the expression of ZmGln1-3 was enhanced under drought stress. Three AS genes (ZmAS1, ZmAS2 and ZmAS3) were differentially regulated by post-silking drought treatment, of which the expression of ZmAS3 was stimulated at late stage of leaf senescence. In NH101, the expression level of ZmAS3 was markedly higher than that in ZD958. In developing grains, there were no significant differences in expression patterns of GS and AS genes between well water and drought treated plants. Drought stress altered maize N partitioning at the whole-plant level, and the up-regulation of GS and AS genes may contribute to the higher leaf nitrogen remobilization when exposed to drought treatments. PMID:26913793

  2. Crystallization and preliminary X-ray diffraction studies of FAD synthetase from Corynebacterium ammoniagenes

    International Nuclear Information System (INIS)

    Native and selenomethionine-labelled FAD synthetase from C. ammoniagenes have been crystallized by the hanging-drop vapour-diffusion method. A MAD data set for SeMet-labelled FAD synthetase was collected to 2.42 Å resolution, while data sets were collected to 1.95 Å resolution for the native crystals. FAD synthetase from Corynebacterium ammoniagenes (CaFADS), a prokaryotic bifunctional enzyme that catalyses the phosphorylation of riboflavin as well as the adenylylation of FMN, has been crystallized using the hanging-drop vapour-diffusion method at 277 K. Diffraction-quality cubic crystals of native and selenomethionine-labelled (SeMet-CaFADS) protein belonged to the cubic space group P213, with unit-cell parameters a = b = c = 133.47 Å and a = b = c = 133.40 Å, respectively. Data sets for native and SeMet-containing crystals were collected to 1.95 and 2.42 Å resolution, respectively

  3. Purification and characterization of fatty acyl-acyl carrier protein synthetase from Vibrio harveyi.

    Science.gov (United States)

    Fice, D; Shen, Z; Byers, D M

    1993-01-01

    A Vibrio harveyi enzyme which catalyzes the ATP-dependent ligation of fatty acids to acyl carrier protein (ACP) has been purified 6,000-fold to apparent homogeneity by anion-exchange, gel filtration, and ACP-Sepharose affinity chromatography. Purified acyl-ACP synthetase migrated as a single 62-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as an 80-kDa protein by gel filtration under reducing conditions. Activity of the purified enzyme was lost within hours in the absence of glycerol and low concentrations of Triton X-100. Acyl-ACP synthetase exhibited Kms for myristic acid, ACP, and ATP of 7 microM, 18 microM, and 0.3 mM, respectively. The enzyme was specific for adenine-containing nucleotides, and AMP was the product of the reaction. No covalent acyl-enzyme intermediate was observed. Enzyme activity was stimulated up to 50% by iodoacetamide but inhibited > 80% by N-ethylmaleimide: inhibition by the latter was prevented by ATP and ACP but not myristic acid. Dithiothreitol and sulfhydryl-directed reagents also influenced enzyme size, activity, and elution pattern on anion-exchange resins. The function of acyl-ACP synthetase has not been established, but it may be related to the capacity of V. harveyi to elongate exogenous fatty acids by an ACP-dependent mechanism. Images PMID:8384617

  4. Directed evolution of adenylosuccinate synthetase from Bacillus subtilis and its application in metabolic engineering.

    Science.gov (United States)

    Wang, Xiaoyue; Wang, Guanglu; Li, Xinli; Fu, Jing; Chen, Tao; Wang, Zhiwen; Zhao, Xueming

    2016-08-10

    Adenylosuccinate synthetase (EC. 6.3.4.4) encoded by purA in Bacillus subtilis, catalyzing the first step of the conversion of IMP to AMP, plays an important role in flux distribution in the purine biosynthetic pathway. In this study, we described the use of site saturation mutagenesis to obtain a desired enzyme activity of adenylosuccinate synthetase and its application in flux regulation. Based on sequence alignment and structural modeling, a library of enzyme variants was created by a semi-rational evolution strategy in position Thr238 and Pro242. Other than purA deletion, the leaky mutation purA(P242N) partially reduced the flux towards AMP derived from IMP and increased the riboflavin synthesis precursor GTP, while also kept the requirement of ATP synthesis for cell growth. PurA(P242N) was introduced into an inosine-producing strain and resulted in an approximately 4.66-fold increase in inosine production, from 0.088±0.009g/L to 0.41±0.051g/L, in minimal medium without hypoxanthine accumulation. These results underline that the directed evolution of adenylosuccinate synthetase could tailor its activities and adjust metabolic flux. This mutation may provide a promising application in purine-based product accumulation, like inosine, guanosine and folate which are directly stemming from purine pathway in B. subtilis. PMID:27234879

  5. Glucosamine synthetase from Escherichia coli: purification, properties and glutamine-utilizing site location

    International Nuclear Information System (INIS)

    L-Glutamine: D-fructose-6-phosphate amidotransferase (glucosamine synthetase) has been purified to homogeneity from Escherichia coli. A subunit molecular weight of 70,800 was estimated by gel electrophoresis in sodium dodecyl sulfate. Pure glucosamine synthetase did not exhibit detectable NH3-dependent activity and did not catalyze the reverse reaction. The enzyme has a K/sub m/ of 2 mM for fructose 6-phosphate, a K/sub m/ of 0.4 mM for glutamine, and a turnover number of 1140 min-1. The amino-terminal sequence confirmed the identification of residues 2-26 of the translated E. coli glmS sequence. Methionine-1 is therefore removed by processing in vivo, leaving cysteine as the NH2-terminal residue. The enzyme was inactivated by the glutamine analog 6-diazo-5-oxo-L-norleucine (DON) and by iodoacetamide. Glucosamine synthetase exhibited half-of-the-sites reactivity when incubated with DON in the absence of fructose 6-phosphate. In its presence, inactivation with [6-14C]DON was accompanied by incorporation of 1 equiv of inhibitor per enzyme subunit. From this behavior, a dimeric structure was tentatively assigned to the native enzyme. The site of reaction with DON was the NH2-terminal cysteine residue as shown by Edman degradation

  6. Structural basis for the binding of succinate to succinyl-CoA synthetase.

    Science.gov (United States)

    Huang, Ji; Fraser, Marie E

    2016-08-01

    Succinyl-CoA synthetase catalyzes the only step in the citric acid cycle that provides substrate-level phosphorylation. Although the binding sites for the substrates CoA, phosphate, and the nucleotides ADP and ATP or GDP and GTP have been identified, the binding site for succinate has not. To determine this binding site, pig GTP-specific succinyl-CoA synthetase was crystallized in the presence of succinate, magnesium ions and CoA, and the structure of the complex was determined by X-ray crystallography to 2.2 Å resolution. Succinate binds in the carboxy-terminal domain of the β-subunit. The succinate-binding site is near both the active-site histidine residue that is phosphorylated in the reaction and the free thiol of CoA. The carboxy-terminal domain rearranges when succinate binds, burying this active site. However, succinate is not in position for transfer of the phosphoryl group from phosphohistidine. Here, it is proposed that when the active-site histidine residue has been phosphorylated by GTP, the phosphohistidine displaces phosphate and triggers the movement of the carboxylate of succinate into position to be phosphorylated. The structure shows why succinyl-CoA synthetase is specific for succinate and does not react appreciably with citrate nor with the other C4-dicarboxylic acids of the citric acid cycle, fumarate and oxaloacetate, but shows some activity with L-malate. PMID:27487822

  7. (p)ppGpp synthetases regulate the pathogenesis of zoonotic Streptococcus suis.

    Science.gov (United States)

    Zhu, Jiawen; Zhang, Tengfei; Su, Zhipeng; Li, Lu; Wang, Dong; Xiao, Ran; Teng, Muye; Tan, Meifang; Zhou, Rui

    2016-10-01

    (p)ppGpp-mediated stringent response is one of the main adaption mechanism in bacteria, and the ability to adapt to environment is linked to the pathogenesis of bacterial pathogens. In the zoonotic pathogen Streptococcus suis, there are two (p)ppGpp synthetases, RelA and RelQ. To investigate the regulatory functions of (p)ppGpp/(p)ppGpp synthetases on the pathogenesis of S. suis, the phenotypes of the [(p)ppGpp(0)] mutant ΔrelAΔrelQ and its parental strain were compared. Light and electron microscopy observation showed that the mutant strain had a longer chain-length than its parental strain. Disruption of relA and relQ led to decreased adhesive and invasive ability to HEp-2 cells, and increased sensitivity to the blood killing and phagocytosis. Mouse infection experiments showed that the mutant strain was attenuated and easier to be cleaned up in vivo. Quantitative reverse transcription PCR (qRT-PCR) analysis indicated that the expressions of virulence related genes involving in morphology and virulence were down-regulated in the mutant strain. Our study demonstrated that the (p)ppGpp synthetases or (p)ppGpp can regulate the pathogenesis of this important zoonotic pathogen. PMID:27524648

  8. Gene rearrangements and evolution of tRNA pseudogenes in the mitochondrial genome of the parrotfish (Teleostei: Perciformes: Scaridae).

    Science.gov (United States)

    Mabuchi, Kohji; Miya, Masaki; Satoh, Takashi P; Westneat, Mark W; Nishida, Mutsumi

    2004-09-01

    Genomic size of animal mitochondrial DNA is usually minimized over time. Thus, when regional duplications occur, they are followed by a rapid elimination of redundant material. In contrast to this general view, we report here long-sustained tRNA pseudogenes in the mitochondrial genome (mitogenome) of teleost fishes of the family Scaridae (parrotfishes). During the course of a molecular phylogenetic study of the suborder Labroidei, we determined the complete nucleotide sequence of the mitogenome for a parrotfish, Chlorurus sordidus, and found a gene rearrangement accompanied by a tRNA pseudogene. In the typical gene order of vertebrates, a tRNA-gene cluster between ND1 and ND2 genes includes tRNA(Ile) (I), tRNA(Gln) (Q), and tRNA(Met) (M) genes in this order (IQM). However, in the mitogenome of the parrotfish, the tRNA(Met) gene was inserted between the tRNA(Ile) and the tRNA(Gln) genes, and the tRNA(Gln) gene was followed by a putative tRNA(Met) pseudogene (psiM). Such a tRNA gene rearrangement including a pseudogene (IMQpsiM) was found in all of the 10 examined species, representing 7 of the 10 currently recognized scarid genera. All sister groups examined (20 species of Labridae and a single species of Odacidae) had the typical gene order of vertebrate mitogenomes. Phylogenetic analysis of the tRNA(Met) genes and the resulting pseudogenes demonstrated that the ancestral tRNA(Met) gene was duplicated in a common ancestor of the parrotfish. Based on the fossil record, these results indicate that the pseudogenes have survived at least 14 million years. Most of the vertebrate mitochondrial gene rearrangements involving the IQM region have held the tRNA(Met) gene just upstream of the ND2 gene, and even in a few exceptional cases, including the present ones, the tRNA pseudogenes have been found in that position. In addition, most of these tRNA(Met) pseudogenes maintained clover-leaf secondary structures, with the remainder sustaining the clover-leaf structure in the

  9. Interferon titer and the 2',5'-oligoadenylate-synthetase activity in rat thymus lymphocytes in conditions of Omeprazol-caused hypergastrinemia

    OpenAIRE

    Kompanets I. V.; Korotkiy О. G.; Karpovets T. P.; Pilipenko S. V.; Nikolska V. V.; Ostapchenko L. I.; Yankovsky D. S.

    2013-01-01

    The aim of this work was the determination of rat thymocytes response to hypergastrinemia evoked by hypoacidity and multiprobiotic «Symbiter® acidophilic concentrated» (symbiter) treatment via the estimation of the interferon (IFN) titer and 2', 5'-oligoadenylate (OA)-synthetase activity in lymphocytes. 2', 5'-OA-synthetase is the IFN-induced enzyme. Methods. The micromethod of IFN titer determination by antiviral activity, spectrophotometrical method of 2', 5'-OA-synthetase activity determin...

  10. The exome sequencing identified the mutation in YARS2 encoding the mitochondrial tyrosyl-tRNA synthetase as a nuclear modifier for the phenotypic manifestation of Leber's hereditary optic neuropathy-associated mitochondrial DNA mutation.

    Science.gov (United States)

    Jiang, Pingping; Jin, Xiaofen; Peng, Yanyan; Wang, Meng; Liu, Hao; Liu, Xiaoling; Zhang, Zengjun; Ji, Yanchun; Zhang, Juanjuan; Liang, Min; Zhao, Fuxin; Sun, Yan-Hong; Zhang, Minglian; Zhou, Xiangtian; Chen, Ye; Mo, Jun Qin; Huang, Taosheng; Qu, Jia; Guan, Min-Xin

    2016-02-01

    Leber's hereditary optic neuropathy (LHON) is the most common mitochondrial disorder. Nuclear modifier genes are proposed to modify the phenotypic expression of LHON-associated mitochondrial DNA (mtDNA) mutations. By using an exome sequencing approach, we identified a LHON susceptibility allele (c.572G>T, p.191Gly>Val) in YARS2 gene encoding mitochondrial tyrosyl-tRNA synthetase, which interacts with m.11778G>A mutation to cause visual failure. We performed functional assays by using lymphoblastoid cell lines derived from members of Chinese families (asymptomatic individuals carrying m.11778G>A mutation, or both m.11778G>A and heterozygous p.191Gly>Val mutations and symptomatic subjects harboring m.11778G>A and homozygous p.191Gly>Val mutations) and controls lacking these mutations. The 191Gly>Val mutation reduced the YARS2 protein level in the mutant cells. The aminoacylated efficiency and steady-state level of tRNA(Tyr) were markedly decreased in the cell lines derived from patients both carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The failure in tRNA(Tyr) metabolism impaired mitochondrial translation, especially for polypeptides with high content of tyrosine codon such as ND4, ND5, ND6 and COX2 in cells lines carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The YARS2 p.191Gly>Val mutation worsened the respiratory phenotypes associated with m.11778G>A mutation, especially reducing activities of complexes I and IV. The respiratory deficiency altered the efficiency of mitochondrial ATP synthesis and increased the production of reactive oxygen species. Thus, mutated YARS2 aggravates mitochondrial dysfunctions associated with the m.11778G>A mutation, exceeding the threshold for the expression of blindness phenotype. Our findings provided new insights into the pathophysiology of LHON that were manifested by interaction between mtDNA mutation and mutated nuclear-modifier YARS2. PMID:26647310

  11. Biosynthesis of Polymyxins B, E, and P Using Genetically Engineered Polymyxin Synthetases in the Surrogate Host Bacillus subtilis.

    Science.gov (United States)

    Kim, Se-Yu; Park, Soo-Young; Choi, Soo-Keun; Park, Seung-Hwan

    2015-07-01

    The development of diverse polymyxin derivatives is needed to solve the toxicity and resistance problems of polymyxins. However, no platform has generated polymyxin derivatives by genetically engineering a polymyxin synthetase, which is a nonribosomal peptide synthetase. In this study, we present a two-step approach for the construction of engineered polymyxin synthetases by substituting the adenylation (A) domains of polymyxin A synthetase, which is encoded by the pmxABCDE gene cluster of Paenibacillus polymyxa E681. First, the seventh L-threonine-specific A-domain region in pmxA was substituted with the Lleucine- specific A-domain region obtained from P. polymyxa ATCC21830 to make polymyxin E synthetase, and then the sixth D-leucine-specific A-domain region (A6-D-Leu-domain) was substituted with the D-phenylalanine-specific A-domain region (A6-D-Phe-domain) obtained from P. polymyxa F4 to make polymyxin B synthetase. This step was performed in Escherichia coli on a pmxA-containing fosmid, using the lambda Red recombination system and the sacB gene as a counter-selectable marker. Next, the modified pmxA gene was fused to pmxBCDE on the chromosome of Bacillus subtilis BSK4dA, and the resulting recombinant strains BSK4-PB and BSK4-PE were confirmed to produce polymyxins B and E, respectively. We also succeeded in constructing the B. subtilis BSK4-PP strain, which produces polymyxin P, by singly substituting the A6-D-Leu-domain with the A6-D-Phe-domain. This is the first report in which polymyxin derivatives were generated by genetically engineering polymyxin synthetases. The two recombinant B. subtilis strains will be useful for improving the commercial production of polymyxins B and E, and they will facilitate the generation of novel polymyxin derivatives. PMID:26059516

  12. Deletion analysis of the expression of rRNA genes and associated tRNA genes carried by a lambda transducing bacteriophage

    International Nuclear Information System (INIS)

    Transducing phage lambda ilv5 carries genes for rRNA's, spacer tRNA's (tRNA1/sup Ile/ and tRNA/sub 1B//sup Ala/), and two other tRNA's (tRNA1/sup Asp/ and tRNA/sup Trp/). We have isolated a mutant of lambda ilv5, lambda ilv5su7, which carries an amber suppressor mutation in the tRNA/sup Trp/ gene. A series of deletion mutants were isolated from the lambda ilv5su7 phage. Genetic and biochemical analyses of these deletion mutants have confirmed our previous conclusion that the genes for tRNA1/sup Asp/ and tRNA/sup Trp/ located at the distal end of the rRNA operon (rrnC) are cotranscribed with other rRNA genes in that operon. In addition, these deletions were used to define roughly the physical location of the promoter(s) of the rRNA operon carried by the lambda ilv5su7 transducing phage

  13. Properties of 5-aminolaevulinate synthetase and its relationship to microsomal mixed-function oxidation in the southern armyworm (Spodoptera eridania).

    Science.gov (United States)

    Brattsten, L B; Wilkinson, C F

    1975-07-01

    1. Activity of 5-aminolaevulinate synthetase was measured in the midgut and other tissues of the last larval instar of the southern armyworm (Spodoptera eridania Cramer, formerly Prodenia eridania Cramer). 2. Optimum conditions for measuring the activity were established with respect to all variables involved and considerable differences from those reported for mammalian enzyme preparations were found. 3. Maximum activity (20 nmol/h per mg of protein) occurs 18-24 h after the fifth moult and thereafter decreases to trace amounts as the larvae age and approach pupation. 4. Synthetase activity was rapidly induced by oral administration (in the diet) of pentamethylbenzene, phenobarbital, diethyl 1,4-dihydro-2,4,6-trimethylpyridine-3, 5-dicarboxylate, and 2-allyl-2-isopropylacetamide. 5. Puromycin inhibited the induction of synthetase by pentamethylbenzene. 6. Induction of 5-aminolaevulinate synthetase correlated well with the induction of microsomal N-demethylation of p-chloro-N-methylaniline, except for phenobarbital, which induced the microsomal oxidase relatively more than the synthetase. PMID:1004

  14. A yeast tRNA mutant that causes pseudohyphal growth exhibits reduced rates of CAG codon translation

    OpenAIRE

    Kemp, Alain J; Betney, Russell; Ciandrini, Luca; Schwenger, Alexandra C M; Romano, M. Carmen; Stansfield, Ian

    2012-01-01

    In Saccharomyces cerevisiae, the SUP70 gene encodes the CAG-decoding tRNAGln CUG. A mutant allele, sup70-65 , induces pseudohyphal growth on rich medium, an inappropriate nitrogen starvation response. This mutant tRNA is also a UAG nonsense suppressor via first base wobble. To investigate the basis of the pseudohyphal phenotype, 10 novel sup70 UAG suppressor alleles were identified, defining positions in the tRNAGln CUG anticodon stem that restrict first base wobble. However, none conferred p...

  15. Use of PCR Amplification of tRNA Gene-Linked Short Tandem Repeats for Genotyping Entamoeba histolytica§

    OpenAIRE

    Ali, Ibne Karim M; Zaki, Mehreen; Clark, C. Graham

    2005-01-01

    We have developed a reliable method for PCR-based genotyping of Entamoeba histolytica based on variation in the numbers of short tandem repeats that are linked to tRNA genes in this species. Species-specific primer pairs were designed that differentiate E. histolytica from E. dispar as well as that reveal intraspecies PCR product length polymorphisms. The primers were tested with samples from different parts of the world, and DNA was extracted from cultured cells as well as liver abscess pus ...

  16. Crystal structure analysis reveals functional flexibility in the selenocysteine-specific tRNA from mouse.

    Directory of Open Access Journals (Sweden)

    Oleg M Ganichkin

    Full Text Available BACKGROUND: Selenocysteine tRNAs (tRNA(Sec exhibit a number of unique identity elements that are recognized specifically by proteins of the selenocysteine biosynthetic pathways and decoding machineries. Presently, these identity elements and the mechanisms by which they are interpreted by tRNA(Sec-interacting factors are incompletely understood. METHODOLOGY/PRINCIPAL FINDINGS: We applied rational mutagenesis to obtain well diffracting crystals of murine tRNA(Sec. tRNA(Sec lacking the single-stranded 3'-acceptor end ((ΔGCCARNA(Sec yielded a crystal structure at 2.0 Å resolution. The global structure of (ΔGCCARNA(Sec resembles the structure of human tRNA(Sec determined at 3.1 Å resolution. Structural comparisons revealed flexible regions in tRNA(Sec used for induced fit binding to selenophosphate synthetase. Water molecules located in the present structure were involved in the stabilization of two alternative conformations of the anticodon stem-loop. Modeling of a 2'-O-methylated ribose at position U34 of the anticodon loop as found in a sub-population of tRNA(Secin vivo showed how this modification favors an anticodon loop conformation that is functional during decoding on the ribosome. Soaking of crystals in Mn(2+-containing buffer revealed eight potential divalent metal ion binding sites but the located metal ions did not significantly stabilize specific structural features of tRNA(Sec. CONCLUSIONS/SIGNIFICANCE: We provide the most highly resolved structure of a tRNA(Sec molecule to date and assessed the influence of water molecules and metal ions on the molecule's conformation and dynamics. Our results suggest how conformational changes of tRNA(Sec support its interaction with proteins.

  17. Phosphoribosylpyrophosphate synthetase of Escherichia coli. Properties of the purified enzyme and primary structure of the prs gene

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Harlow, Kenneth W.; King, Cheryl J.;

    1986-01-01

    Phosphoribosylpyrophosphate (P-Rib-PP) synthetase of Escherichia coli has been purified to near homogeneity from a strain harboring the prs gene, encoding P-Rib-PP synthetase, on a multicopy plasmid. Analysis of the enzyme showed that it required inorganic phosphate for activity and for stability...

  18. REGULATION OF RAT HEPATIC DELTA-AMINOLEVULINIC ACID SYNTHETASE AND HEME OXYGENASE ACTIVITIES: EVIDENCE FOR CONTROL BY HEME AND AGAINST MEDIATION BY PROSTHETIC IRON

    Science.gov (United States)

    The effects of in vivo administration of 6 compounds on the activity of delta-aminolevulinic acid (ALA) synthetase and heme oxygenase were determined. The order of decreasing potency in reducing ALA synthetase activity was heme, bilirubin, protoporphyrin IX, bilirubin dimethyl es...

  19. δ-(L-α-Aminoadipyl)-L-cysteinyl-D-valine synthetase, that mediates the first committed step in penicillin biosynthesis, is a cytosolic enzyme

    NARCIS (Netherlands)

    Lende, Ted R. van der; Kamp, Mart van de; Berg, Marco van den; Sjollema, Klaas; Bovenberg, Roel A.L.; Veenhuis, Marten; Konings, Wil N.; Driessen, Arnold J.M.

    2002-01-01

    Penicillin biosynthesis by Penicillium chrysogenum is a compartmentalized process. The first catalytic step is mediated by δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACV synthetase), a high molecular mass enzyme that condenses the amino acids L-α-aminoadipate, L-cysteine, and L-valine into

  20. delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase, that mediates the first committed step in penicillin biosynthesis, is a cytosolic enzyme

    NARCIS (Netherlands)

    van der Lende, T.R.; de Kamp, M.; den Berg, M.van; Sjollema, K.; Bovenberg, R.A.L.; Veenhuis, M; Konings, W.N; Driessen, A.J.M.

    2002-01-01

    Penicillin biosynthesis by Penicillium chrysogenum is a compartmentalized process. The first catalytic step is mediated by delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACV synthetase), a high molecular mass enzyme that condenses the amino acids L-alpha-aminoadipate, L-cysteme, and L-

  1. Glial glutamate transporter and glutamine synthetase regulate GABAergic synaptic strength in the spinal dorsal horn.

    Science.gov (United States)

    Jiang, Enshe; Yan, Xisheng; Weng, Han-Rong

    2012-05-01

    Decreased GABAergic synaptic strength ('disinhibition') in the spinal dorsal horn is a crucial mechanism contributing to the development and maintenance of pathological pain. However, mechanisms leading to disinhibition in the spinal dorsal horn remain elusive. We investigated the role of glial glutamate transporters (GLT-1 and GLAST) and glutamine synthetase in maintaining GABAergic synaptic activity in the spinal dorsal horn. Electrically evoked GABAergic inhibitory post-synaptic currents (eIPSCs), spontaneous IPSCs (sIPSCs) and miniature IPSCs were recorded in superficial spinal dorsal horn neurons of spinal slices from young adult rats. We used (2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA), to block both GLT-1 and GLAST and dihydrokainic acid to block only GLT-1. We found that blockade of both GLAST and GLT-1 and blockade of only GLT-1 in the spinal dorsal horn decreased the amplitude of GABAergic eIPSCs, as well as both the amplitude and frequency of GABAergic sIPSCs or miniature IPSCs. Pharmacological inhibition of glial glutamine synthetase had similar effects on both GABAergic eIPSCs and sIPSCs. We provided evidence demonstrating that the reduction in GABAergic strength induced by the inhibition of glial glutamate transporters is due to insufficient GABA synthesis through the glutamate-glutamine cycle between astrocytes and neurons. Thus, our results indicate that deficient glial glutamate transporters and glutamine synthetase significantly attenuate GABAergic synaptic strength in the spinal dorsal horn, which may be a crucial synaptic mechanism underlying glial-neuronal interactions caused by dysfunctional astrocytes in pathological pain conditions. PMID:22339645

  2. Molecular cloning and characterization of an S-adenosylmethionine synthetase gene from Chorispora bungeana.

    Science.gov (United States)

    Ding, Chenchen; Chen, Tao; Yang, Yu; Liu, Sha; Yan, Kan; Yue, Xiule; Zhang, Hua; Xiang, Yun; An, Lizhe; Chen, Shuyan

    2015-11-10

    S-adenosylmethionine synthetase (SAMS) catalyzes the formation of S-adenosylmethionine (SAM) which is a molecule essential for polyamines and ethylene biosynthesis, methylation modifications of protein, DNA and lipids. SAMS also plays an important role in abiotic stress response. Chorispora bungeana (C. bungeana) is an alpine subnival plant species which possesses strong tolerance to cold stress. Here, we cloned and characterized an S-adenosylmethionine synthetase gene, CbSAMS (C. bungeana S-adenosylmethionine synthetase), from C. bungeana, which encodes a protein of 393 amino acids containing a methionine binding motif GHPDK, an ATP binding motif GAGDQG and a phosphate binding motif GGGAFSGDK. Furthermore, an NES (nuclear export signal) peptide was identified through bioinformatics analysis. To explore the CbSAMS gene expression regulation, we isolated the promoter region of CbSAMS gene 1919bp upstream the ATG start codon, CbSAMSp, and analyzed its cis-acting elements by bioinformatics method. It was revealed that a transcription start site located at 320 bp upstream the ATG start codon and cis-acting elements related to light, ABA, auxin, ethylene, MeJA, low temperature and drought had been found in the CbSAMSp sequence. The gene expression pattern of CbSAMS was then analyzed by TR-qPCR and GUS assay method. The result showed that CbSAMS is expressed in all examined tissues including callus, roots, petioles, leaves, and flowers with a significant higher expression level in roots and flowers. Furthermore, the expression level of CbSAMS was induced by low temperature, ethylene and NaCl. Subcellular localization revealed that CbSAMS was located in the cytoplasm and nucleus but has a significant higher level in the nucleus. These results indicated a potential role of CbSAMS in abiotic stresses and plant growth in C. bungeana. PMID:26205258

  3. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of DHNA synthetase from Geobacillus kaustophilus

    International Nuclear Information System (INIS)

    DHNA synthetase from G. kaustophilus has been cloned, expressed, purified and crystallized. The aerobic Gram-positive bacterium Geobacillus kaustophilus is a bacillus species that was isolated from deep-sea sediment from the Mariana Trench. 1,4-Dihydroxy-2-naphthoate (DHNA) synthetase plays a vital role in the biosynthesis of menaquinone (vitamin K2) in this bacterium. DHNA synthetase from Geobacillus kaustophilus was crystallized in the orthorhombic space group C2221, with unit-cell parameters a = 77.01, b = 130.66, c = 131.69 Å. The crystal diffracted to a resolution of 2.2 Å. Preliminary studies and molecular-replacement calculations reveal the presence of three monomers in the asymmetric unit

  4. Purification, gene cloning, and characterization of γ-butyrobetainyl CoA synthetase from Agrobacterium sp. 525a.

    Science.gov (United States)

    Fujimitsu, Hiroshi; Matsumoto, Akira; Takubo, Sayaka; Fukui, Akiko; Okada, Kazuma; Mohamed Ahmed, Isam A; Arima, Jiro; Mori, Nobuhiro

    2016-08-01

    The report is the first of purification, overproduction, and characterization of a unique γ-butyrobetainyl CoA synthetase from soil-isolated Agrobacterium sp. 525a. The primary structure of the enzyme shares 70-95% identity with those of ATP-dependent microbial acyl-CoA synthetases of the Rhizobiaceae family. As distinctive characteristics of the enzyme of this study, ADP was released in the catalytic reaction process, whereas many acyl CoA synthetases are annotated as an AMP-forming enzyme. The apparent Km values for γ-butyrobetaine, CoA, and ATP were, respectively, 0.69, 0.02, and 0.24 mM. PMID:27125317

  5. Purification and characterization of fatty acyl-acyl carrier protein synthetase from Vibrio harveyi.

    OpenAIRE

    Fice, D; Z. Shen; Byers, D M

    1993-01-01

    A Vibrio harveyi enzyme which catalyzes the ATP-dependent ligation of fatty acids to acyl carrier protein (ACP) has been purified 6,000-fold to apparent homogeneity by anion-exchange, gel filtration, and ACP-Sepharose affinity chromatography. Purified acyl-ACP synthetase migrated as a single 62-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as an 80-kDa protein by gel filtration under reducing conditions. Activity of the purified enzyme was lost within hours in the ...

  6. Purification and characterization of 3-deoxy-D-manno-octulosonate 8-phosphate synthetase from Escherichia coli.

    OpenAIRE

    Ray, P H

    1980-01-01

    3-Deoxy-D-manno-octulosonate (KDO)-8-phosphate synthetase has been purified 450-fold from frozen Escherichia coli B cells. The purified enzyme catalyzed the stoichiometric formation of KDO-8-phosphate and Pi from phosphoenolpyruvate (PEP) and D-arabinose-5-phosphate. The enzyme showed no metal requirement for activity and was inhibited by 1 mM Cd2+, Cu2+, Zn2+, and Hg2+. The inhibition by Hg2+ could be reversed by dithiothreitol. The optimum temperature for enzyme activity was determined to b...

  7. A radiochemical method for carbamoyl-phosphate synthetase I: application to rats fed a hyperproteic diet

    OpenAIRE

    Arriarán, Sofía; Agnelli, Silvia; Fernández López, José Antonio; Remesar Betlloch, Xavier; Alemany, Marià

    2012-01-01

    A method for the measurement of carbamoyl-phosphate synthetase I activity in animal tissues has been developed using the livers of rats under normal and hyperproteic diets. The method is based on the incorporation of 14C-ammonium bicarbonate to carbamoyl-phosphate in the presence of ATP-Mg and N-acetyl-glutamate. The reaction is stopped by chilling, lowering the pH and adding ethanol. Excess bicarbonate is flushed out under a gentle stream of cold CO2. The only label remaining in the medium w...

  8. Determinant nucleotides of yeast tRNA(Asp) interact directly with aspartyl-tRNA synthetase.

    OpenAIRE

    Rudinger, J; Puglisi, J D; Pütz, J.; Schatz, D; Eckstein, F; Florentz, C; Giegé, R

    1992-01-01

    The interaction of wild-type and mutant yeast tRNA(Asp) transcripts with yeast aspartyl-tRNA synthetase (AspRS; EC 6.1.1.12) has been probed by using iodine cleavage of phosphorothioate-substituted transcripts. AspRS protects phosphates in the anticodon (G34, U35), D-stem (U25), and acceptor end (G73) that correspond to determinant nucleotides for aspartylation. This protection, as well as that in anticodon stem (C29, U40, G41) and D-stem (U11 to U13), is consistent with direct interaction of...

  9. Comparative properties of glutamine synthetases I and II in Rhizobium and Agrobacterium spp.

    OpenAIRE

    Fuchs, R L; Keister, D L

    1980-01-01

    Some properties of glutamine synthetase I (GSI) and GSII are described for a fast-growing Rhizobium sp. (Rhizobium trifolii T1), a slow-growing Rhizobium sp. (Rhizobium japonicum USDA 83), and Agrobacterium tumefaciens C58. GSII of the fast-growing Rhizobium sp. and GSII of the Agrobacterium sp. were considerably more heat labile than GSII of the slow-growing Rhizobium sp. As previously shown in R. japonicum 61A76, GSI became adenylylated rapidly in all species tested in response to ammonium....

  10. Heme ligand identification and redox properties of the cytochrome c synthetase, CcmF†

    OpenAIRE

    Francisco, Brian San; Bretsnyder, Eric C.; Rodgers, Kenton R.; Kranz, Robert G.

    2011-01-01

    Cytochrome c maturation in many bacteria, archaea, and plant mitochondria involves the integral membrane protein CcmF, which is thought to function as a cytochrome c synthetase by facilitating the final covalent attachment of heme to the apocytochrome c. We previously reported that the E. coli CcmF protein contains a b-type heme that is stably and stoichiometrically associated with the protein and is not the heme attached to apocytochrome c. Here, we show that mutation of either of two conser...

  11. The structures of cytosolic and plastid-located glutamine synthetases from Medicago truncatula reveal a common and dynamic architecture

    Energy Technology Data Exchange (ETDEWEB)

    Torreira, Eva [Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Seabra, Ana Rita [IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto (Portugal); Marriott, Hazel; Zhou, Min [University of Oxford, South Parks Road, Oxford OX1 3QZ (United Kingdom); Llorca, Óscar [Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Robinson, Carol V. [University of Oxford, South Parks Road, Oxford OX1 3QZ (United Kingdom); Carvalho, Helena G. [IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto (Portugal); Fernández-Tornero, Carlos, E-mail: cftornero@cib.csic.es [Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Pereira, Pedro José Barbosa, E-mail: cftornero@cib.csic.es [IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto (Portugal); Centro de Investigaciones Biológicas – CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain)

    2014-04-01

    The experimental models of dicotyledonous cytoplasmic and plastid-located glutamine synthetases unveil a conserved eukaryotic-type decameric architecture, with subtle structural differences in M. truncatula isoenzymes that account for their distinct herbicide resistance. The first step of nitrogen assimilation in higher plants, the energy-driven incorporation of ammonia into glutamate, is catalyzed by glutamine synthetase. This central process yields the readily metabolizable glutamine, which in turn is at the basis of all subsequent biosynthesis of nitrogenous compounds. The essential role performed by glutamine synthetase makes it a prime target for herbicidal compounds, but also a suitable intervention point for the improvement of crop yields. Although the majority of crop plants are dicotyledonous, little is known about the structural organization of glutamine synthetase in these organisms and about the functional differences between the different isoforms. Here, the structural characterization of two glutamine synthetase isoforms from the model legume Medicago truncatula is reported: the crystallographic structure of cytoplasmic GSII-1a and an electron cryomicroscopy reconstruction of plastid-located GSII-2a. Together, these structural models unveil a decameric organization of dicotyledonous glutamine synthetase, with two pentameric rings weakly connected by inter-ring loops. Moreover, rearrangement of these dynamic loops changes the relative orientation of the rings, suggesting a zipper-like mechanism for their assembly into a decameric enzyme. Finally, the atomic structure of M. truncatula GSII-1a provides important insights into the structural determinants of herbicide resistance in this family of enzymes, opening new avenues for the development of herbicide-resistant plants.

  12. The structures of cytosolic and plastid-located glutamine synthetases from Medicago truncatula reveal a common and dynamic architecture

    International Nuclear Information System (INIS)

    The experimental models of dicotyledonous cytoplasmic and plastid-located glutamine synthetases unveil a conserved eukaryotic-type decameric architecture, with subtle structural differences in M. truncatula isoenzymes that account for their distinct herbicide resistance. The first step of nitrogen assimilation in higher plants, the energy-driven incorporation of ammonia into glutamate, is catalyzed by glutamine synthetase. This central process yields the readily metabolizable glutamine, which in turn is at the basis of all subsequent biosynthesis of nitrogenous compounds. The essential role performed by glutamine synthetase makes it a prime target for herbicidal compounds, but also a suitable intervention point for the improvement of crop yields. Although the majority of crop plants are dicotyledonous, little is known about the structural organization of glutamine synthetase in these organisms and about the functional differences between the different isoforms. Here, the structural characterization of two glutamine synthetase isoforms from the model legume Medicago truncatula is reported: the crystallographic structure of cytoplasmic GSII-1a and an electron cryomicroscopy reconstruction of plastid-located GSII-2a. Together, these structural models unveil a decameric organization of dicotyledonous glutamine synthetase, with two pentameric rings weakly connected by inter-ring loops. Moreover, rearrangement of these dynamic loops changes the relative orientation of the rings, suggesting a zipper-like mechanism for their assembly into a decameric enzyme. Finally, the atomic structure of M. truncatula GSII-1a provides important insights into the structural determinants of herbicide resistance in this family of enzymes, opening new avenues for the development of herbicide-resistant plants

  13. Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins

    OpenAIRE

    Englert, Markus; Beier, Hildburg

    2005-01-01

    Pre-tRNA splicing is an essential process in all eukaryotes. It requires the concerted action of an endonuclease to remove the intron and a ligase for joining the resulting tRNA halves as studied best in the yeast Saccharomyces cerevisiae. Here, we report the first characterization of an RNA ligase protein and its gene from a higher eukaryotic organism that is an essential component of the pre-tRNA splicing process. Purification of tRNA ligase from wheat germ by successive column chromatograp...

  14. Expression of acyl-CoA synthetase 5 reflects the state of villus architecture in human small intestine

    DEFF Research Database (Denmark)

    Gassler, Nikolaus; Kopitz, Jürgen; Tehrani, Arman;

    2004-01-01

    -CoA synthetase 5 pattern correlate with conversion of intestinal epithelial cells to a gastric phenotype. These results suggest that deranged acyl-CoA synthetase 5 expression, synthesis, and activity are closely related to the state of villus architecture and epithelial homeostasis in human small intestine.......Several disorders of the small intestine are associated with disturbances in villus architecture. Thus, an understanding of the molecular mechanisms associated with the differentiation of villi represents an important step in the improvement of the understanding of small intestinal pathology...

  15. Functional Analysis of Long-chain Acyl-CoA Synthetase 1 in 3T3-L1 Adipocytes*

    OpenAIRE

    Lobo, Sandra; Wiczer, Brian M.; Bernlohr, David A

    2009-01-01

    ACSL1 (acyl-CoA synthetase 1), the major acyl-CoA synthetase of adipocytes, has been proposed to function in adipocytes as mediating free fatty acid influx, esterification, and storage as triglyceride. To test this hypothesis, ACSL1 was stably silenced (knockdown (kd)) in 3T3-L1 cells, differentiated into adipocytes, and evaluated for changes in lipid metabolism. Surprisingly, ACSL1-silenced adipocytes exhibited no significant changes in basal or insulin-stimulated long-chain fatty acid uptak...

  16. Activity of interferon-dependent 2',5'-oligoadenylate synthetase in rat lymphoid cells under transformed environment conditions

    Science.gov (United States)

    Ostapchenko, L. I.; Mikhailik, I. V.; Prokopova, K. V.

    It is detected that interferon-dependent 2',5'-oligoadenylate synthetase is a sensitive index of immunocompetent cells functional state under transformed environment conditions. Microgravitation and ionising radiation induce increase of investigated enzyme activity in rat lymphocytes, which can be a result of lymphoid cells compensatory mechanisms starting in response to stress factors action. Administration of interferon inductors permits to stimulate the 2',5'-oligoadenylate synthetase, which enables one to correct pathological changes in the cells and to intensify adaptive reactions of immune systems.

  17. A yeast tRNA mutant that causes pseudohyphal growth exhibits reduced rates of CAG codon translation.

    Science.gov (United States)

    Kemp, Alain J; Betney, Russell; Ciandrini, Luca; Schwenger, Alexandra C M; Romano, M Carmen; Stansfield, Ian

    2013-01-01

    In Saccharomyces cerevisiae, the SUP70 gene encodes the CAG-decoding tRNA(Gln)(CUG). A mutant allele, sup70-65, induces pseudohyphal growth on rich medium, an inappropriate nitrogen starvation response. This mutant tRNA is also a UAG nonsense suppressor via first base wobble. To investigate the basis of the pseudohyphal phenotype, 10 novel sup70 UAG suppressor alleles were identified, defining positions in the tRNA(Gln)(CUG) anticodon stem that restrict first base wobble. However, none conferred pseudohyphal growth, showing altered CUG anticodon presentation cannot itself induce pseudohyphal growth. Northern blot analysis revealed the sup70-65 tRNA(Gln)(CUG) is unstable, inefficiently charged, and 80% reduced in its effective concentration. A stochastic model simulation of translation predicted compromised expression of CAG-rich ORFs in the tRNA(Gln)(CUG)-depleted sup70-65 mutant. This prediction was validated by demonstrating that luciferase expression in the mutant was 60% reduced by introducing multiple tandem CAG (but not CAA) codons into this ORF. In addition, the sup70-65 pseudohyphal phenotype was partly complemented by overexpressing CAA-decoding tRNA(Gln)(UUG), an inefficient wobble-decoder of CAG. We thus show that introducing codons decoded by a rare tRNA near the 5' end of an ORF can reduce eukaryote translational expression, and that the mutant tRNA(CUG)(Gln) constitutive pseudohyphal differentiation phenotype correlates strongly with reduced CAG decoding efficiency. PMID:23146061

  18. Crystallographic capture of a radical S-adenosylmethionine enzyme in the act of modifying tRNA.

    Science.gov (United States)

    Schwalm, Erica L; Grove, Tyler L; Booker, Squire J; Boal, Amie K

    2016-04-15

    RlmN is a dual-specificity RNA methylase that modifies C2 of adenosine 2503 (A2503) in 23S rRNA and C2 of adenosine 37 (A37) in several Escherichia coli transfer RNAs (tRNAs). A related methylase, Cfr, modifies C8 of A2503 via a similar mechanism, conferring resistance to multiple classes of antibiotics. Here, we report the x-ray structure of a key intermediate in the RlmN reaction, in which a Cys(118)→Ala variant of the protein is cross-linked to a tRNA(Glu)substrate through the terminal methylene carbon of a formerly methylcysteinyl residue and C2 of A37. RlmN contacts the entire length of tRNA(Glu), accessing A37 by using an induced-fit strategy that completely unfolds the tRNA anticodon stem-loop, which is likely critical for recognition of both tRNA and ribosomal RNA substrates. PMID:27081063

  19. Three-Dimensional Algebraic Models of the tRNA Code and 12 Graphs for Representing the Amino Acids

    Directory of Open Access Journals (Sweden)

    Marco V. José

    2014-08-01

    Full Text Available Three-dimensional algebraic models, also called Genetic Hotels, are developed to represent the Standard Genetic Code, the Standard tRNA Code (S-tRNA-C, and the Human tRNA code (H-tRNA-C. New algebraic concepts are introduced to be able to describe these models, to wit, the generalization of the 2n-Klein Group and the concept of a subgroup coset with a tail. We found that the H-tRNA-C displayed broken symmetries in regard to the S-tRNA-C, which is highly symmetric. We also show that there are only 12 ways to represent each of the corresponding phenotypic graphs of amino acids. The averages of statistical centrality measures of the 12 graphs for each of the three codes are carried out and they are statistically compared. The phenotypic graphs of the S-tRNA-C display a common triangular prism of amino acids in 10 out of the 12 graphs, whilst the corresponding graphs for the H-tRNA-C display only two triangular prisms. The graphs exhibit disjoint clusters of amino acids when their polar requirement values are used. We contend that the S-tRNA-C is in a frozen-like state, whereas the H-tRNA-C may be in an evolving state.

  20. Fluctuations between multiple EF-G-induced chimeric tRNA states during translocation on the ribosome

    Science.gov (United States)

    Adio, Sarah; Senyushkina, Tamara; Peske, Frank; Fischer, Niels; Wintermeyer, Wolfgang; Rodnina, Marina V.

    2015-06-01

    The coupled translocation of transfer RNA and messenger RNA through the ribosome entails large-scale structural rearrangements, including step-wise movements of the tRNAs. Recent structural work has visualized intermediates of translocation induced by elongation factor G (EF-G) with tRNAs trapped in chimeric states with respect to 30S and 50S ribosomal subunits. The functional role of the chimeric states is not known. Here we follow the formation of translocation intermediates by single-molecule fluorescence resonance energy transfer. Using EF-G mutants, a non-hydrolysable GTP analogue, and fusidic acid, we interfere with either translocation or EF-G release from the ribosome and identify several rapidly interconverting chimeric tRNA states on the reaction pathway. EF-G engagement prevents backward transitions early in translocation and increases the fraction of ribosomes that rapidly fluctuate between hybrid, chimeric and posttranslocation states. Thus, the engagement of EF-G alters the energetics of translocation towards a flat energy landscape, thereby promoting forward tRNA movement.

  1. Autosomal-Recessive Mutations in the tRNA Splicing Endonuclease Subunit TSEN15 Cause Pontocerebellar Hypoplasia and Progressive Microcephaly.

    Science.gov (United States)

    Breuss, Martin W; Sultan, Tipu; James, Kiely N; Rosti, Rasim O; Scott, Eric; Musaev, Damir; Furia, Bansri; Reis, André; Sticht, Heinrich; Al-Owain, Mohammed; Alkuraya, Fowzan S; Reuter, Miriam S; Abou Jamra, Rami; Trotta, Christopher R; Gleeson, Joseph G

    2016-07-01

    The tRNA splicing endonuclease is a highly evolutionarily conserved protein complex, involved in the cleavage of intron-containing tRNAs. In human it consists of the catalytic subunits TSEN2 and TSEN34, as well as the non-catalytic TSEN54 and TSEN15. Recessive mutations in the corresponding genes of the first three are known to cause pontocerebellar hypoplasia (PCH) types 2A-C, 4, and 5. Here, we report three homozygous TSEN15 variants that cause a milder version of PCH2. The affected individuals showed progressive microcephaly, delayed developmental milestones, intellectual disability, and, in two out of four cases, epilepsy. None, however, displayed the central visual failure seen in PCH case subjects where other subunits of the TSEN are mutated, and only one was affected by the extensive motor defects that are typical in other forms of PCH2. The three amino acid substitutions impacted the protein level of TSEN15 and the stoichiometry of the interacting subunits in different ways, but all resulted in an almost complete loss of in vitro tRNA cleavage activity. Taken together, our results demonstrate that mutations in any known subunit of the TSEN complex can cause PCH and progressive microcephaly, emphasizing the importance of its function during brain development. PMID:27392077

  2. Structure, Mechanism, and Specificity of a Eukaryal tRNA Restriction Enzyme Involved in Self-Nonself Discrimination

    Directory of Open Access Journals (Sweden)

    Anupam K. Chakravarty

    2014-04-01

    Full Text Available tRNA restriction by anticodon nucleases underlies cellular stress responses and self-nonself discrimination in a wide range of taxa. Anticodon breakage inhibits protein synthesis, which, in turn, results in growth arrest or cell death. The eukaryal ribotoxin PaT secreted by Pichia acaciae inhibits growth of Saccharomyces cerevisiae via cleavage of tRNAGln(UUG. We find that recombinant PaT incises a synthetic tRNAGln(UUG stem-loop RNA by transesterification at a single site 3′ of the wobble uridine, yielding 2′,3′-cyclic phosphate and 5′-OH ends. Incision is suppressed by replacement of the wobble nucleobase with adenine or guanine. The crystal structure of PaT reveals a distinctive fold and active site, essential components of which are demonstrated by mutagenesis. Pichia acaciae evades self-toxicity via a distinctive intracellular immunity protein, ImmPaT, which binds PaT and blocks nuclease activity. Our results highlight the evolutionary diversity of tRNA restriction and immunity systems.

  3. The alpha-subunit of Leishmania F1 ATP synthase hydrolyzes ATP in presence of tRNA.

    Science.gov (United States)

    Goswami, Srikanta; Adhya, Samit

    2006-07-14

    Import of tRNAs into the mitochondria of the kinetoplastid protozoon Leishmania requires the tRNA-dependent hydrolysis of ATP leading to the generation of membrane potential through the pumping of protons. Subunit RIC1 of the inner membrane RNA import complex is a bi-functional protein that is identical to the alpha-subunit of F1F0 ATP synthase and specifically binds to a subset (Type I) of importable tRNAs. We show that recombinant, purified RIC1 is a Type I tRNA-dependent ATP hydrolase. The activity was insensitive to oligomycin, sensitive to mutations within the import signal of the tRNA, and required the cooperative interaction between the ATP-binding and C-terminal domains of RIC1. The ATPase activity of the intact complex was inhibited by anti-RIC1 antibody, while knockdown of RIC1 in Leishmania tropica resulted in deficiency of the tRNA-dependent ATPase activity of the mitochondrial inner membrane. Moreover, RIC1 knockdown extracts failed to generate a membrane potential across reconstituted proteoliposomes, as shown by a rhodamine 123 uptake assay, but activity was restored by adding back purified RIC1. These observations identify RIC1 as a novel form of the F1 ATP synthase alpha-subunit that acts as the major energy transducer for tRNA import. PMID:16735512

  4. Defects in tRNA modification associated with neurological and developmental dysfunctions in Caenorhabditis elegans elongator mutants.

    Directory of Open Access Journals (Sweden)

    Changchun Chen

    2009-07-01

    Full Text Available Elongator is a six subunit protein complex, conserved from yeast to humans. Mutations in the human Elongator homologue, hELP1, are associated with the neurological disease familial dysautonomia. However, how Elongator functions in metazoans, and how the human mutations affect neural functions is incompletely understood. Here we show that in Caenorhabditis elegans, ELPC-1 and ELPC-3, components of the Elongator complex, are required for the formation of the 5-carbamoylmethyl and 5-methylcarboxymethyl side chains of wobble uridines in tRNA. The lack of these modifications leads to defects in translation in C. elegans. ELPC-1::GFP and ELPC-3::GFP reporters are strongly expressed in a subset of chemosensory neurons required for salt chemotaxis learning. elpc-1 or elpc-3 gene inactivation causes a defect in this process, associated with a posttranscriptional reduction of neuropeptide and a decreased accumulation of acetylcholine in the synaptic cleft. elpc-1 and elpc-3 mutations are synthetic lethal together with those in tuc-1, which is required for thiolation of tRNAs having the 5'methylcarboxymethyl side chain. elpc-1; tuc-1 and elpc-3; tuc-1 double mutants display developmental defects. Our results suggest that, by its effect on tRNA modification, Elongator promotes both neural function and development.

  5. Crystallization and preliminary crystallographic characterization of glutamine synthetase from Medicago truncatula

    International Nuclear Information System (INIS)

    The enzyme glutamine synthetase from M. truncatula has been expressed, purified and crystallized. The crystals belonged to the monoclinic space group P21 and diffracted to 2.35 Å resolution. The condensation of ammonium and glutamate into glutamine catalyzed by glutamine synthetase (GS) is a fundamental step in nitrogen metabolism in all kingdoms of life. In plants, this is preceded by the reduction of inorganic nitrogen to an ammonium ion and therefore effectively articulates nitrogen fixation and metabolism. Although the three-dimensional structure of the dodecameric bacterial GS was determined quite some time ago, the quaternary architecture of the plant enzyme has long been assumed to be octameric, mostly on the basis of low-resolution electron-microscopy studies. Recently, the crystallographic structure of a monocotyledonous plant GS was reported that revealed a homodecameric organization. In order to unambiguously establish the quaternary architecture of GS from dicotyledonous plants, GS1a from the model legume Medicago truncatula was overexpressed, purified and crystallized. The collection of synchrotron diffraction data to 2.35 Å resolution allowed the determination of the three-dimensional structure of this enzyme by molecular replacement

  6. Reaction Mechanism of Mycobacterium Tuberculosis Glutamine Synthetase Using Quantum Mechanics/Molecular Mechanics Calculations.

    Science.gov (United States)

    Moreira, Cátia; Ramos, Maria J; Fernandes, Pedro Alexandrino

    2016-06-27

    This paper is devoted to the understanding of the reaction mechanism of mycobacterium tuberculosis glutamine synthetase (mtGS) with atomic detail, using computational quantum mechanics/molecular mechanics (QM/MM) methods at the ONIOM M06-D3/6-311++G(2d,2p):ff99SB//B3LYP/6-31G(d):ff99SB level of theory. The complete reaction undergoes a three-step mechanism: the spontaneous transfer of phosphate from ATP to glutamate upon ammonium binding (ammonium quickly loses a proton to Asp54), the attack of ammonia on phosphorylated glutamate (yielding protonated glutamine), and the deprotonation of glutamine by the leaving phosphate. This exothermic reaction has an activation free energy of 21.5 kcal mol(-1) , which is consistent with that described for Escherichia coli glutamine synthetase (15-17 kcal mol(-1) ). The participating active site residues have been identified and their role and energy contributions clarified. This study provides an insightful atomic description of the biosynthetic reaction that takes place in this enzyme, opening doors for more accurate studies for developing new anti-tuberculosis therapies. PMID:27225077

  7. Sirtuin-dependent reversible lysine acetylation of glutamine synthetases reveals an autofeedback loop in nitrogen metabolism.

    Science.gov (United States)

    You, Di; Yin, Bin-Cheng; Li, Zhi-Hai; Zhou, Ying; Yu, Wen-Bang; Zuo, Peng; Ye, Bang-Ce

    2016-06-14

    In cells of all domains of life, reversible lysine acetylation modulates the function of proteins involved in central cellular processes such as metabolism. In this study, we demonstrate that the nitrogen regulator GlnR of the actinomycete Saccharopolyspora erythraea directly regulates transcription of the acuA gene (SACE_5148), which encodes a Gcn5-type lysine acetyltransferase. We found that AcuA acetylates two glutamine synthetases (GlnA1 and GlnA4) and that this lysine acetylation inactivated GlnA4 (GSII) but had no significant effect on GlnA1 (GSI-β) activity under the conditions tested. Instead, acetylation of GlnA1 led to a gain-of-function that modulated its interaction with the GlnR regulator and enhanced GlnR-DNA binding. It was observed that this regulatory function of acetylated GSI-β enzymes is highly conserved across actinomycetes. In turn, GlnR controls the catalytic and regulatory activities (intracellular acetylation levels) of glutamine synthetases at the transcriptional and posttranslational levels, indicating an autofeedback loop that regulates nitrogen metabolism in response to environmental change. Thus, this GlnR-mediated acetylation pathway provides a signaling cascade that acts from nutrient sensing to acetylation of proteins to feedback regulation. This work presents significant new insights at the molecular level into the mechanisms underlying the regulation of protein acetylation and nitrogen metabolism in actinomycetes. PMID:27247389

  8. Synthetic cycle of the initiation module of a formylating nonribosomal peptide synthetase.

    Science.gov (United States)

    Reimer, Janice M; Aloise, Martin N; Harrison, Paul M; Schmeing, T Martin

    2016-01-14

    Nonribosomal peptide synthetases (NRPSs) are very large proteins that produce small peptide molecules with wide-ranging biological activities, including environmentally friendly chemicals and many widely used therapeutics. NRPSs are macromolecular machines, with modular assembly-line logic, a complex catalytic cycle, moving parts and many active sites. In addition to the core domains required to link the substrates, they often include specialized tailoring domains, which introduce chemical modifications and allow the product to access a large expanse of chemical space. It is still unknown how the NRPS tailoring domains are structurally accommodated into megaenzymes or how they have adapted to function in nonribosomal peptide synthesis. Here we present a series of crystal structures of the initiation module of an antibiotic-producing NRPS, linear gramicidin synthetase. This module includes the specialized tailoring formylation domain, and states are captured that represent every major step of the assembly-line synthesis in the initiation module. The transitions between conformations are large in scale, with both the peptidyl carrier protein domain and the adenylation subdomain undergoing huge movements to transport substrate between distal active sites. The structures highlight the great versatility of NRPSs, as small domains repurpose and recycle their limited interfaces to interact with their various binding partners. Understanding tailoring domains is important if NRPSs are to be utilized in the production of novel therapeutics. PMID:26762462

  9. Glutamine synthetase in Medicago truncatula, unveiling new secrets of a very old enzyme

    Directory of Open Access Journals (Sweden)

    Ana Rita Seabra

    2015-07-01

    Full Text Available Glutamine Synthetase (GS catalyses the first step at which nitrogen is brought into cellular metabolism and is also involved in the reassimilation of ammonium released by a number of metabolic pathways. Due to its unique position in plant nitrogen metabolism, GS plays essential roles in all aspects of plant development, from germination to senescence, and is a key component of nitrogen use efficiency (NUE and plant yield. Understanding the mechanisms regulating GS activity is therefore of utmost importance and a great effort has been dedicated to understand how GS is regulated in different plant species. The present review summarizes exciting recent developments concerning the structure and regulation of glutamine synthetase isoenzymes, using the model legume Medicago truncatula. These include the understanding of the structural determinants of both the cytosolic and plastid located isoenzymes, the existence of a seed-specific GS gene unique to M. truncatula and closely related species and the discovery that GS isoenzymes are regulated by nitric oxide at the post-translational level. The data is discussed and integrated with the potential roles of the distinct GS isoenzymes within the whole plant context.

  10. Poly specific trans-acyltransferase machinery revealed via engineered acyl-CoA synthetases.

    Science.gov (United States)

    Koryakina, Irina; McArthur, John; Randall, Shan; Draelos, Matthew M; Musiol, Ewa M; Muddiman, David C; Weber, Tilmann; Williams, Gavin J

    2013-01-18

    Polyketide synthases construct polyketides with diverse structures and biological activities via the condensation of extender units and acyl thioesters. Although a growing body of evidence suggests that polyketide synthases might be tolerant to non-natural extender units, in vitro and in vivo studies aimed at probing and utilizing polyketide synthase specificity are severely limited to only a small number of extender units, owing to the lack of synthetic routes to a broad variety of acyl-CoA extender units. Here, we report the construction of promiscuous malonyl-CoA synthetase variants that can be used to synthesize a broad range of malonyl-CoA extender units substituted at the C2-position, several of which contain handles for chemoselective ligation and are not found in natural biosynthetic systems. We highlighted utility of these enzymes by probing the acyl-CoA specificity of several trans-acyltransferases, leading to the unprecedented discovery of poly specificity toward non-natural extender units, several of which are not found in naturally occurring biosynthetic pathways. These results reveal that polyketide biosynthetic machinery might be more tolerant to non-natural substrates than previously established, and that mutant synthetases are valuable tools for probing the specificity of biosynthetic machinery. Our data suggest new synthetic biology strategies for harnessing this promiscuity and enabling the regioselective modification of polyketides. PMID:23083014

  11. Antipeptide antibodies that can distinguish specific subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.)

    Science.gov (United States)

    Cai, X.; Henry, R. L.; Takemoto, L. J.; Guikema, J. A.; Wong, P. P.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    The amino acid sequences of the beta and gamma subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.) root nodules are very similar. However, there are small regions within the sequences that are significantly different between the two polypeptides. The sequences between amino acids 2 and 9 and between 264 and 274 are examples. Three peptides (gamma 2-9, gamma 264-274, and beta 264-274) corresponding to these sequences were synthesized. Antibodies against these peptides were raised in rabbits and purified with corresponding peptide-Sepharose affinity chromatography. Western blot analysis of polyacrylamide gel electrophoresis of bean nodule proteins demonstrated that the anti-beta 264-274 antibodies reacted specifically with the beta polypeptide and the anti-gamma 264-274 and anti-gamma 2-9 antibodies reacted specifically with the gamma polypeptide of the native and denatured glutamine synthetase. These results showed the feasibility of using synthetic peptides in developing antibodies that are capable of distinguishing proteins with similar primary structures.

  12. Identification of the Escherichia coli ADP-glucose synthetase inhibitor binding site(s)

    International Nuclear Information System (INIS)

    The photoaffinity labeling agent 8-azido adenylate (AMP) is an inhibitor site specific probe of the E. coli ADPG synthetase. In the absence of light, 8-azido AMP exhibits the typical reversible allosteric kinetics of the physiological inhibitor AMP. In the presence of light (254 nm), [2-3H]8-azido AMP specifically and covalently incorporates into the enzyme. Photoincorporation is linearly related to loss of catalytic activity up to at least 65% inactivation. The substrate ADP-glucose (ADPG) provides nearly 100% protection from 8-azido AMP photoinactivation, while the substrate AMP provides approximately 50% protection and the inhibitor AMP provides approximately 30% protection. These three adenylate allosteric effects of E. coli ADPG synthetase also protect it from photoincorporation of 8-azido AMP. The reaction site(s) of [2-3H]-azido AMP with the enzyme was identified by reverse phase HPLC isolation and chemical characterization of CNBr and mouse submaxillary arginyl protease generated peptides containing the labeled analog. This site is the same as the major binding region of the substrate site specific probe, 8-azido ADP-[14C]glucose. Conformational analysis of this region predicts that it is a part of a Rossmann fold, the super-secondary structure found in many adenine nucleotide binding proteins. Two minor reaction regions of the enzyme with [2-3H]8-azido AMP were also identified. The three modified peptide regions may be juxtaposed in the enzyme's tertiary structure

  13. CcsBA is a cytochrome c synthetase that also functions in heme transport

    Science.gov (United States)

    Frawley, Elaine R.; Kranz, Robert G.

    2009-01-01

    Little is known about trafficking of heme from its sites of synthesis to sites of heme-protein assembly. We describe an integral membrane protein that allows trapping of endogenous heme to elucidate trafficking mechanisms. We show that CcsBA, a representative of a superfamily of integral membrane proteins involved in cytochrome c biosynthesis, exports and protects heme from oxidation. CcsBA has 10 transmembrane domains (TMDs) and reconstitutes cytochrome c synthesis in the Escherichia coli periplasm; thus, CcsBA is a cytochrome c synthetase. Purified CcsBA contains heme in an “external heme binding domain” for which two external histidines are shown to serve as axial ligands that protect the heme iron from oxidation. This is likely the active site of the synthetase. Furthermore, two conserved histidines in TMDs are required for heme to travel to the external heme binding domain. Remarkably, the function of CcsBA with mutations in these TMD histidines is corrected by exogenous imidazole, a result analogous to correction of heme binding by myoglobin when its proximal histidine is mutated. These data suggest that CcsBA has a heme binding site within the bilayer and that CcsBA is a heme channel. PMID:19509336

  14. Characterization of two members among the five ADP-forming acyl coenzyme A (Acyl-CoA) synthetases reveals the presence of a 2-(Imidazol-4-yl)acetyl-CoA synthetase in Thermococcus kodakarensis.

    Science.gov (United States)

    Awano, Tomotsugu; Wilming, Anja; Tomita, Hiroya; Yokooji, Yuusuke; Fukui, Toshiaki; Imanaka, Tadayuki; Atomi, Haruyuki

    2014-01-01

    The genome of Thermococcus kodakarensis, along with those of most Thermococcus and Pyrococcus species, harbors five paralogous genes encoding putative α subunits of nucleoside diphosphate (NDP)-forming acyl coenzyme A (acyl-CoA) synthetases. The substrate specificities of the protein products for three of these paralogs have been clarified through studies on the individual enzymes from Pyrococcus furiosus and T. kodakarensis. Here we have examined the biochemical properties of the remaining two acyl-CoA synthetase proteins from T. kodakarensis. The TK0944 and TK2127 genes encoding the two α subunits were each coexpressed with the β subunit-encoding TK0943 gene. In both cases, soluble proteins with an α2β2 structure were obtained and their activities toward various acids in the ADP-forming reaction were examined. The purified TK0944/TK0943 protein (ACS IIITk) accommodated a broad range of acids that corresponded to those generated in the oxidative metabolism of Ala, Val, Leu, Ile, Met, Phe, and Cys. In contrast, the TK2127/TK0943 protein exhibited relevant levels of activity only toward 2-(imidazol-4-yl)acetate, a metabolite of His degradation, and was thus designated 2-(imidazol-4-yl)acetyl-CoA synthetase (ICSTk), a novel enzyme. Kinetic analyses were performed on both proteins with their respective substrates. In T. kodakarensis, we found that the addition of histidine to the medium led to increases in intracellular ADP-forming 2-(imidazol-4-yl)acetyl-CoA synthetase activity, and 2-(imidazol-4-yl)acetate was detected in the culture medium, suggesting that ICSTk participates in histidine catabolism. The results presented here, together with those of previous studies, have clarified the substrate specificities of all five known NDP-forming acyl-CoA synthetase proteins in the Thermococcales. PMID:24163338

  15. Purification and Properties of a Prokaryote Type Glutamine Synthetase from the Bialaphos Producer Streptomyces hygroscopicus SF1293

    NARCIS (Netherlands)

    Kumada, Yoichi; Takano, Eriko; Nagaoka, Kozo

    1990-01-01

    A prokaryote type glutamine synthetase (GS) was purified from a bialaphos (BA)-producing organism, Streptomyces hygroscopicus SF1293 (SF1293). The GS (GS I) consisted of a 55,000 dalton subunit, and its N-terminal amino acid sequence was similar to that of S. coelicolor GS. GS I was highly sensitive

  16. Acyl-CoA synthetase activity links wild-type but not mutant a-Synuclein to brain arachidonate metabolism

    DEFF Research Database (Denmark)

    Golovko, Mikhail; Rosenberger, Thad; Færgeman, Nils J.;

    2006-01-01

    an established steady-state kinetic model. Liver was used as a negative control, and no changes were observed between groups. In Snca-/- brains, there was a marked reduction in 20:4n-6-CoA mass and in microsomal acyl-CoA synthetase (Acsl) activity toward 20:4n-6. Microsomal Acsl activity was...

  17. Organization and expression of genes in the genomic region surrounding the glutamine synthetase gene Gln1 from Lotus japonicus

    DEFF Research Database (Denmark)

    Thykjaer, T; Danielsen, D; She, Q;

    1997-01-01

    within the 23326-bp genomic region analysed. The LjGln1 gene encodes a cytosolic glutamine synthetase and the LjKrm (Kinesin repeat motif) gene encodes a polypeptide with similarity to a repeated motif present in the microtubule-associated kinesin light chain protein. Transcripts of the glutamine...

  18. Structure of the gene encoding phosphoribosylpyrophosphate synthetase (prsA) in Salmonella typhimurium

    DEFF Research Database (Denmark)

    Bower, Stanley G.; Hove-Jensen, Bjarne; Switzer, Robert L.

    1988-01-01

    The Salmonella typhimurium gene prsA, which encodes phosphoribosylpyrophosphate synthetase, has been cloned, and the nucleotide sequence has been determined. The amino acid sequence derived from the S. typhimurium gene is 99% identical to the derived Escherichia coli sequence and 47% identical to...

  19. The carB gene encoding the large subunit of carbamoylphosphate synthetase from Lactococcus lactis is transcribed monocistronically

    DEFF Research Database (Denmark)

    Martinussen, Jan; Hammer, Karin

    1998-01-01

    The biosynthesis of carbamoylphosphate is catalysed by the heterodimeric enzyme carbamoylphosphate synthetase (CPSase). The genes encoding the two subunits in procaryotes are normally transcribed as an operon, whereas in Lactococcus lactis, the gene encoding the large subunit (carB) is shown to b...

  20. Glucosamine synthetase from Escherichia coli: kinetic mechanism and inhibition by N3-fumaroyl-L-2,3-diaminopropionic derivatives

    International Nuclear Information System (INIS)

    N3-(4-Methoxyfumaroyl)-L-2,3-diaminopropionic acid (FMDP; 1, R = OMe), a member of a new class of glutamine analogues, has been investigated as an inhibitor of pure Escherichia coli glucosamine synthetase. Product and dead-end inhibition studies indicate an ordered association to the enzyme with the sugar molecule binding prior to substrate or inhibitor. The inactivation exhibits pseudo-first-order kinetics, is irreversible, and occurs faster in the presence of fructose 6-phosphate, a behavior previously reported for the partially purified enzyme from Salmonella typhimurium. Inhibition occurs with partial covalent incorporation of L-FMDP into glucosamine synthetase. In the presence of fructose 6-phosphate, enzyme inactivation with [2-3H]-DL-FMDP is associated with the incorporation of 0.75 equiv of inhibitor and with the modification of 0.78 thiol residue per enzyme subunit. This result is the first evidence for covalent entrapment of the entire inhibitor molecule following FMDP-mediated glucosamine synthetase inactivation. Preliminary inactivation with 6-diazo-5-oxo-L-norleucine, known to alkylate selectively the NH2-terminal cysteine residue, completely prevents radioactivity incorporation. Therefore, this inhibitor is postulated to covalently modify glucosamine synthetase through direct addition of the thiol nucleophile from the terminal cysteine residue to the Michael acceptor 1, so acting as an affinity label rather than a mechanism-based inhibitor

  1. Phenotypic Changes Resulting from Distinct Point Mutations in the Azospirillum brasilense glnA Gene, Encoding Glutamine Synthetase

    OpenAIRE

    Van Dommelen, Anne; Keijers, Veerle; Wollebrants, An; Vanderleyden, Jozef

    2003-01-01

    Sequencing the glnA genes of two chemically induced Azospirillum brasilense glutamine synthetase mutants revealed an Arg→Cys mutation, corresponding to the glutamate binding site, in one mutant and an Asp→Asn mutation, corresponding to the ammonium binding site, in the second mutant. The phenotypic changes in these mutants are discussed in relation to their genotypes.

  2. Physiological role of taurine - from organism to organelle

    DEFF Research Database (Denmark)

    Lambert, Ian Henry; Kristensen, David Møbjerg Boslev; Holm, Jacob Bak;

    2015-01-01

    Taurine is often referred to as a semi-essential amino acid as newborn mammals have a limited ability to synthesize taurine and have to rely on dietary supply. Taurine is not thought to be incorporated into proteins as no aminoacyl tRNA synthetase has yet been identified and is not oxidized in...... mammalian cells. However, taurine contributes significantly to the cellular pool of organic osmolytes and has accordingly been acknowledged for its role in cell volume restoration following osmotic perturbation. This review describes taurine homeostasis in cells and organelles with emphasis on taurine...... biophysics/membrane dynamics, regulation of transport proteins involved in active taurine uptake and passive taurine release as well as physiological processes, for example, development, lung function, mitochondrial function, antioxidative defence and apoptosis which seem to be affected by a shift in the...

  3. New insights into regulation of the tryptophan biosynthetic operon in Gram-positive bacteria.

    Science.gov (United States)

    Gutierrez-Preciado, A; Jensen, R A; Yanofsky, C; Merino, E

    2005-08-01

    The tryptophan operon of Bacillus subtilis serves as an excellent model for investigating transcription regulation in Gram-positive bacteria. In this article, we extend this knowledge by analyzing the predicted regulatory regions in the trp operons of other fully sequenced Gram-positive bacteria. Interestingly, it appears that in eight of the organisms examined, transcription of the trp operon appears to be regulated by tandem T-box elements. These regulatory elements have recently been described in the trp operons of two bacterial species. Single T-box elements are commonly found in Gram-positive bacteria in operons encoding aminoacyl tRNA synthetases and proteins performing other functions. Different regulatory mechanisms appear to be associated with variations of trp gene organization within the trp operon. PMID:15953653

  4. The Cm56 tRNA modification in archaea is catalyzed either by a specific 2′-O-methylase, or a C/D sRNP

    Science.gov (United States)

    RENALIER, MARIE-HÉLÈNE; JOSEPH, NICOLE; GASPIN, CHRISTINE; THEBAULT, PATRICIA; MOUGIN, ANNIE

    2005-01-01

    We identified the first archaeal tRNA ribose 2′-O-methylase, aTrm56, belonging to the Cluster of Orthologous Groups (COG) 1303 that contains archaeal genes only. The corresponding protein exhibits a SPOUT S-adenosylmethionine (AdoMet)-dependent methyltransferase domain found in bacterial and yeast G18 tRNA 2′-O-methylases (SpoU, Trm3). We cloned the Pyrococcus abyssi PAB1040 gene belonging to this COG, expressed and purified the corresponding protein, and showed that in vitro, it specifically catalyzes the AdoMet-dependent 2′-O-ribose methylation of C at position 56 in tRNA transcripts. This tRNA methylation is present only in archaea, and the gene for this enzyme is present in all the archaeal genomes sequenced up to now, except in the crenarchaeon Pyrobaculum aerophilum. In this archaea, the C56 2′-O-methylation is provided by a C/D sRNP. Our work is the first demonstration that, within the same kingdom, two different mechanisms are used to modify the same nucleoside in tRNAs. PMID:15987815

  5. Role of the primer activation signal in tRNA annealing onto the HIV-1 genome studied by single-molecule FRET microscopy

    NARCIS (Netherlands)

    N. Beerens (Nancy); M.D.E. Jepsen (Mette); V. Nechyporuk-Zloy (Volodymyr); A.C. Krüger (Asger); J.-L. Darlix (Jean-Luc); J. Kjems (Jørgen); V. Birkedal (Victoria)

    2013-01-01

    textabstractHIV-1 reverse transcription is primed by a cellular tRNAlys3 molecule that binds to the primer binding site (PBS) in the genomic RNA. An additional interaction between the tRNA molecule and the primer activation signal (PAS) is thought to regulate the initiation of reverse transcription.

  6. A novel strategy for the identification of genomic islands by comparative analysis of the contents and contexts of tRNA sites in closely related bacteria.

    Science.gov (United States)

    Ou, Hong-Yu; Chen, Ling-Ling; Lonnen, James; Chaudhuri, Roy R; Thani, Ali Bin; Smith, Rebecca; Garton, Natalie J; Hinton, Jay; Pallen, Mark; Barer, Michael R; Rajakumar, Kumar

    2006-01-01

    We devised software tools to systematically investigate the contents and contexts of bacterial tRNA and tmRNA genes, which are known insertion hotspots for genomic islands (GIs). The strategy, based on MAUVE-facilitated multigenome comparisons, was used to examine 87 Escherichia coli MG1655 tRNA and tmRNA genes and their orthologues in E.coli EDL933, E.coli CFT073 and Shigella flexneri Sf301. Our approach identified 49 GIs occupying approximately 1.7 Mb that mapped to 18 tRNA genes, missing 2 but identifying a further 30 GIs as compared with Islander [Y. Mantri and K. P. Williams (2004), Nucleic Acids Res., 32, D55-D58]. All these GIs had many strain-specific CDS, anomalous GC contents and/or significant dinucleotide biases, consistent with foreign origins. Our analysis demonstrated marked conservation of sequences flanking both empty tRNA sites and tRNA-associated GIs across all four genomes. Remarkably, there were only 2 upstream and 5 downstream deletions adjacent to the 328 loci investigated. In silico PCR analysis based on conserved flanking regions was also used to interrogate hotspots in another eight completely or partially sequenced E.coli and Shigella genomes. The tools developed are ideal for the analysis of other bacterial species and will lead to in silico and experimental discovery of new genomic islands. PMID:16414954

  7. Radiosensitivity of glutathione-synthetase deficient human fibroblasts in vitro: Indication of a correlation between OER and glutathione-synthetase activity

    International Nuclear Information System (INIS)

    5-oxoprolinuria is an autosomal recessive disease associated with a glutathione-synthetase (GSH-S) deficiency. Previous results obtained with a fibroplast cell strain originating from such a patient (VP) and exhibiting a decreased glutathione (GSH) level, showed a reduced oxygen enhancement ratio (OER) (1.5; control: 2.9). In order to assess the radioprotective role of the intrinsic GSH, radiosensitivity of 6 fibroblast cell strains with different GSH-S activity have been studied using colony assay as endpoint. 3 strains originated from 5-oxoprolinuria patients (OB, EB and AZ); 3 other strains were got from parents of patients (RB, JB and SP). Among those 6 cell strains, the GSH level was found to be depleted only in AZ and SP. However, a reduced OER is observed for the 6 cell strains (1.7, 2.0 and 2.0 for the patients; 1.9, 2.2 and 2.7 for parents; controls: 2.9). As far as survival of GSH-S deficient cells is concerned, OER seems to be correlated with GSH-S activity: the lower the GSH-S activity, the lower the OER. These results are discussed taking into account other unpublished results obtained from experiments designed to investigate the role of GSH in radio-induced enzymatic repair

  8. Unique Characteristics of the Pyrrolysine System in the 7th Order of Methanogens: Implications for the Evolution of a Genetic Code Expansion Cassette

    Directory of Open Access Journals (Sweden)

    Guillaume Borrel

    2014-01-01

    Full Text Available Pyrrolysine (Pyl, the 22nd proteogenic amino acid, was restricted until recently to few organisms. Its translational use necessitates the presence of enzymes for synthesizing it from lysine, a dedicated amber stop codon suppressor tRNA, and a specific amino-acyl tRNA synthetase. The three genomes of the recently proposed Thermoplasmata-related 7th order of methanogens contain the complete genetic set for Pyl synthesis and its translational use. Here, we have analyzed the genomic features of the Pyl-coding system in these three genomes with those previously known from Bacteria and Archaea and analyzed the phylogeny of each component. This shows unique peculiarities, notably an amber   tRNAPyl with an imperfect anticodon stem and a shortened tRNAPyl synthetase. Phylogenetic analysis indicates that a Pyl-coding system was present in the ancestor of the seventh order of methanogens and appears more closely related to Bacteria than to Methanosarcinaceae, suggesting the involvement of lateral gene transfer in the spreading of pyrrolysine between the two prokaryotic domains. We propose that the Pyl-coding system likely emerged once in Archaea, in a hydrogenotrophic and methanol-H2-dependent methylotrophic methanogen. The close relationship between methanogenesis and the Pyl system provides a possible example of expansion of a still evolving genetic code, shaped by metabolic requirements.

  9. Variation in the Spacer Regions Separating tRNA Genes in Renibacterium salmoninarum Distinguishes Recent Clinical Isolates from the Same Location

    Science.gov (United States)

    Alexander, Sarah M.; Grayson, T. Hilton; Chambers, Edel M.; Cooper, Lynne F.; Barker, Gavin A.; Gilpin, Martyn L.

    2001-01-01

    A means for distinguishing between clinical isolates of Renibacterium salmoninarum that is based on the PCR amplification of length polymorphisms in the tRNA intergenic spacer regions (tDNA-ILPs) was investigated. The method used primers specific to nucleotide sequences of R. salmoninarum tRNA genes and tRNA intergenic spacer regions that had been generated by using consensus tRNA gene primers. Twenty-one PCR products were sequenced from five isolates of R. salmoninarum from the United States, England, and Scotland, and four complete tRNA genes and spacer regions were identified. Sixteen specific PCR primers were designed and tested singly and in all possible pairwise combinations for their potential to discriminate between isolates from recent clinical outbreaks of bacterial kidney disease (BKD) in the United Kingdom. Fourteen of the isolates were cultured from kidney samples taken from fish displaying clinical signs of BKD on five farms, and some of the isolates came from the same farm and at the same time. The tDNA-ILP profiles separated 22 clinical isolates into nine groups and highlighted that some farms may have had more than one source of infection. The grouping of isolates improved on the discriminatory power of previously reported typing methods based on randomly amplified polymorphic DNA analysis and restriction fragment length profiles developed using insertion sequence IS994. Our method enabled us to make divisions between closely related clinical isolates of R. salmoninarum that have identical exact tandem repeat (ETR-A) loci, rRNA intergenic spacer sequences, and IS994 profiles. PMID:11136759

  10. Clues to tRNA Evolution from the Distribution of Class II tRNAs and Serine Codons in the Genetic Code.

    Science.gov (United States)

    Bernhardt, Harold S

    2016-01-01

    We have previously proposed that tRNA(Gly) was the first tRNA and glycine was the first amino acid incorporated into the genetic code. The next two amino acids incorporated would have been the other two small hydrophilic amino acids serine and aspartic acid, which occurred through the duplication of the tRNA(Gly) sequence, followed by mutation of its anticodon by single C to U transition mutations, possibly through spontaneous deamination. Interestingly, however, tRNA(Ser) has a different structure than most other tRNAs, possessing a long variable arm; because of this tRNA(Ser) is classified as a class II tRNA. Also, serine codons are found not only in the bottom right-hand corner of the genetic code table next to those for glycine and aspartic acid, but also in the top row of the table, next to those for two of the most hydrophobic amino acids, leucine and phenylalanine. In the following, I propose that the class II tRNA structure of tRNA(Ser) and the arrangement of serine codons in the genetic code provide clues to the early evolution of tRNA and the genetic code. In addition, I address Di Giulio's recent criticism of our proposal that tRNA(Gly) was the first tRNA, and discuss how early peptides produced from a restricted amino acid alphabet of glycine, serine and aspartic acid might have possessed proteolytic activity, which is possibly important for the early recycling of amino acid monomers. PMID:26927183

  11. Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA and HIV-1 nef Genes in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Siti Aisyah Mualif

    Full Text Available Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef, HIV-1 p24 (ca, and HIV-1 vif in NiCo21(DE3 E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

  12. Variation in the spacer regions separating tRNA genes in Renibacterium salmoninarum distinguishes recent clinical isolates from the same location.

    Science.gov (United States)

    Alexander, S M; Grayson, T H; Chambers, E M; Cooper, L F; Barker, G A; Gilpin, M L

    2001-01-01

    A means for distinguishing between clinical isolates of Renibacterium salmoninarum that is based on the PCR amplification of length polymorphisms in the tRNA intergenic spacer regions (tDNA-ILPs) was investigated. The method used primers specific to nucleotide sequences of R. salmoninarum tRNA genes and tRNA intergenic spacer regions that had been generated by using consensus tRNA gene primers. Twenty-one PCR products were sequenced from five isolates of R. salmoninarum from the United States, England, and Scotland, and four complete tRNA genes and spacer regions were identified. Sixteen specific PCR primers were designed and tested singly and in all possible pairwise combinations for their potential to discriminate between isolates from recent clinical outbreaks of bacterial kidney disease (BKD) in the United Kingdom. Fourteen of the isolates were cultured from kidney samples taken from fish displaying clinical signs of BKD on five farms, and some of the isolates came from the same farm and at the same time. The tDNA-ILP profiles separated 22 clinical isolates into nine groups and highlighted that some farms may have had more than one source of infection. The grouping of isolates improved on the discriminatory power of previously reported typing methods based on randomly amplified polymorphic DNA analysis and restriction fragment length profiles developed using insertion sequence IS994. Our method enabled us to make divisions between closely related clinical isolates of R. salmoninarum that have identical exact tandem repeat (ETR-A) loci, rRNA intergenic spacer sequences, and IS994 profiles. PMID:11136759

  13. Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA) and HIV-1 nef Genes in Escherichia coli.

    Science.gov (United States)

    Mualif, Siti Aisyah; Teow, Sin-Yeang; Omar, Tasyriq Che; Chew, Yik Wei; Yusoff, Narazah Mohd; Ali, Syed A

    2015-01-01

    Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW) rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef), HIV-1 p24 (ca), and HIV-1 vif in NiCo21(DE3) E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes. PMID:26147991

  14. Activity of CMP-2-keto-3-deoxyoctulosonic acid synthetase in Escherichia coli strains expressing the capsular K5 polysaccharide implication for K5 polysaccharide biosynthesis.

    OpenAIRE

    Finke, A.; Roberts, I.; Boulnois, G; Pzzani, C; Jann, K

    1989-01-01

    The activity of the cytoplasmic CMP-2-keto-3-deoxyoctulosonic acid synthetase (CMP-KDO synthetase), which is low in Escherichia coli rough strains such as E. coli K-12 and in uncapsulated strains such as E. coli O111, was significantly elevated in encapsulated E. coli O10:K5 and O18:K5. This enzyme activity was even higher in an E. coli clone expressing the K5 capsule. This and the following findings suggest a correlation between elevated CMP-KDO synthetase activity and the biosynthesis of th...

  15. Phosphoribosylpyrophosphate synthetase of Escherichia coli. Properties of the purified enzyme and primary structure of the prs gene

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Harlow, Kenneth W.; King, Cheryl J.;

    1986-01-01

    Phosphoribosylpyrophosphate (P-Rib-PP) synthetase of Escherichia coli has been purified to near homogeneity from a strain harboring the prs gene, encoding P-Rib-PP synthetase, on a multicopy plasmid. Analysis of the enzyme showed that it required inorganic phosphate for activity and for stability...... ADP. The nucleotide sequence of the E. coli prs gene has been determined and the coding segment established. The deduced amino acid sequence of P-Rib-PP synthetase contained 314 amino acid residues and the molecular weight was calculated as 34,060. The initiation site of transcription was determined...

  16. ALKBH8-mediated formation of a novel diastereomeric pair of wobble nucleosides in mammalian tRNA

    DEFF Research Database (Denmark)

    van den Born, E.; Vagbo, C. B.; Songe-Moller, L.;

    2011-01-01

    Mammals have nine different homologues (ALKBH1-9) of the Escherichia coli DNA repair demethylase AlkB. ALKBH2 is a genuine DNA repair enzyme, but the in vivo function of the other ALKBH proteins has remained elusive. It was recently shown that ALKBH8 contains an additional transfer RNA ( tRNA......) methyltransferase domain, which generates the wobble nucleoside 5-methoxycarbonylmethyluridine (mcm(5)U) from its precursor 5-carboxymethyluridine (cm(5)U). In this study, we report that (R)- and (S)-5-methoxycarbonylhydroxymethyluridine (mchm(5)U), hydroxylated forms of mcm(5)U, are present in mammalian tRNA......(UCG)(Arg), and tRNA(UCC)(Gly), respectively, representing the first example of a diastereomeric pair of modified RNA nucleosides. Through in vitro and in vivo studies, we show that both diastereomers of mchm(5)U are generated from mcm(5)U, and that the AlkB domain of ALKBH8 specifically hydroxylates mcm(5)U into...

  17. Interaction of Ru(Ⅱ) Complex with Yeast tRNA Studied by Isothermal Titration Calorimetry

    Institute of Scientific and Technical Information of China (English)

    徐宏; 刘敛洪; 刘志刚; 梁毅; 张鹏; 杜芬; 周兵瑞; 计亮年

    2005-01-01

    The interaction of metal complex with RNA has been studied by isothermal titration calorimetry (ITC) for the first time. ITC experiments show that complex [Ru(phen)2MPIP]2+ {phen= 1,10-phenanthroline, MP[P-2-(4-methylphenyl)imidazo[4,5-f]-1, 10-phenanthroline} interacts with yeast tRNA in terms of a model for a singleset of identical sites through intercalation, which is consistent with our previous observation obtained from spectroscopic methods, and this binding process was driven by a moderately favorable enthalpy decrease in combination with a moderately favorable entropy increase, suggesting that ITC is an effective method for deep studying the interactions of metal complexes with RNA.

  18. Diversity of nonribosomal peptide synthetase genes in the microbial metagenomes of marine sponges.

    Science.gov (United States)

    Pimentel-Elardo, Sheila Marie; Grozdanov, Lubomir; Proksch, Sebastian; Hentschel, Ute

    2012-06-01

    Genomic mining revealed one major nonribosomal peptide synthetase (NRPS) phylogenetic cluster in 12 marine sponge species, one ascidian, an actinobacterial isolate and seawater. Phylogenetic analysis predicts its taxonomic affiliation to the actinomycetes and hydroxy-phenyl-glycine as a likely substrate. Additionally, a phylogenetically distinct NRPS gene cluster was discovered in the microbial metagenome of the sponge Aplysina aerophoba, which shows highest similarities to NRPS genes that were previously assigned, by ways of single cell genomics, to a Chloroflexi sponge symbiont. Genomic mining studies such as the one presented here for NRPS genes, contribute to on-going efforts to characterize the genomic potential of sponge-associated microbiota for secondary metabolite biosynthesis. PMID:22822366

  19. Cloning and expression of the Thiobacillus ferrooxidans glutamine synthetase gene in Escherichia coli

    International Nuclear Information System (INIS)

    The glutamine synthetase (GS) gene glnA of Thiobacillus ferrooxidans was cloned on recombinant plasmid pMEB100 which enabled Escherichia coli glnA deletion mutants to utilize (NH4)2SO4 as the sole source of nitrogen. High levels of GS-specific activity were obtained in the E. coli glnA deletion mutants containing the T. ferrooxidans GS gene. The cloned T. ferrooxidans DNA fragment containing the glnA gene activated histidase activity in an E. coli glnA glnL glnG deletion mutant containing the Klebsiella aerogenes hut operon. Plasmid pMEB100 also enabled the E. coli glnA glnL glnG deletion mutant to utilize arginine or low levels of glutamine as the sole source of nitrogen. There was no detectable DNA homology between the T. ferrooxidans glnA gene and the E. coli glnA gene

  20. Evolution of the 2'-5'-Oligoadenylate Synthetase family in eukaryotes and bacteria

    DEFF Research Database (Denmark)

    Kjær, Karina Hansen; Poulsen, Jesper Buchhave; Reitamm, Tonu;

    2009-01-01

    The 2′-5′-oligoadenylate synthetase (OAS) belongs to a nucleotidyl transferase family that includes poly(A) polymerases and CCA-adding enzymes. In mammals and birds, the OAS functions in the interferon system but it is also present in an active form in sponges, which are devoid of the interferon...... system. In view of these observations, we have pursued the idea that OAS genes could be present in other metazoans and in unicellular organisms as well. We have identified a number of OAS1 genes in annelids, mollusks, a cnidarian, chordates, and unicellular eukaryotes and also found a family of proteins...... the OASL may have evolved from an ancestor of cartilaginous fishes, and that the OAS2 and the OAS3 genes evolved from a mammalian ancestor. OAS proteins function in the interferon system in mammals. This system is only found in jawed vertebrates. We therefore suggest that the original function of OAS...

  1. Radiosensitizing and cytotoxic properties of misonidazole on glutathione synthetase deficient human fibroblasts

    International Nuclear Information System (INIS)

    The cytotoxic and radiosensitizing effects of misonidazole have been studied on glutathione synthetase deficient fibroblasts and on their controls. At any concentration from 0.1 to 4 mM, deficient cells are more sensitive to the cytotoxic effect of misonidazole than the control cells. The differential effect between the two cell strains concerns both the shoulder and the slope of the survival curve, thus suggesting that NPSH play a role in the determination of misonidazole cytotoxicity. Like oxygen, misonidazole clearly sensitizes deficient cells to a lesser extent than control cells. For both cell strains, the maximum sensitizing effect of misonidazole is very close to that of oxygen (1.5 and 1.5 for deficient cells, 2.8 and 2.9 for control cells, respectively). The sensitizing effect of misonidazole appears in the same concentration range for both cell strains, with a maximal effect at lower concentrations for deficient cells. (author)

  2. Effect of glutamine synthetase inhibition on brain and interorgan ammonia metabolism in bile duct ligated rats

    DEFF Research Database (Denmark)

    Fries, Andreas W; Dadsetan, Sherry; Keiding, Susanne;

    2014-01-01

    Ammonia has a key role in the development of hepatic encephalopathy (HE). In the brain, glutamine synthetase (GS) rapidly converts blood-borne ammonia into glutamine which in high concentrations may cause mitochondrial dysfunction and osmolytic brain edema. In astrocyte-neuron cocultures and brains...... of healthy rats, inhibition of GS by methionine sulfoximine (MSO) reduced glutamine synthesis and increased alanine synthesis. Here, we investigate effects of MSO on brain and interorgan ammonia metabolism in sham and bile duct ligated (BDL) rats. Concentrations of glutamine, glutamate, alanine, and...... aspartate and incorporation of (15)NH4(+) into these amino acids in brain, liver, muscle, kidney, and plasma were similar in sham and BDL rats treated with saline. Methionine sulfoximine reduced glutamine concentrations in liver, kidney, and plasma but not in brain and muscle; MSO reduced incorporation of...

  3. Diversity of Nonribosomal Peptide Synthetase Genes in the Microbial Metagenomes of Marine Sponges

    Directory of Open Access Journals (Sweden)

    Ute Hentschel

    2012-05-01

    Full Text Available Genomic mining revealed one major nonribosomal peptide synthetase (NRPS phylogenetic cluster in 12 marine sponge species, one ascidian, an actinobacterial isolate and seawater. Phylogenetic analysis predicts its taxonomic affiliation to the actinomycetes and hydroxy-phenyl-glycine as a likely substrate. Additionally, a phylogenetically distinct NRPS gene cluster was discovered in the microbial metagenome of the sponge Aplysina aerophoba, which shows highest similarities to NRPS genes that were previously assigned, by ways of single cell genomics, to a Chloroflexi sponge symbiont. Genomic mining studies such as the one presented here for NRPS genes, contribute to on-going efforts to characterize the genomic potential of sponge-associated microbiota for secondary metabolite biosynthesis.

  4. Plant growth is influenced by glutamine synthetase-catalyzed nitrogen metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Langston-Unkefer, P.J.

    1991-06-11

    Ammonia assimilation has been implicated as participating in regulation of nitrogen fixation in free-living bacteria. In fact, these simple organisms utilize an integrated regulation of carbon and nitrogen metabolism; we except to observe an integration of nitrogen and carbon fixation in plants; how could these complex systems grow efficiently and compete in the ecosystem without coordinating these two crucial activities We have been investigating the role of ammonia assimilation regulating the complex symbiotic nitrogen fixation of legumes. Just as is observed in the simple bacterial systems, perturbation of ammonia assimilation in legumes results in increased overall nitrogen fixation. The perturbed plants have increased growth and total nitrogen fixation capability. Because we have targeted the first enyzme in ammonia assimilation, glutamine synthetase, this provides a marker that could be used to assist selection or screening for increased biomass yield. 45 refs., 4 tabs.

  5. Targeted Disruption of Nonribosomal Peptide Synthetase pes3 Augments the Virulence of Aspergillus fumigatus

    DEFF Research Database (Denmark)

    O'Hanlon, Karen A.; Cairns, Timothy; Stack, Deirdre;

    2011-01-01

    Nonribosomal peptide synthesis (NRPS) is a documented virulence factor for the opportunistic pathogen Aspergillus fumigatus and other fungi. Secreted or intracellularly located NRP products include the toxic molecule gliotoxin and the iron-chelating siderophores triacetylfusarinine C and...... contrast to other NRP synthetases, deletion of pes3 significantly increases the virulence of A. fumigatus, whereby the pes3 deletion strain (A. fumigatus Δpes3) exhibited heightened virulence (increased killing) in invertebrate (P <0.001) and increased fungal burden (P = 0.008) in a corticosteroid model of...... metabolite profiling revealed that Pes3 does not produce a secreted or intracellularly stored NRP in A. fumigatus. Macrophage infections and histological analysis of infected murine tissue indicate that Δpes3 heightened virulence appears to be mediated by aberrant innate immune recognition of the fungus...

  6. Glutamine Synthetase in Legumes: Recent Advances in Enzyme Structure and Functional Genomics

    Directory of Open Access Journals (Sweden)

    Marco Betti

    2012-06-01

    Full Text Available Glutamine synthetase (GS is the key enzyme involved in the assimilation of ammonia derived either from nitrate reduction, N2 fixation, photorespiration or asparagine breakdown. A small gene family is encoding for different cytosolic (GS1 or plastidic (GS2 isoforms in legumes. We summarize here the recent advances carried out concerning the quaternary structure of GS, as well as the functional relationship existing between GS2 and processes such as nodulation, photorespiration and water stress, in this latter case by means of proline production. Functional genomic analysis using GS2-minus mutant reveals the key role of GS2 in the metabolic control of the plants and, more particularly, in carbon metabolism.

  7. Peculiarities and impacts of expression of bacterial cyanophycin synthetases in plants.

    Science.gov (United States)

    Nausch, Henrik; Huckauf, Jana; Broer, Inge

    2016-02-01

    Cyanophycin (CP) can be successfully produced in plants by the ectopic expression of the CphA synthetase from Thermosynechococcus elongatus BP-1 (Berg et al. 2000), yielding up to 6.8 % of dry weight (DW) in tobacco leaf tissue and 7.5 % in potato tubers (Huehns et al. 2008, 2009). Though, high amounts of the polymer lead to phenotypical abnormalities in both crops. The extension of abnormalities and the maximum amount of CP tolerated depend on the compartment that CP production is localized at the tissue/crop in which CP was produced (Huehns et al. 2008, 2009; Neumann et al. 2005). It cannot be ascribed to a depletion of arginine, lysine, or aspartate, the substrates for CP synthesis. PMID:26658983

  8. Inhibition of Glutamine Synthetase: A Potential Drug Target in Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Sherry L. Mowbray

    2014-08-01

    Full Text Available Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. Globally, tuberculosis is second only to AIDS in mortality and the disease is responsible for over 1.3 million deaths each year. The impractically long treatment schedules (generally 6–9 months and unpleasant side effects of the current drugs often lead to poor patient compliance, which in turn has resulted in the emergence of multi-, extensively- and totally-drug resistant strains. The development of new classes of anti-tuberculosis drugs and new drug targets is of global importance, since attacking the bacterium using multiple strategies provides the best means to prevent resistance. This review presents an overview of the various strategies and compounds utilized to inhibit glutamine synthetase, a promising target for the development of drugs for TB therapy.

  9. The defective expression of gtpbp3 related to tRNA modification alters the mitochondrial function and development of zebrafish.

    Science.gov (United States)

    Chen, Danni; Li, Feng; Yang, Qingxian; Tian, Miao; Zhang, Zengming; Zhang, Qinghai; Chen, Ye; Guan, Min-Xin

    2016-08-01

    Human mitochondrial DNA (mtDNA) mutations have been associated with a wide spectrum of clinical abnormalities. However, nuclear modifier gene(s) modulate the phenotypic expression of pathogenic mtDNA mutations. In our previous investigation, we identified the human GTPBP3 related to mitochondrial tRNA modification, acting as a modifier to influence of deafness-associated mtDNA mutation. Mutations in GTPBP3 have been found to be associated with other human diseases. However, the pathophysiology of GTPBP3-associated disorders is still not fully understood. Here, we reported the generation and characterization of Gtpbp3 depletion zebrafish model using antisense morpholinos. Zebrafish gtpbp3 has three isoforms localized at mitochondria. Zebrafish gtpbp3 is expressed at various embryonic stages and in multiple tissues. In particular, the gtpbp3 was expressed more abundantly in adult zebrafish ovary and testis. The expression of zebrafish gtpbp3 can functionally restore the growth defects caused by the mss1/gtpbp3 mutation in yeast. A marked decrease of mitochondrial ATP generation accompanied by increased levels of apoptosis and reactive oxygen species were observed in gtpbp3 knockdown zebrafish embryos. The Gtpbp3 morphants exhibited defective in embryonic development including bleeding, melenin, oedema and curved tails within 5days post fertilization, as compared with uninjected controls. The co-injection of wild type gtpbp3 mRNA partially rescued these defects in Gtpbp3 morphants. These data suggest that zebrafish Gtpbp3 is a structural and functional homolog of human and yeast GTPBP3. The mitochondrial dysfunction caused by defective Gtpbp3 may alter the embryonic development in the zebrafish. In addition, this zebrafish model of mitochondrial disease may provide unique opportunities for studying defective tRNA modification, mitochondrial biogenesis, and pathophysiology of mitochondrial disorders. PMID:27184967

  10. Role of codon usage and tRNA changes in rat cytomegalovirus latency and (re)activation.

    Science.gov (United States)

    Kanduc, Darja

    2016-06-01

    Herpesviruses can remain in their hosts by establishing a latent infection with a low pattern of viral gene expression. Passage from latency to reactivation may occur under particular conditions such as immunosuppressive treatments or during fetal development, and often is accompanied by heavy pathologic sequelae. To investigate the molecular basis underlying herpesvirus latency and (re)activation, codon usage of rat cytomegalovirus was comparatively analyzed with respect to the rat codon usage. Two major points stand out as follows: (i) six codons - GCG (Ala), CCG (Pro), CGG (Arg), CGC (Arg), TCG (Ser), and ACG (Thr) - are rare in rat genes and intensively used in rat cytomegalovirus coding sequences; (ii) in many instances, the codons seldom used by the host are clustered along viral sequences coding for single amino acid repeats such as poly-Ala and poly-Thr stretches. The results indicate that rare host codons and their iteration along viral sequences might represent major constraints that lock rat cytomegalovirus translation in its host during the viral latent phase. Consequently, the data also suggest a link between rat cytomegalovirus quiescence/activation and the functional tRNA coadaptation phenomenon. Indeed, increases in minor tRNA species corresponding to rare rat codons mark rat cell proliferation and might rescue difficult viral translational contexts. Ala isoaccepting-tRNA (CGC) is reported as an example. On the whole, the present findings may contribute to explain how the molecular mechanisms that normally control host gene expression can silence/(re)activate viral gene expression, and might address research toward new approaches in anti-viral therapeutics. PMID:26875974

  11. Global translational impacts of the loss of the tRNA modification t6A in yeast

    Directory of Open Access Journals (Sweden)

    Patrick C. Thiaville

    2015-12-01

    Full Text Available The universal tRNA modification t6A is found at position 37 of nearly all tRNAs decoding ANN codons. The absence of t6A37 leads to severe growth defects in baker’s yeast, phenotypes similar to those caused by defects in mcm5s2U34 synthesis. Mutants in mcm5s2U34 can be suppressed by overexpression of tRNALysUUU, but we show t6A phenotypes could not be suppressed by expressing any individual ANN decoding tRNA, and t6A and mcm5s2U are not determinants for each other’s formation. Our results suggest that t6A deficiency, like mcm5s2U deficiency, leads to protein folding defects, and show that the absence of t6A led to stress sensitivities (heat, ethanol, salt and sensitivity to TOR pathway inhibitors. Additionally, L-homoserine suppressed the slow growth phenotype seen in t6A-deficient strains, and proteins aggregates and Advanced Glycation End-products (AGEs were increased in the mutants. The global consequences on translation caused by t6A absence were examined by ribosome profiling. Interestingly, the absence of t6A did not lead to global translation defects, but did increase translation initiation at upstream non-AUG codons and increased frame-shifting in specific genes. Analysis of codon occupancy rates suggests that one of the major roles of t6A is to homogenize the process of elongation by slowing the elongation rate at codons decoded by high abundance tRNAs and I34:C3 pairs while increasing the elongation rate of rare tRNAs and G34:U3 pairs. This work reveals that the consequences of t6A absence are complex and multilayered and has set the stage to elucidate the molecular basis of the observed phenotypes.

  12. Peptidyl transferase antibiotics perturb the relative positioning of the 3'-terminal adenosine of P/P'-site-bound tRNA and 23S rRNA in the ribosome

    DEFF Research Database (Denmark)

    Kirillov, S V; Porse, B T; Garrett, R A

    1999-01-01

    A range of antibiotic inhibitors that act within the peptidyl transferase center of the ribosome were examined for their capacity to perturb the relative positioning of the 3' end of P/P'-site-bound tRNA and the Escherichia coli ribosome. The 3'-terminal adenosines of deacylated tRNA and N......-ribosome complexes. It is concluded that the antibiotics perturb the relative positioning of the 3' end of the P/P'-site-bound tRNA and the peptidyl transferase loop region of 23S rRNA....

  13. Heme ligand identification and redox properties of the cytochrome c synthetase, CcmF†

    Science.gov (United States)

    Francisco, Brian San; Bretsnyder, Eric C.; Rodgers, Kenton R.; Kranz, Robert G.

    2011-01-01

    Cytochrome c maturation in many bacteria, archaea, and plant mitochondria involves the integral membrane protein CcmF, which is thought to function as a cytochrome c synthetase by facilitating the final covalent attachment of heme to the apocytochrome c. We previously reported that the E. coli CcmF protein contains a b-type heme that is stably and stoichiometrically associated with the protein and is not the heme attached to apocytochrome c. Here, we show that mutation of either of two conserved transmembrane histidines (His261 or His491) impairs stoichiometric b-heme binding in CcmF and results in spectral perturbations in the remaining heme. Exogeneous imidazole is able to correct cytochrome c maturation for His261 and His491 substitutions with small side chains (Ala or Gly), suggesting that a “cavity” is formed in these CcmF mutants in which imidazole binds and acts as a functional ligand to the b-heme. The results of resonance Raman spectroscopy on wild-type CcmF are consistent with a hexacoordinate low spin b-heme with at least one endogeneous axial His ligand. Analysis of purified recombinant CcmF proteins from diverse prokaryotes reveals that the b-heme in CcmF is widely conserved. We have also determined the reduction potential of the CcmF b-heme (Em,7 = -147 mV). We discuss these results in the context of CcmF structure and functions as a heme reductase and cytochrome c synthetase. PMID:22066495

  14. Cloning and characterization of the prs gene encoding phosphoribosylpyrophosphate synthetase of Escherichia coli

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    1985-01-01

    The gene, prs, encoding phosphoribosylpyrophosphate (PRPP) synthetase of Escherichia coli was isolated from a library of E. coli genes cloned in the bacteriophage λD69 vector. A strain with a temperature-lethal defect in PRPP synthetase, (prs-2), was used as the host and cloning was performed by...... lysogenic complementation. The prs gene resided on a 5.6 kilobase-pair (kbp) DNA fragment generated by hydrolysis with restriction endonuclease BamHI. The nearby gene pth, encoding peptidyl-tRNA hydrolase, was also on this fragment. Subcloning of the fragment in the multi-copy plasmid pBR322 and subsequent...... deletion of parts of the insert resulted in a 1.7 kbp DNA fragment containing the entire prs gene. Bacterial strains harbouring prs-bearing plasmids showed up to 50-fold increased PRPP synthetase activity. The PRPP synthetase subunit was identified by analysis of plasmid-harbouring minicells and the...

  15. The Populus Superoxide Dismutase Gene Family and Its Responses to Drought Stress in Transgenic Poplar Overexpressing a Pine Cytosolic Glutamine Synthetase (GS1a)

    OpenAIRE

    Molina-Rueda, Juan Jesús; Tsai, Chung Jui; Kirby, Edward G.

    2013-01-01

    Background Glutamine synthetase (GS) plays a central role in plant nitrogen assimilation, a process intimately linked to soil water availability. We previously showed that hybrid poplar (Populus tremula X alba, INRA 717-1B4) expressing ectopically a pine cytosolic glutamine synthetase gene (GS1a) display enhanced tolerance to drought. Preliminary transcriptome profiling revealed that during drought, members of the superoxide dismutase (SOD) family were reciprocally regulated in GS poplar when...

  16. Nostophycin Biosynthesis Is Directed by a Hybrid Polyketide Synthase-Nonribosomal Peptide Synthetase in the Toxic Cyanobacterium Nostoc sp. Strain 152▿†

    OpenAIRE

    Fewer, David P.; Österholm, Julia; Rouhiainen, Leo; Jokela, Jouni; Wahlsten, Matti; Sivonen, Kaarina

    2011-01-01

    Cyanobacteria are a rich source of natural products with interesting pharmaceutical properties. Here, we report the identification, sequencing, annotation, and biochemical analysis of the nostophycin (npn) biosynthetic gene cluster. The npn gene cluster spans 45.1 kb and consists of three open reading frames encoding a polyketide synthase, a mixed polyketide nonribosomal peptide synthetase, and a nonribosomal peptide synthetase. The genetic architecture and catalytic domain organization of th...

  17. Heterologous expression in Escherichia coli of the first module of the nonribosomal peptide synthetase for chloroeremomycin, a vancomycin-type glycopeptide antibiotic

    OpenAIRE

    Trauger, John W.; Walsh, Christopher T.

    2000-01-01

    The gene cluster from Amycolotopsis orientalis responsible for biosynthesis of the vancomycin-type glycopeptide antibiotic chloroeremomycin was recently sequenced, indicating that this antibiotic derives from a seven-residue peptide synthesized by a three-subunit (CepA, CepB, and CepC) modular nonribosomal peptide synthetase. Expression of all or parts of the peptide synthetase in Escherichia coli would facilitate biochemical characterization of its substrate specificity, an important step to...

  18. The carbamate kinase-like carbamoyl phosphate synthetase of the hyperthermophilic archaeon Pyrococcus furiosus, a missing link in the evolution of carbamoyl phosphate biosynthesis

    OpenAIRE

    Durbecq, Virginie; Legrain, Christianne; Roovers, Martine; Piérard, André; Glansdorff, Nicolas

    1997-01-01

    Microbial carbamoyl phosphate synthetases (CPS) use glutamine as nitrogen donor and are composed of two subunits (or domains), one exhibiting glutaminase activity, the other able to synthesize carbamoyl phosphate (CP) from bicarbonate, ATP, and ammonia. The pseudodimeric organization of this synthetase suggested that it has evolved by duplication of a smaller kinase, possibly a carbamate kinase (CK). In contrast to other prokaryotes the hyperthermophilic archaeon Pyrococcus furiosus was found...

  19. Improvement of an l-Leucine-Producing Mutant of Brevibacterium lactofermentum 2256 by Genetically Desensitizing It to α-Acetohydroxy Acid Synthetase

    OpenAIRE

    Tsuchida, Takayasu; Momose,Haruo

    1986-01-01

    Genetic improvement of l-leucine productivity in strain 218, an ile− 2-thiazolealanine-resistant mutant of Brevibacterium lactofermentum 2256, was attempted. In strain 218, which produced 28 mg of l-leucine per ml from 13% glucose, α-isopropylmalate synthetase was genetically desensitized and derepressed to the effect of l-leucine, whereas α-acetohydroxy acid synthetase remained unaltered, although it could be derepressed phenotypically by limiting the isoleucine concentration in the culture....

  20. The CcmFH complex is the system I holocytochrome c synthetase: engineering cytochrome c maturation independent of CcmABCDE

    OpenAIRE

    Francisco, Brian San; Sutherland, Molly C.; Kranz, Robert G.

    2014-01-01

    Cytochrome c maturation (ccm) in many bacteria, archaea, and plant mitochondria requires eight membrane proteins, CcmABCDEFGH, called system I. This pathway delivers and attaches heme covalently to two cysteines (of Cys-Xxx-Xxx-Cys-His) in the cytochrome c. All models propose that CcmFH facilitates covalent attachment of heme to the apocytochrome; namely, that it is the synthetase. However, holocytochrome c synthetase activity has not been directly demonstrated for CcmFH. We report formation ...

  1. Characterization of Two Members among the Five ADP-Forming Acyl Coenzyme A (Acyl-CoA) Synthetases Reveals the Presence of a 2-(Imidazol-4-yl)Acetyl-CoA Synthetase in Thermococcus kodakarensis

    OpenAIRE

    Awano, Tomotsugu; Wilming, Anja; Tomita, Hiroya; Yokooji, Yuusuke; Fukui, Toshiaki; Imanaka, Tadayuki; Atomi, Haruyuki

    2014-01-01

    The genome of Thermococcus kodakarensis, along with those of most Thermococcus and Pyrococcus species, harbors five paralogous genes encoding putative α subunits of nucleoside diphosphate (NDP)-forming acyl coenzyme A (acyl-CoA) synthetases. The substrate specificities of the protein products for three of these paralogs have been clarified through studies on the individual enzymes from Pyrococcus furiosus and T. kodakarensis. Here we have examined the biochemical properties of the remaining t...

  2. Interferon titer and the 2',5'-oligoadenylate-synthetase activity in rat thymus lymphocytes in conditions of Omeprazol-caused hypergastrinemia

    Directory of Open Access Journals (Sweden)

    Kompanets I. V.

    2013-01-01

    Full Text Available The aim of this work was the determination of rat thymocytes response to hypergastrinemia evoked by hypoacidity and multiprobiotic «Symbiter® acidophilic concentrated» (symbiter treatment via the estimation of the interferon (IFN titer and 2', 5'-oligoadenylate (OA-synthetase activity in lymphocytes. 2', 5'-OA-synthetase is the IFN-induced enzyme. Methods. The micromethod of IFN titer determination by antiviral activity, spectrophotometrical method of 2', 5'-OA-synthetase activity determination. Results. It was shown that the IFN production by cultivated thymocytes is amplified while the 2', 5'-OA-synthetase activity decreases in these cells in conditions of hypoacidity caused by the 28-days omeprazol treatment. The treatment of animals by symbiter against a background of hypoacidity causes the augmentation of IFN production by thymocytes, but does not stimulate the 2', 5'-OA-synthetase activity. The IFN production by thymocytes in response to IFN inducers (PHA and cycloferone in vitro is intensified comparatively to the control at hypoacidity and symbiter treatment. Conclusions. The multiprobiotic symbiter exhibits interferonogenic properties. The IFN synthesis in response to induction in vitro is intensified in comparison with healthy animals at both hypoacidity and symbiter treatment while the 2', 5'-OA-synthetase acivity in thymocytes decreases.

  3. In vitro and in vivo effect of interleukin-2 on the 2',5'-oligoadenylate synthetase activity of peripheral mononuclear blood cells

    International Nuclear Information System (INIS)

    The in vitro and in vivo influence of interleukin-2 (IL-2) on 2',5'-oligoadenylate (2-5A) synthetase activity and natural killer (NK) activity of peripheral mononuclear blood cells (PBMCs) was investigated. Incubation of PBMCs in vitro with IL-2 resulted in a considerable secretion of interferon-gamma (IFN-gamma) and in a significant elevation of 2-5A synthetase activity, as well as NK activity. Neutralizing monoclonal anti-IFN-gamma antibodies inhibited the elevation of 2-5A synthetase activity, but not the IL-2-induced augmentation of NK activity. Additionally, 2-5A synthetase and NK activity of PBMCs was measured in a child with neuroblastoma that was treated with recombinant IL-2 by continuous intravenous application. During the treatment, NK activity against the NK-sensitive cell line K 562 and against autologous tumor cells was not augmented. However, a significant increase of 2-5A synthetase activity in PBMCs was observed during IL-2 treatment, whereas there was no detectable serum level of IFN-gamma. We conclude that measuring 2-5A synthetase activity in patients treated with IL-2 may be helpful in monitoring the immunomodulatory effects of IL-2 on immune effector cells

  4. Loss of (2'-5')oligoadenylate synthetase activity by production of antisense RNA results in lack of protection by interferon from viral infections

    International Nuclear Information System (INIS)

    An expression vector was constructed that carries part of the human BK papovavirus with 0.5 kilobases of (2'-5')oligoadenylate (2-5A) synthetase cDNA inserted in inverted orientation downstream from the virion proteins (VP) promoter and the neomycin-resistance gene neo under the control of a simian virus 40 promoter. Cells transfected with this vector and selected for resistance to the neomycin derivative G418 synthesized RNA complementary to 2-5A synthetase mRNA. These cells lacked 2-5A synthetase activity, and the enzyme was not inducible by interferon. In contrast, 2-5A synthetase was induced in cells transfected with a control vector without the cDNA insert. Such cells were protected by interferon from RNA viruses, whereas cells lacking 2-5A synthetase were not protected from encephalomyocarditis virus, vesicular stomatitis virus, and Sindbis virus but were fully protected from influenza virus. These findings show that a high level of 2-5A synthetase is required for interferon-induced protection from the cytoplasmic RNA viruses tested

  5. Three-dimensional structure of phosphoribosyl pyrophosphate synthetase from E. coli at 2.71 Å resolution

    International Nuclear Information System (INIS)

    Phosphoribosyl pyrophosphate synthetase from Escherichia coli was cloned, purified, and crystallized. Single crystals of the enzyme were grown under microgravity. The X-ray diffraction data set was collected at the Spring-8 synchrotron facility and used to determine the three-dimensional structure of the enzyme by the molecular-replacement method at 2.71 Å resolution. The active and regulatory sites in the molecule of E. coli phosphoribosyl pyrophosphate synthetase were revealed by comparison with the homologous protein from Bacillus subtilis, the structure of which was determined in a complex with functional ligands. The conformations of polypeptide-chain fragments surrounding and composing the active and regulatory sites were shown to be identical in both proteins

  6. Three-dimensional structure of phosphoribosyl pyrophosphate synthetase from E. coli at 2.71 Å resolution

    Energy Technology Data Exchange (ETDEWEB)

    Timofeev, V. I., E-mail: inna@ns.crys.ras.ru, E-mail: tostars@mail.ru, E-mail: ugama@yandex.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Abramchik, Yu. A. [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Zhukhlistova, N. E. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Muravieva, T. I.; Esipov, R. S. [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Kuranova, I. P. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    2016-01-15

    Phosphoribosyl pyrophosphate synthetase from Escherichia coli was cloned, purified, and crystallized. Single crystals of the enzyme were grown under microgravity. The X-ray diffraction data set was collected at the Spring-8 synchrotron facility and used to determine the three-dimensional structure of the enzyme by the molecular-replacement method at 2.71 Å resolution. The active and regulatory sites in the molecule of E. coli phosphoribosyl pyrophosphate synthetase were revealed by comparison with the homologous protein from Bacillus subtilis, the structure of which was determined in a complex with functional ligands. The conformations of polypeptide-chain fragments surrounding and composing the active and regulatory sites were shown to be identical in both proteins.

  7. Fatty acid biosynthesis VII. Substrate control of chain-length of products synthesised by rat liver fatty acid synthetase

    DEFF Research Database (Denmark)

    Hansen, Heinz Johs. Max; Carey, E.M.; Dils, R.

    1970-01-01

    - 1. Gas-liquid and paper chromatography have been used to determine the chain-lengths of fatty acids synthesised by purified rat liver fatty acid synthetase from [1-14C]acetyl-CoA, [1,3-14C2]malonyl-CoA and from [1-14C]acetyl-CoA plus partially purified rat liver acetyl-CoA carboxylase. - 2. A...

  8. Formyltetrahydrofolate Synthetase Gene Diversity in the Guts of Higher Termites with Different Diets and Lifestyles ▿ †

    OpenAIRE

    Ottesen, Elizabeth A.; Leadbetter, Jared R.

    2011-01-01

    In this study, we examine gene diversity for formyl-tetrahydrofolate synthetase (FTHFS), a key enzyme in homoacetogenesis, recovered from the gut microbiota of six species of higher termites. The “higher” termites (family Termitidae), which represent the majority of extant termite species and genera, engage in a broader diversity of feeding and nesting styles than the “lower” termites. Previous studies of termite gut homoacetogenesis have focused on wood-feeding lower termites, from which the...

  9. Thermodynamic properties distinguish human mitochondrial aspartyl-tRNA synthetase from bacterial homolog with same 3D architecture

    OpenAIRE

    Neuenfeldt, Anne; Lorber, Bernard; Ennifar, Eric; Gaudry, Agnès; Sauter, Claude; Sissler, Marie; Florentz, Catherine

    2012-01-01

    In the mammalian mitochondrial translation apparatus, the proteins and their partner RNAs are coded by two genomes. The proteins are nuclear-encoded and resemble their homologs, whereas the RNAs coming from the rapidly evolving mitochondrial genome have lost critical structural information. This raises the question of molecular adaptation of these proteins to their peculiar partner RNAs. The crystal structure of the homodimeric bacterial-type human mitochondrial aspartyl-tRNA synthetase (DRS)...

  10. Nicotinamide mononucleotide synthetase is the key enzyme for an alternative route of NAD biosynthesis in Francisella tularensis

    OpenAIRE

    Sorci, Leonardo; Martynowski, Dariusz; Rodionov, Dmitry A; Eyobo, Yvonne; Zogaj, Xhavit; Klose, Karl E.; Nikolaev, Evgeni V.; Magni, Giulio; Zhang, Hong; Osterman, Andrei L.

    2009-01-01

    Enzymes involved in the last 2 steps of nicotinamide adenine dinucleotide (NAD) cofactor biosynthesis, which catalyze the adenylylation of the nicotinic acid mononucleotide (NaMN) precursor to nicotinic acid dinucleotide (NaAD) followed by its amidation to NAD, constitute promising drug targets for the development of new antibiotics. These enzymes, NaMN adenylyltransferase (gene nadD) and NAD synthetase (gene nadE), respectively, are indispensable and conserved in nearly all bacterial pathoge...

  11. Mechanism-based inhibitors of MenE, an acyl-CoA synthetase involved in bacterial menaquinone biosynthesis†

    OpenAIRE

    Lu, Xuequan; Zhang, Huaning; Tonge, Peter J.; Tan, Derek S.

    2008-01-01

    Menaquinone (vitamin K2) is an essential component of the electron transfer chain in many pathogens, including Mycobacterium tuberculosis and Staphylococcus aureus, and menaquinone biosynthesis is a potential target for antibiotic drug discovery. We report herein a series of mechanism-based inhibitors of MenE, an acyl-CoA synthetase that catalyzes adenylation and thioesterification of o-succinylbenzoic acid (OSB) during menaquinone biosynthesis. The most potent compound inhibits MenE with an ...

  12. Arabidopsis acyl-acyl carrier protein synthetase AAE15 with medium chain fatty acid specificity is functional in cyanobacteria

    OpenAIRE

    Kaczmarzyk, Danuta; Hudson, Elton P.; Fulda, Martin

    2016-01-01

    Cyanobacteria are potential hosts for the biosynthesis of oleochemical compounds. The metabolic precursors for such compounds are fatty acids and their derivatives, which require chemical activation to become substrates in further conversion steps. We characterized the acyl activating enzyme AAE15 of Arabidopsis encoded by At4g14070, which is a homologue of a cyanobacterial acyl-ACP synthetase (AAS). We expressed AAE15 in insect cells and demonstrated its AAS activity with medium chain fatty ...

  13. Formyltetrahydrofolate Synthetase Sequences from Salt Marsh Plant Roots Reveal a Diversity of Acetogenic Bacteria and Other Bacterial Functional Groups

    OpenAIRE

    Leaphart, A. B.; Friez, M. J.; Lovell, C R

    2003-01-01

    Sixty-two partial formyltetrahydrofolate synthetase (FTHFS) structural gene sequences were recovered from roots of salt marsh plants, including Spartina alterniflora, Salicornia virginica, and Juncus roemerianus. Only S. alterniflora roots yielded sequences grouping with FTHFS sequences from known acetogens. Most other FTHFS or FTHFS-like sequences grouped with those from sulfate-reducing bacteria. Several sequences that grouped with Sphingomonas paucimobilis ligH were also recovered.

  14. The carB Gene Encoding the Large Subunit of Carbamoylphosphate Synthetase from Lactococcus lactis Is Transcribed Monocistronically

    OpenAIRE

    Martinussen, Jan; Hammer, Karin

    1998-01-01

    The biosynthesis of carbamoylphosphate is catalyzed by the heterodimeric enzyme carbamoylphosphate synthetase. The genes encoding the two subunits of this enzyme in procaryotes are normally transcribed as an operon, but the gene encoding the large subunit (carB) in Lactococcus lactis is shown to be transcribed as an isolated unit. Carbamoylphosphate is a precursor in the biosynthesis of both pyrimidine nucleotides and arginine. By mutant analysis, L. lactis is shown to possess only one carB g...

  15. Acetyl-CoA synthetase 2 promotes acetate utilization and maintains cancer cell growth under metabolic stress

    OpenAIRE

    Schug, Zachary T.; Peck, Barrie; Jones, Dylan T; Zhang, Qifeng; Grosskurth, Shaun; Alam, Israt S.; Goodwin, Louise M.; Smethurst, Elizabeth; Mason, Susan; Blyth, Karen; McGarry, Lynn; James, Daniel; Shanks, Emma; Kalna, Gabriela; Saunders, Rebecca E.

    2015-01-01

    Summary A functional genomics study revealed that the activity of acetyl-CoA synthetase 2 (ACSS2) contributes to cancer cell growth under low-oxygen and lipid-depleted conditions. Comparative metabolomics and lipidomics demonstrated that acetate is used as a nutritional source by cancer cells in an ACSS2-dependent manner, and supplied a significant fraction of the carbon within the fatty acid and phospholipid pools. ACSS2 expression is upregulated under metabolically stressed conditions and A...

  16. Effect of growth temperature on folding of carbamoylphosphate synthetases of Salmonella typhimurium and a cold-sensitive derivative.

    OpenAIRE

    Han, B D; Nolan, W G; Hopkins, H P; Jones, R. T.; Ingraham, J L; Abdelal, A T

    1990-01-01

    The properties of homogeneous preparations of carbamoylphosphate synthetase (CPSase) from wild-type Salmonella typhimurium and a cold-sensitive derivative grown at different growth temperatures were examined. For the cold-sensitive mutant, the affinity for glutamine of the form of CPSase synthesized at 20 degrees C was lower than that of the form of the enzyme synthesized at 37 degrees C, regardless of the assay temperature. Thus, the cold sensitivity of the mutant reflects an effect of tempe...

  17. Elongation in translation as a dynamic interaction among the ribosome, tRNA, and elongation factors EF-G and EF-Tu.

    Science.gov (United States)

    Agirrezabala, Xabier; Frank, Joachim

    2009-08-01

    The ribosome is a complex macromolecular machine that translates the message encoded in the messenger RNA and synthesizes polypeptides by linking the individual amino acids carried by the cognate transfer RNAs (tRNAs). The protein elongation cycle, during which the tRNAs traverse the ribosome in a coordinated manner along a path of more than 100 A, is facilitated by large-scale rearrangements of the ribosome. These rearrangements go hand in hand with conformational changes of tRNA as well as elongation factors EF-Tu and EF-G - GTPases that catalyze tRNA delivery and translocation, respectively. This review focuses on the structural data related to the dynamics of the ribosomal machinery, which are the basis, in conjunction with existing biochemical, kinetic, and fluorescence resonance energy transfer data, of our knowledge of the decoding and translocation steps of protein elongation. PMID:20025795

  18. Dexamethasone enhances glutamine synthetase activity and reduces N-methyl-D-aspartate neurotoxicity in mixed cultures of neurons and astrocytes

    Directory of Open Access Journals (Sweden)

    Edith Debroas

    2015-05-01

    Full Text Available Astrocytes are claimed to protect neurons against excitotoxicity by clearing glutamate from the extracellular space and rapidly converting it into glutamine. Glutamine, is then released into the extracellular medium, taken up by neurons and transformed back into glutamate which is then stored into synaptic vesicles. Glutamine synthetase (GS, the key enzyme that governs this glutamate/glutamine cycle, is known to be upregulated by glucocorticoids. In the present work we have thus studied in parallel the effects of dexamethasone on glutamine synthetase activity and NMDA-induced neuronal death in cultures derived from the brain cortex of murine embryos. We showed that dexamethasone was able to markedly enhance GS activity in cultures of astrocytes but not in near pure neuronal cultures. The pharmacological characteristics of the dexamethasone action strongly suggest that it corresponds to a typical receptor-mediated effect. We also observed that long lasting incubation (72 h of mixed astrocyte-neuron cultures in the presence of 100 nM dexamethasone significantly reduced the toxicity of NMDA treatment. Furthermore we demonstrated that methionine sulfoximine, a selective inhibitor of GS, abolished the dexamethasone-induced increase in GS activity and also markedly potentiated NMDA toxicity. Altogether these results suggest that dexamethasone may promote neuroprotection through a stimulation of astrocyte glutamine synthetase.

  19. Purification, crystallization and preliminary X-ray diffraction analysis of the seryl-tRNA synthetase from Candida albicans

    International Nuclear Information System (INIS)

    The seryl-tRNA synthetase from C. albicans was crystallized by the sitting-drop vapour-diffusion method using ammonium sulfate as precipitant. The crystals belonged to the hexagonal space group P6122 and diffraction data were collected to 2.0 Å resolution at a synchrotron source. The seryl-tRNA synthetase (SerRS) from Candida albicans exists naturally as two isoforms resulting from ambiguity in the natural genetic code. Both enzymes were crystallized by the sitting-drop vapour-diffusion method using 3.2–3.4 M ammonium sulfate as precipitant. The crystals belonged to the hexagonal space group P6122 and contained one monomer per asymmetric unit, despite the synthetase existing as a homodimer (with a molecular weight of ∼116 kDa) in solution. Diffraction data were collected to 2.0 Å resolution at a synchrotron source and the crystal structures of unliganded SerRS and of its complexes with ATP and with a seryl-adenylate analogue were solved by molecular replacement. The structure of C. albicans SerRS represents the first reported structure of a eukaryotic cytoplasmic SerRS

  20. Defective i6A37 modification of mitochondrial and cytosolic tRNAs results from pathogenic mutations in TRIT1 and its substrate tRNA.

    Directory of Open Access Journals (Sweden)

    John W Yarham

    2014-06-01

    Full Text Available Identifying the genetic basis for mitochondrial diseases is technically challenging given the size of the mitochondrial proteome and the heterogeneity of disease presentations. Using next-generation exome sequencing, we identified in a patient with severe combined mitochondrial respiratory chain defects and corresponding perturbation in mitochondrial protein synthesis, a homozygous p.Arg323Gln mutation in TRIT1. This gene encodes human tRNA isopentenyltransferase, which is responsible for i6A37 modification of the anticodon loops of a small subset of cytosolic and mitochondrial tRNAs. Deficiency of i6A37 was previously shown in yeast to decrease translational efficiency and fidelity in a codon-specific manner. Modelling of the p.Arg323Gln mutation on the co-crystal structure of the homologous yeast isopentenyltransferase bound to a substrate tRNA, indicates that it is one of a series of adjacent basic side chains that interact with the tRNA backbone of the anticodon stem, somewhat removed from the catalytic center. We show that patient cells bearing the p.Arg323Gln TRIT1 mutation are severely deficient in i6A37 in both cytosolic and mitochondrial tRNAs. Complete complementation of the i6A37 deficiency of both cytosolic and mitochondrial tRNAs was achieved by transduction of patient fibroblasts with wild-type TRIT1. Moreover, we show that a previously-reported pathogenic m.7480A>G mt-tRNASer(UCN mutation in the anticodon loop sequence A36A37A38 recognised by TRIT1 causes a loss of i6A37 modification. These data demonstrate that deficiencies of i6A37 tRNA modification should be considered a potential mechanism of human disease caused by both nuclear gene and mitochondrial DNA mutations while providing insight into the structure and function of TRIT1 in the modification of cytosolic and mitochondrial tRNAs.

  1. Secondary structure and feature of mitochondrial tRNA genes of the Ussurian tube-nosed bat Murina ussuriensis (Chiroptera: Vespertilionidae)

    OpenAIRE

    Kwang Bae Yoon; Yung Chul Park

    2015-01-01

    The complete mitogenome (NC_021119) of the Ussurian tube-nosed bat Murina ussuriensis (Chiroptera: Vespertilionidae) was annotated and characterized in our recent publication (http://www.ncbi.nlm.nih.gov/nuccore/NC_021119). Here we provide additional information on methods in detail for obtaining the complete sequence of M. ussuriensis mitogenome. In addition, we describe characteristics of 22 tRNA genes and secondary structure and feature of 22 tRNAs of M. ussuriensis mitogenome.

  2. Secondary structure and feature of mitochondrial tRNA genes of the Ussurian tube-nosed bat Murina ussuriensis (Chiroptera: Vespertilionidae

    Directory of Open Access Journals (Sweden)

    Kwang Bae Yoon

    2015-09-01

    Full Text Available The complete mitogenome (NC_021119 of the Ussurian tube-nosed bat Murina ussuriensis (Chiroptera: Vespertilionidae was annotated and characterized in our recent publication (http://www.ncbi.nlm.nih.gov/nuccore/NC_021119. Here we provide additional information on methods in detail for obtaining the complete sequence of M. ussuriensis mitogenome. In addition, we describe characteristics of 22 tRNA genes and secondary structure and feature of 22 tRNAs of M. ussuriensis mitogenome.

  3. An uncommon clinical presentation of relapsing dilated cardiomyopathy with identification of sequence variations in MYNPC3, KCNH2 and mitochondrial tRNA cysteine

    OpenAIRE

    Guillen Sacoto, Maria J.; Chapman, Kimberly A; Deneen Heath; Mary Beth Seprish; Zand, Dina J.

    2015-01-01

    We describe a young girl with dilated cardiomyopathy, long QT syndrome, and possible energy deficiency. Two major sequence changes were identified by whole exome sequencing (WES) and mitochondrial DNA analysis that were interpreted as potentially causative. Changes were identified in the KCNH2 gene and mitochondrial tRNA for cysteine. A variation was also seen in MYPBC3. Since the launch of WES as a clinically available technology in 2010, there has been concern regarding the identification o...

  4. Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA) and HIV-1 nef Genes in Escherichia coli

    OpenAIRE

    Mualif, Siti Aisyah; Teow, Sin-Yeang; Omar, Tasyriq Che; Chew, Yik Wei; Yusoff, Narazah Mohd; Syed A. Ali

    2015-01-01

    Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expres...

  5. EST data: 59 [RED

    Lifescience Database Archive (English)

    Full Text Available CB0250 AU166273 AU166274 DPlate 001 C08 5' TAG ATG TAT GTG TAT CAT GC 3' 5' GAC ATT GTG TAC ACC ... NA SYNTHETASE (CONTAINS: GLUTAMYL-TRNA SYNTHETASE (EC ... 6.1.1.17) (GLUTAMATE--TRNA LIGASE), AND PROLYL-TRN ... A SYNTHETASE (EC ... 6.1.1.15) (PROLINE--TRNA LIGASE)). &HSGTS_1(X54326 ...

  6. Identification and sequence analysis of metazoan tRNA 3'-end processing enzymes tRNase Zs.

    Directory of Open Access Journals (Sweden)

    Zhikang Wang

    Full Text Available tRNase Z is the endonuclease responsible for removing the 3'-trailer sequences from precursor tRNAs, a prerequisite for the addition of the CCA sequence. It occurs in the short (tRNase Z(S and long (tRNase Z(L forms. Here we report the identification and sequence analysis of candidate tRNase Zs from 81 metazoan species. We found that the vast majority of deuterostomes, lophotrochozoans and lower metazoans have one tRNase Z(S and one tRNase Z(L genes, whereas ecdysozoans possess only a single tRNase Z(L gene. Sequence analysis revealed that in metazoans, a single nuclear tRNase Z(L gene is likely to encode both the nuclear and mitochondrial forms of tRNA 3'-end processing enzyme through mechanisms that include alternative translation initiation from two in-frame start codons and alternative splicing. Sequence conservation analysis revealed a variant PxKxRN motif, PxPxRG, which is located in the N-terminal region of tRNase Z(Ss. We also identified a previously unappreciated motif, AxDx, present in the C-terminal region of both tRNase Z(Ss and tRNase Z(Ls. The AxDx motif consisting mainly of a very short loop is potentially close enough to form hydrogen bonds with the loop containing the PxKxRN or PxPxRG motif. Through complementation analysis, we demonstrated the likely functional importance of the AxDx motif. In conclusion, our analysis supports the notion that in metazoans a single tRNase Z(L has evolved to participate in both nuclear and mitochondrial tRNA 3'-end processing, whereas tRNase Z(S may have evolved new functions. Our analysis also unveils new evolutionarily conserved motifs in tRNase Zs, including the C-terminal AxDx motif, which may have functional significance.

  7. Crystal structure of tRNA m(1)A58 methyltransferase TrmI from Aquifex aeolicus in complex with S-adenosyl-L-methionine.

    Science.gov (United States)

    Kuratani, Mitsuo; Yanagisawa, Tatsuo; Ishii, Ryohei; Matsuno, Michiyo; Si, Shu-Yi; Katsura, Kazushige; Ushikoshi-Nakayama, Ryoko; Shibata, Rie; Shirouzu, Mikako; Bessho, Yoshitaka; Yokoyama, Shigeyuki

    2014-09-01

    The N (1)-methyladenosine residue at position 58 of tRNA is found in the three domains of life, and contributes to the stability of the three-dimensional L-shaped tRNA structure. In thermophilic bacteria, this modification is important for thermal adaptation, and is catalyzed by the tRNA m(1)A58 methyltransferase TrmI, using S-adenosyl-L-methionine (AdoMet) as the methyl donor. We present the 2.2 Å crystal structure of TrmI from the extremely thermophilic bacterium Aquifex aeolicus, in complex with AdoMet. There are four molecules per asymmetric unit, and they form a tetramer. Based on a comparison of the AdoMet binding mode of A. aeolicus TrmI to those of the Thermus thermophilus and Pyrococcus abyssi TrmIs, we discuss their similarities and differences. Although the binding modes to the N6 amino group of the adenine moiety of AdoMet are similar, using the side chains of acidic residues as well as hydrogen bonds, the positions of the amino acid residues involved in binding are diverse among the TrmIs from A. aeolicus, T. thermophilus, and P. abyssi. PMID:24894648

  8. A model for mis-sense error in protein synthesis: mis-charged cognate tRNA versus mis-reading of codon

    CERN Document Server

    Dutta, Annwesha

    2015-01-01

    The sequence of amino acid monomers in the primary structure of protein is decided by the corresponding sequence of codons (triplets of nucleic acid monomers) on the template messenger RNA (mRNA). The polymerization of a protein, by incorporation of the successive amino acid monomers, is carried out by a molecular machine called ribosome. Transfer RNA (tRNA) molecules, each species of which is "charged" with a specific amino acid, enters the ribosome and participates in the reading of the codon by the ribosome. Both mis-reading of mRNA codon and prior mis-charging of a tRNA can lead to "mis-sense" error, i.e,. erroneous substitution of a correct amino acid monomer by an incorrect one during the synthesis of a protein. We develop a theoretical model of protein synthesis that allows for both types of contributions to the "mis-sense" error. We report exact analytical formulae for several quantities that characterize the interplay of mis-charging of tRNA and mis-reading of mRNA. The average rate of elongation of ...

  9. Crystal structure of the two-subunit tRNA m1A58 methyltransferase TRM6-TRM61 from Saccharomyces cerevisiae

    Science.gov (United States)

    Wang, Mingxing; Zhu, Yuwei; Wang, Chongyuan; Fan, Xiaojiao; Jiang, Xuguang; Ebrahimi, Mohammad; Qiao, Zhi; Niu, Liwen; Teng, Maikun; Li, Xu

    2016-01-01

    The N1 methylation of adenine at position 58 (m1A58) of tRNA is an important post-transcriptional modification, which is vital for maintaining the stability of the initiator methionine tRNAiMet. In eukaryotes, this modification is performed by the TRM6-TRM61 holoenzyme. To understand the molecular mechanism that underlies the cooperation of TRM6 and TRM61 in the methyl transfer reaction, we determined the crystal structure of TRM6-TRM61 holoenzyme from Saccharomyces cerevisiae in the presence and absence of its methyl donor S-Adenosyl-L-methionine (SAM). In the structures, two TRM6-TRM61 heterodimers assemble as a heterotetramer. Both TRM6 and TRM61 subunits comprise an N-terminal β-barrel domain linked to a C-terminal Rossmann-fold domain. TRM61 functions as the catalytic subunit, containing a methyl donor (SAM) binding pocket. TRM6 diverges from TRM61, lacking the conserved motifs used for binding SAM. However, TRM6 cooperates with TRM61 forming an L-shaped tRNA binding regions. Collectively, our results provide a structural basis for better understanding the m1A58 modification of tRNA occurred in Saccharomyces cerevisiae. PMID:27582183

  10. On the assembly of dodecameric glutamine synthetase from stable chaperonin complexes.

    Science.gov (United States)

    Fisher, M T

    1993-07-01

    For many in vitro protein-folding reactions, the fraction of correctly folded product declines as the initial protein concentration increases due primarily to misfolding and aggregation reactions. Under optimal conditions and in the presence of ATP, chaperonins (groEL and groES) enhanced the renaturation of dodecameric glutamine synthetase (GS) with yields of active enzyme between 75 and 85% of the original activity (Fisher, M.T. (1992) Biochemistry 31, 3955-3963). In spite of this enhancement, a concentration-dependent decline in recoverable activity was observed when increasing concentrations of unfolded GS were rapidly mixed with renaturation buffer containing a 2-fold molar excess (GS subunits:groEL oligomer) of chaperonins. When a stable groEL-GS complex, formed under optimal conditions, was concentrated 4-fold by centrifugal ultrafiltration prior to ATP addition, the amount of total active GS (percent of the original activity) recovered remained at optimal levels and no longer showed a concentration-dependent decline. The GS subunits that are initially bound and then released from groEL by ATP are assembly-competent. It is proposed that the subunits are no longer able to kinetically equilibrate with folding intermediates that misfold or aggregate. If a stable groEL-protein substrate complex can be amassed without loss of activity, this will facilitate studies on molecular aspects of chaperonin release mechanisms and oligomeric protein assembly. PMID:8100224

  11. Genetic identification of essential indels and domains in carbamoyl phosphate synthetase II of Toxoplasma gondii.

    Science.gov (United States)

    Fox, Barbara A; Ristuccia, Jessica G; Bzik, David J

    2009-04-01

    New treatments need to be developed for the significant human diseases of toxoplasmosis and malaria to circumvent problems with current treatments and drug resistance. Apicomplexan parasites causing these lethal diseases are deficient in pyrimidine salvage, suggesting that selective inhibition of de novo pyrimidine biosynthesis can lead to a severe loss of uridine 5'-monophosphate (UMP) and thymidine 5'-monophosphate (dTMP) pools, thereby inhibiting parasite RNA and DNA synthesis. Disruption of Toxoplasma gondii carbamoyl phosphate synthetase II (CPSII) induces a severe uracil auxotrophy with no detectable parasite replication in vitro and complete attenuation of virulence in mice. Here we show that a CPSII cDNA minigene efficiently complements the uracil auxotrophy of CPSII-deficient mutants, restoring parasite growth and virulence. Our complementation assays reveal that engineered mutations within, or proximal to, the catalytic triad of the N-terminal glutamine amidotransferase (GATase) domain inactivate the complementation activity of T. gondii CPSII and demonstrate a critical dependence on the apicomplexan CPSII GATase domain in vivo. Surprisingly, indels present within the T. gondii CPSII GATase domain as well as the C-terminal allosteric regulatory domain are found to be essential. In addition, several mutations directed at residues implicated in allosteric regulation in Escherichia coli CPS either abolish or markedly suppress complementation and further define the functional importance of the allosteric regulatory region. Collectively, these findings identify novel features of T. gondii CPSII as potential parasite-selective targets for drug development. PMID:18992249

  12. Modulation of phenolic metabolism under stress conditions in a Lotus japonicus mutant lacking plastidic glutamine synthetase.

    Science.gov (United States)

    García-Calderón, Margarita; Pons-Ferrer, Teresa; Mrázova, Anna; Pal'ove-Balang, Peter; Vilková, Mária; Pérez-Delgado, Carmen M; Vega, José M; Eliášová, Adriana; Repčák, Miroslav; Márquez, Antonio J; Betti, Marco

    2015-01-01

    This paper was aimed to investigate the possible implications of the lack of plastidic glutamine synthetase (GS2) in phenolic metabolism during stress responses in the model legume Lotus japonicus. Important changes in the transcriptome were detected in a GS2 mutant called Ljgln2-2, compared to the wild type, in response to two separate stress conditions, such as drought or the result of the impairment of the photorespiratory cycle. Detailed transcriptomic analysis showed that the biosynthesis of phenolic compounds was affected in the mutant plants in these two different types of stress situations. For this reason, the genes and metabolites related to this metabolic route were further investigated using a combined approach of gene expression analysis and metabolite profiling. A high induction of the expression of several genes for the biosynthesis of different branches of the phenolic biosynthetic pathway was detected by qRT-PCR. The extent of induction was always higher in Ljgln2-2, probably reflecting the higher stress levels present in this genotype. This was paralleled by accumulation of several kaempferol and quercetine glycosides, some of them described for the first time in L. japonicus, and of high levels of the isoflavonoid vestitol. The results obtained indicate that the absence of GS2 affects different aspects of phenolic metabolism in L. japonicus plants in response to stress. PMID:26442073

  13. Diversity of Nonribosomal Peptide Synthetases Involved in the Biosynthesis of Lipopeptide Biosurfactants

    Directory of Open Access Journals (Sweden)

    Niran Roongsawang

    2010-12-01

    Full Text Available Lipopeptide biosurfactants (LPBSs consist of a hydrophobic fatty acid portion linked to a hydrophilic peptide chain in the molecule. With their complex and diverse structures, LPBSs exhibit various biological activities including surface activity as well as anti-cellular and anti-enzymatic activities. LPBSs are also involved in multi-cellular behaviors such as swarming motility and biofilm formation. Among the bacterial genera, Bacillus (Gram-positive and Pseudomonas (Gram-negative have received the most attention because they produce a wide range of effective LPBSs that are potentially useful for agricultural, chemical, food, and pharmaceutical industries. The biosynthetic mechanisms and gene regulation systems of LPBSs have been extensively analyzed over the last decade. LPBSs are generally synthesized in a ribosome-independent manner with megaenzymes called nonribosomal peptide synthetases (NRPSs. Production of active‑form NRPSs requires not only transcriptional induction and translation but also post‑translational modification and assemblage. The accumulated knowledge reveals the versatility and evolutionary lineage of the NRPSs system. This review provides an overview of the structural and functional diversity of LPBSs and their different biosynthetic mechanisms in Bacillus and Pseudomonas, including both typical and unique systems. Finally, successful genetic engineering of NRPSs for creating novel lipopeptides is also discussed.

  14. Intestinal acyl-CoA synthetase 5: Activation of long chain fatty acids and behind

    Directory of Open Access Journals (Sweden)

    Christina Klaus

    2013-01-01

    Full Text Available The intestinal mucosa is characterized by a high complexity in terms of structure and functions and allows for a controlled demarcation towards the gut lumen. On the one hand it is responsible for pulping and selective absorption of alimentary substances ensuring the immunological tolerance, on the other hand it prevents the penetration of micro-organisms as well as bacterial outgrowth. The continuous regeneration of surface epithelia along the crypt-villus-axis in the small intestine is crucial to assuring these various functions. The core phenomena of intestinal epithelia regeneration comprise cell proliferation, migration, differentiation, and apoptosis. These partly contrarily oriented processes are molecularly balanced through numerous interacting signaling pathways like Wnt/β-catenin, Notch and Hedgehog, and regulated by various modifying factors. One of these modifiers is acyl-CoA synthetase 5 (ACSL5. It plays a key role in de novo lipid synthesis, fatty acid degradation and membrane modifications, and regulates several intestinal processes, primarily through different variants of protein lipidation, e.g., palmitoylation. ACSL5 was shown to interact with proapoptotic molecules, and besides seems to inhibit proliferation along the crypt-villus-axis. Because of its proapoptotic and antiproliferative characteristics it could be of significant relevance for intestinal homeostasis, cellular disorder and tumor development.

  15. Porcine 2', 5'-oligoadenylate synthetases inhibit Japanese encephalitis virus replication in vitro.

    Science.gov (United States)

    Zheng, Sheng; Zhu, Dan; Lian, Xue; Liu, Weiting; Cao, Ruibing; Chen, Puyan

    2016-05-01

    The 2', 5'-oligoadenylate synthetases (OAS) are antiviral proteins and several isoforms have been identified as flavivirus-resistance biomarkers in human and mouse. The expression kinetics and antiviral functions of porcine OAS family (OAS1, OAS2, and OASL) in PK-15 cells following infection by Japanese encephalitis virus (JEV) were evaluated in the present study. The endogenous expression of the three OAS genes was efficiently induced by IFN-α treatment in PK-15 cells. However, expression of pOAS1 and pOAS2 responded more quickly than pOASL. Infection by JEV also induced the expression of the pOAS isoforms, but at a significantly lower level than that observed following IFN-α stimulation. Transient overexpression of pOASL and pOAS1 inhibited JEV replication more efficiently than OAS2 overexpression. Interestingly, knockdown of pOAS2 expression by siRNA treatment led to the highest increase in JEV multiplication. Co-silencing of RNase L and each pOAS revealed that the anti-JEV function of pOAS1 and pOAS2 were RNase L dependent, while the antiviral activity of pOASL was not. In conclusion, all pOAS isoforms play a significant role in the response to JEV infection, and are differentially induced by different stimuli. The alternative pathways of antiviral activity stimulated by OASL require further study. PMID:26437676

  16. Physiological Studies of Glutamine Synthetases I and III from Synechococcus sp. WH7803 Reveal Differential Regulation.

    Science.gov (United States)

    Domínguez-Martín, María Agustina; Díez, Jesús; García-Fernández, José M

    2016-01-01

    The marine picocyanobacterium Synechococcus sp. WH7803 possesses two glutamine synthetases (GSs; EC 6.3.1.2), GSI encoded by glnA and GSIII encoded by glnN. This is the first work addressing the physiological regulation of both enzymes in a marine cyanobacterial strain. The increase of GS activity upon nitrogen starvation was similar to that found in other model cyanobacteria. However, an unusual response was found when cells were grown under darkness: the GS activity was unaffected, reflecting adaptation to the environment where they thrive. On the other hand, we found that GSIII did not respond to nitrogen availability, in sharp contrast with the results observed for this enzyme in other cyanobacteria thus far studied. These features suggest that GS activities in Synechococcus sp. WH7803 represent an intermediate step in the evolution of cyanobacteria, in a process of regulatory streamlining where GSI lost the regulation by light, while GSIII lost its responsiveness to nitrogen. This is in good agreement with the phylogeny of Synechococcus sp. WH7803 in the context of the marine cyanobacterial radiation. PMID:27446010

  17. Glutamine synthetase in Durum Wheat: Genotypic Variation and Relationship with Grain Protein Content.

    Science.gov (United States)

    Nigro, Domenica; Fortunato, Stefania; Giove, Stefania L; Paradiso, Annalisa; Gu, Yong Q; Blanco, Antonio; de Pinto, Maria C; Gadaleta, Agata

    2016-01-01

    Grain protein content (GPC), is one of the most important trait in wheat and its characterized by a very complex genetic control. The identification of wheat varieties with high GPC (HGPC), as well as the characterization of central enzymes involved in these processes, are important for more sustainable agricultural practices. In this study, we focused on Glutamine synthetase (GS) as a candidate to study GPC in wheat. We analyzed GS expression and its enzymatic activity in different tissues and phenological stages in 10 durum wheat genotypes with different GPC. Although each genotype performed quite differently from the others, both because their genetic variability and their adaptability to specific environmental conditions, the highest GS activity and expression were found in genotypes with HGPC and vice versa the lowest ones in genotypes with low GPC (LGPC). Moreover, in genotypes contrasting in GPC bred at different nitrogen regimes (0, 60, 140 N Unit/ha) GS behaved differently in diverse organs. Nitrogen supplement increased GS expression and activity in roots of all genotypes, highlighting the key role of this enzyme in nitrogen assimilation and ammonium detoxification in roots. Otherwise, nitrogen treatments decreased GS expression and activity in the leaves of HGPC genotypes and did not affect GS in the leaves of LGPC genotypes. Finally, no changes in GS and soluble protein content occurred at the filling stage in the caryopses of all analyzed genotypes. PMID:27468287

  18. An Arabidopsis embryonic lethal mutant with reduced expression of alanyl—t RNA synthetase gene

    Institute of Scientific and Technical Information of China (English)

    SUNJIANGE; XIAOLIYAO; 等

    1998-01-01

    In present paper,one of the T-DNA insertional embryonic lethal mutant of Arabidopsis is identified and designated as acd mutant.The embryo developmant of this mutant is arrested in globular stage,The cell division pattern is abnormal during early embryogenesis and results in distubed cellular differentiation.Most of mutant embryos are finally degenerated and aborted in globular stage,However,a few of them still can germinate in agar palte and produce seedlings with shoter hypoctyl and distorted shoot meristem.To understand the molecular basis of the phenotype of this mutant,the joint fragment of T-DNA/plant DNA is isolated by plasmid rescue and Dig-labeled as probe for cDNA library screening.According to the sequence analysis and similarity searching,a 936 bp cDNA sequence(EMBL accession #:Y12555)from selectoed positive clone shows a 99.8%(923/925bp) sequence homolgy with Alanyl-tRNA Synthetase(AlaRS) gene of Arabidopsis thaliana.Furthermore,the data of in situ hybridization experiment indicate that the expression of Ala RS gene is weak in early embryogenesis and declines along with globular embryodevelopment in this mutant Accordingly,the reduced expression of Ala RS gene may be closely related to the morphological changes in early embryogenesis of this lethal mutant.

  19. A plant malonyl-CoA synthetase enhances lipid content and polyketide yield in yeast cells.

    Science.gov (United States)

    Wang, Yechun; Chen, Hui; Yu, Oliver

    2014-06-01

    Malonyl-CoA is the essential building block of natural products such as fatty acids, polyketides, and flavonoids. Engineering the biosynthesis of fatty acids is important for biofuel production while that of polyketides provides precursors of medicines and nutritional supplements. However, microorganisms maintain a small amount of cellular malonyl-CoA, which could limit production of lipid and polyketides under certain conditions. Malonyl-CoA concentration is regulated by multiple pathways and signals, and changes in intracellular malonyl-CoA often lead to complex alterations in metabolism. In the present work, overexpression of a plant malonyl-CoA synthetase gene (AAE13) in Saccharomyces cerevisiae resulted in 1.6- and 2.4-fold increases in lipid and resveratrol accumulation simultaneously. We also demonstrated that AAE13 partially complemented the temperature-sensitive acc1 mutant, replacing this key enzyme in central metabolism. Mechanistic analysis by CoA quantification and transcriptomic measurement suggested that increases in malonyl-CoA concentration were coupled with drastic reductions in other major CoA compounds and clear suppression of tricarboxylic acid cycle-related genes. These results suggest that malonyl-CoA is a critical target for fatty acid and polyketide engineering and that overexpression of malonyl-CoA synthetic enzymes needs to be combined with upregulation of CoA synthesis to maintain metastasis of central metabolism. PMID:24682482

  20. Role of tRNAPro in pretransfer editing of alanine by prolyl-tRNA synthetase

    Directory of Open Access Journals (Sweden)

    Boyarshin K. S.

    2013-09-01

    Full Text Available Aim. To characterize the process of tRNA-dependent pretransfer edi- ting of alanine by prolyl-tRNA synthetase of bacteria Enterococcus faecalis (ProRSEf. Methods. Velocity of the editing processes in vitro was determined by ATP hydrolysis by ProRSEf. Pretransfer and posttransfer editing were experimentally separated by site-directed mutagenesis. Results. tRNA-dependent pretransfer editing is characterized by three-fold larger velocity then tRNA-independent editing. Effectivity of the process depends on the presence of 2'-hydroxyle group of A76 tRNAPro. In the absence of tRNAPro selective release of alanyl-AMP occurs simultaneously with tRNA-independent pretransfer editing. Released alanyl-AMP can be re-bound and hydrolyzed. Conclusions. tRNA-dependent pretransfer editing of alanine by ProRSEf is the catalytic mechanism, mediated by 2'-hydroxyl group of A76 tRNAPro. In the absence of tRNAPro tRNA-independent pretransfer editing and selective release of alanyl-AMP occur.