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Sample records for human trna synthetase

  1. Gain-Of-Function Mutational Activation of Human TRNA Synthetase Procytokine

    SciTech Connect

    Yang, X.L.; Kapoor, M.; Otero, F.J.; Slike, B.M.; Tsuruta, H.; Frausto, R.; Bates, A.; Ewalt, K.L.; Cheresh, D.A.; Schimmel, P.; /Scripps Res. Inst. /SLAC, SSRL

    2009-04-30

    Disease-causing mutations occur in genes for aminoacyl tRNA synthetases. That some mutations are dominant suggests a gain of function. Native tRNA synthetases, such as tyrosyl-tRNA synthetase (TyrRS) and tryptophanyl-tRNA synthetase, catalyze aminoacylation and are also procytokines that are activated by natural fragmentation. In principle, however, gain-of-function phenotypes could arise from mutational activation of synthetase procytokines. From crystal structure analysis, we hypothesized that a steric block of a critical Glu-Leu-Arg (ELR) motif in full-length TyrRS suppresses the cytokine activity of a natural fragment. To test this hypothesis, we attempted to uncover ELR in the procytokine by mutating a conserved tyrosine (Y341) that tethers ELR. Site-specific proteolytic cleavage and small-angle X-ray scattering established subtle opening of the structure by the mutation. Strikingly, four different assays demonstrated mutational activation of cytokine functions. The results prove the possibilities for constitutive gain-of-function mutations in tRNA synthetases.

  2. Complex organisation of the 5'-end of the human glycine tRNA synthetase gene.

    PubMed

    Mudge, S J; Williams, J H; Eyre, H J; Sutherland, G R; Cowan, P J; Power, D A

    1998-03-16

    Glycine tRNA synthetase (glyRS) catalyses the addition of the amino acid glycine to its cognate tRNA molecules. In the silk moth worm Bombyx mori, this gene is subject to complex transcriptional regulation because of the predominance of glycine in silk. In vertebrates, glycine is a major constituent of collagen but there have been no studies of glyRS regulation. In this study we have isolated and mapped a genomic clone containing the 5'-end of glyRS. Primer extension studies identified only one transcriptional start point (TSP) in three different cell lines. Expression of the transcript identified may be regulated translationally because it contains five potential initiation codons, three of which are in good context for initiation. The most 3' of the potential initiation codons has previously been predicted to be the initiating codon for cytoplasmic glyRS. Two of the upstream codons are in-frame with this codon, and both are predicted to extend the N-terminus of glyRS to include a mitochondrial targeting sequence. Sequencing of genomic DNA surrounding the TSP showed features common to the promoters of housekeeping genes, as well as a canonical TATA box at the unusual position of +9. Surprisingly, promoter activity in vitro was not specified by a 1.9 kb genomic fragment containing the TSP and TATA box, but by a contiguous 0.4 kb fragment immediately downstream. These studies suggest that the transcription of glyRS from a single start point requires downstream promoter elements.

  3. The isolated carboxy-terminal domain of human mitochondrial leucyl-tRNA synthetase rescues the pathological phenotype of mitochondrial tRNA mutations in human cells

    PubMed Central

    Perli, Elena; Giordano, Carla; Pisano, Annalinda; Montanari, Arianna; Campese, Antonio F; Reyes, Aurelio; Ghezzi, Daniele; Nasca, Alessia; Tuppen, Helen A; Orlandi, Maurizia; Di Micco, Patrizio; Poser, Elena; Taylor, Robert W; Colotti, Gianni; Francisci, Silvia; Morea, Veronica; Frontali, Laura; Zeviani, Massimo; d'Amati, Giulia

    2014-01-01

    Mitochondrial (mt) diseases are multisystem disorders due to mutations in nuclear or mtDNA genes. Among the latter, more than 50% are located in transfer RNA (tRNA) genes and are responsible for a wide range of syndromes, for which no effective treatment is available at present. We show that three human mt aminoacyl-tRNA syntethases, namely leucyl-, valyl-, and isoleucyl-tRNA synthetase are able to improve both viability and bioenergetic proficiency of human transmitochondrial cybrid cells carrying pathogenic mutations in the mt-tRNAIle gene. Importantly, we further demonstrate that the carboxy-terminal domain of human mt leucyl-tRNA synthetase is both necessary and sufficient to improve the pathologic phenotype associated either with these “mild” mutations or with the “severe” m.3243A>G mutation in the mt-tRNALeu(UUR) gene. Furthermore, we provide evidence that this small, non-catalytic domain is able to directly and specifically interact in vitro with human mt-tRNALeu(UUR) with high affinity and stability and, with lower affinity, with mt-tRNAIle. Taken together, our results sustain the hypothesis that the carboxy-terminal domain of human mt leucyl-tRNA synthetase can be used to correct mt dysfunctions caused by mt-tRNA mutations. PMID:24413190

  4. Long-Range Structural Effects of a Charcot-Marie-Tooth Disease-Causing Mutation in Human Glycyl-TRNA Synthetase

    SciTech Connect

    Xie, W.; Nangle, L.A.; Zhang, W.; Schimmel, P.; Yang, X.-L.

    2009-06-04

    Functional expansion of specific tRNA synthetases in higher organisms is well documented. These additional functions may explain why dominant mutations in glycyl-tRNA synthetase (GlyRS) and tyrosyl-tRNA synthetase cause Charcot-Marie-Tooth (CMT) disease, the most common heritable disease of the peripheral nervous system. At least 10 disease-causing mutant alleles of GlyRS have been annotated. These mutations scatter broadly across the primary sequence and have no apparent unifying connection. Here we report the structure of wild type and a CMT-causing mutant (G526R) of homodimeric human GlyRS. The mutation is at the site for synthesis of glycyl-adenylate, but the rest of the two structures are closely similar. Significantly, the mutant form diffracts to a higher resolution and has a greater dimer interface. The extra dimer interactions are located {approx}30 {angstrom} away from the G526R mutation. Direct experiments confirm the tighter dimer interaction of the G526R protein. The results suggest the possible importance of subtle, long-range structural effects of CMT-causing mutations at the dimer interface. From analysis of a third crystal, an appended motif, found in higher eukaryote GlyRSs, seems not to have a role in these long-range effects.

  5. Aminoacyl tRNA synthetases and their connections to disease.

    PubMed

    Park, Sang Gyu; Schimmel, Paul; Kim, Sunghoon

    2008-08-12

    Aminoacylation of transfer RNAs establishes the rules of the genetic code. The reactions are catalyzed by an ancient group of 20 enzymes (one for each amino acid) known as aminoacyl tRNA synthetases (AARSs). Surprisingly, the etiology of specific diseases-including cancer, neuronal pathologies, autoimmune disorders, and disrupted metabolic conditions-is connected to specific aminoacyl tRNA synthetases. These connections include heritable mutations in the genes for tRNA synthetases that are causally linked to disease, with both dominant and recessive disease-causing mutations being annotated. Because some disease-causing mutations do not affect aminoacylation activity or apparent enzyme stability, the mutations are believed to affect functions that are distinct from aminoacylation. Examples include enzymes that are secreted as procytokines that, after activation, operate in pathways connected to the immune system or angiogenesis. In addition, within cells, synthetases form multiprotein complexes with each other or with other regulatory factors and in that way control diverse signaling pathways. Although much has been uncovered in recent years, many novel functions, disease connections, and interpathway connections of tRNA synthetases have yet to be worked out.

  6. Crystallization and preliminary X-ray analysis of a native human tRNA synthetase whose allelic variants are associated with Charcot–Marie–Tooth disease

    SciTech Connect

    Xie, Wei; Schimmel, Paul; Yang, Xiang-Lei

    2006-12-01

    Crystallization and preliminary X-ray analysis of a native human tRNA synthetase whose allelic variants are associated with Charcot–Marie–Tooth Disease. Glycyl-tRNA synthetase (GlyRS) is one of a group of enzymes that catalyze the synthesis of aminoacyl-tRNAs for translation. Mutations of human and mouse GlyRSs are causally associated with Charcot–Marie–Tooth disease, the most common genetic disorder of the peripheral nervous system. As the first step towards a structure–function analysis of this disease, native human GlyRS was expressed, purified and crystallized. The crystal belonged to space group P4{sub 3}2{sub 1}2 or its enantiomorphic space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 91.74, c = 247.18 Å, and diffracted X-rays to 3.0 Å resolution. The asymmetric unit contained one GlyRS molecule and had a solvent content of 69%.

  7. A prokaryote and human tRNA synthetase provide an essential RNA splicing function in yeast mitochondria

    PubMed Central

    Houman, Fariba; Rho, Seung Bae; Zhang, Jiansu; Shen, Xiaoyu; Wang, Chien-Chia; Schimmel, Paul; Martinis, Susan A.

    2000-01-01

    Mitochondrial leucyl-tRNA synthetase (LeuRS) in the yeast Saccharomyces cerevisiae provides two essential functions. In addition to aminoacylation, LeuRS functions in RNA splicing. The details of how it came to act in splicing are not known. Here we show that Mycobacterium tuberculosis and human mitochondrial LeuRSs can substitute in splicing for the S. cerevisiae mitochondrial LeuRS. Mutations of yeast mitochondrial LeuRS that had previously been shown to abolish splicing activity also eliminate splicing by the M. tuberculosis enzyme. These results suggest the role of LeuRS in splicing in yeast mitochondria results from features of the enzyme that are broadly conserved in evolution. These features are not likely to be designed for splicing per se, but instead have been adopted in yeast for that purpose. PMID:11087829

  8. Aminoacyl tRNA Synthetase Deficiency Promotes Angiogenesis via the Unfolded Protein Response Pathway

    PubMed Central

    Castranova, Daniel; Davis, Andrew E.; Lo, Brigid D.; Miller, Mayumi F.; Paukstelis, Paul J.; Swift, Matthew R.; Pham, Van N.; Torres-Vázquez, Jesús; Bell, Kameha; Shaw, Kenna M.; Kamei, Makoto; Weinstein, Brant M.

    2016-01-01

    Objective Understanding the mechanisms regulating normal and pathologic angiogenesis is of great scientific and clinical interest. In this report, we show that mutations in two different aminoacyl tRNA synthetases, threonyl tRNA synthetase (tarsy58) or isoleucyl tRNA synthetase (iarsy68), lead to similar increased branching angiogenesis in developing zebrafish. Approach and Results The Unfolded Protein Response (UPR) pathway is activated by aminoacyl tRNA synthetase deficiencies, and we show that UPR genes atf4, atf6, and xbp1, as well as the key pro-angiogenic ligand vascular endothelial growth factor (vegfaa), are all up-regulated in tarsy58 and iarsy68 mutants. Finally, we show that the PERK-ATF4 arm of the UPR pathway is necessary for both the elevated vegfaa levels and increased angiogenesis observed in tarsy58 mutants. Conclusions Our results suggest that endoplasmic reticulum (ER) stress acts as a pro-angiogenic signal via UPR pathway-dependent up-regulation of vegfaa. PMID:26821951

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

    PubMed Central

    Alriyami, Maha Z.; Jones, Martin R.; Johnsen, Robert C.; Banerjee, Yajnavalka; Baillie, David L.

    2014-01-01

    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. PMID:25606464

  10. Unique domain appended to vertebrate tRNA synthetase is essential for vascular development

    PubMed Central

    Xu, Xiaoling; Shi, Yi; Zhang, Hui-Min; Swindell, Eric C.; Marshall, Alan G.; Guo, Min; Kishi, Shuji; Yang, Xiang-Lei

    2012-01-01

    New domains were progressively added to cytoplasmic aminoacyl transfer RNA (tRNA) synthetases during evolution. One example is the UNE-S domain, appended to seryl-tRNA synthetase (SerRS) in species that developed closed circulatory systems. Here we show using solution and crystal structure analyses and in vitro and in vivo functional studies that UNE-S harbours a robust nuclear localization signal (NLS) directing SerRS to the nucleus where it attenuates vascular endothelial growth factor A expression. We also show that SerRS mutants previously linked to vasculature abnormalities either deleted the NLS or have the NLS sequestered in an alternative conformation. A structure-based second-site mutation, designed to release the sequestered NLS, restored normal vasculature. Thus, the essential function of SerRS in vascular development depends on UNE-S. These results are the first to show an essential role for a tRNA synthetase-associated appended domain at the organism level, and suggest that acquisition of UNE-S has a role in the establishment of the closed circulatory systems of vertebrates. PMID:22353712

  11. Expanding tRNA recognition of a tRNA synthetase by a single amino acid change

    PubMed Central

    Feng, Liang; Tumbula-Hansen, Debra; Toogood, Helen; Söll, Dieter

    2003-01-01

    Aspartyl-tRNA synthetase (AspRS) occurs in two types: the discriminating enzyme (D-AspRS) forms only Asp-tRNAAsp, whereas the nondiscriminating enzyme (ND-AspRS) also synthesizes Asp-tRNAAsn, which is a required intermediate for protein synthesis in many organisms. We attempted to expand the tRNA recognition of the discriminating Thermococcus kodakaraensis AspRS to that of a ND-AspRS by in vitro mutagenesis. An alignment of 26 archaeal AspRS proteins revealed two positions (26 and 85 in the T. kodakaraensis sequence) whose amino acid identity changes according to the enzymes' tRNA specificity. In their anticodon-binding domain, D-AspRS proteins contain W26 (or Q26) and K85, compared with H26 and P85 in the ND-AspRSs. T. kodakaraensis AspRS gained the ability to form Asp-tRNAAsn in vitro when the W26H or K85P changes were introduced independently or in combination. In the aminoacylation of tRNAAsn or tRNAAsp transcripts, the mutant enzymes displayed at least a 100- to 500-fold change in tRNA specificity, as judged by the ratio of the kcat/Km values of Asp-tRNAAsp vs. Asp-tRNAAsn formation. That T. kodakaraensis mutant AspRSs mischarge tRNAAsn was also manifested in the higher level (1.7%) of aspartylation of unfractionated Pyrococcus tRNA compared with that achieved by the wild-type enzyme (0.9%). Northern blot analysis of the Asp-tRNA separated by acid/urea gel electrophoresis confirmed the in vitro synthesis of Asp-tRNAAsn. A structure-based model points to a direct interaction of K85 in T. kodakaraensis AspRS with the anticodon nucleotide C36 of tRNAAsp. Thus, a switch between D-AspRS and ND-AspRS enzymes could have evolved with only limited amino acid changes. PMID:12730374

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

    PubMed

    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

    2016-07-08

    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.

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

    PubMed Central

    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

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

    PubMed Central

    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 been elucidated, limiting our understanding of which genes harbor variants causing mitochondrial–nuclear disruption and of the pathways and processes that are impacted by mitochondrial–nuclear coevolution. Here we identify an amino acid polymorphism in the Drosophila melanogaster nuclear-encoded mitochondrial tyrosyl–tRNA synthetase that interacts epistatically with a polymorphism in the D. simulans mitochondrial-encoded tRNATyr to significantly delay development, compromise bristle formation, and decrease fecundity. The incompatible genotype specifically decreases the activities of oxidative phosphorylation complexes I, III, and IV that contain mitochondrial-encoded subunits. Combined with the identity of the interacting alleles, this pattern indicates that mitochondrial protein translation is affected by this interaction. Our findings suggest that interactions between mitochondrial tRNAs and their nuclear-encoded tRNA synthetases may be targets of compensatory molecular evolution. Human mitochondrial diseases are often genetically complex and variable in penetrance, and the mitochondrial–nuclear interaction we document provides a plausible mechanism to explain this complexity. PMID:23382693

  15. A dispensable peptide from Acidithiobacillus ferrooxidans tryptophanyl-tRNA synthetase affects tRNA binding.

    PubMed

    Zúñiga, Roberto; Salazar, Juan; Canales, Mauricio; Orellana, Omar

    2002-12-18

    The activation domain of class I aminoacyl-tRNA synthetases, which contains the Rossmann fold and the signature sequences HIGH and KMSKS, is generally split into two halves by the connective peptides (CP1, CP2) whose amino acid sequences are idiosyncratic. CP1 has been shown to participate in the binding of tRNA as well as the editing of the reaction intermediate aminoacyl-AMP or the aminoacyl-tRNA. No function has been assigned to CP2. The amino acid sequence of Acidithiobacillus ferrooxidans TrpRS was predicted from the genome sequence. Protein sequence alignments revealed that A. ferrooxidans TrpRS contains a 70 amino acids long CP2 that is not found in any other bacterial TrpRS. However, a CP2 in the same relative position was found in the predicted sequence of several archaeal TrpRSs. A. ferrooxidans TrpRS is functional in vivo in Escherichia coli. A deletion mutant of A. ferrooxidans trpS lacking the coding region of CP2 was constructed. The in vivo activity of the mutant TrpRS in E. coli, as well as the kinetic parameters of the in vitro activation of tryptophan by ATP, were not altered by the deletion. However, the K(m) value for tRNA was seven-fold higher upon deletion, reducing the efficiency of aminoacylation. Structural modeling suggests that CP2 binds to the inner corner of the L shape of tRNA.

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

    PubMed

    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.

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

    PubMed Central

    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 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 simw501 mtDNA-encoded transfer RNA (tRNA) for tyrosine (tRNATyr) 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 simw501 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 tRNATyr 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. Structure and Activity of an Aminoacyl-tRNA Synthetase that Charges tRNA with Nitro-Tryptophan

    SciTech Connect

    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.

  19. Dynamics of the Active Sites of Dimeric Seryl tRNA Synthetase from Methanopyrus kandleri.

    PubMed

    Dutta, Saheb; Nandi, Nilashis

    2015-08-27

    Aminoacyl tRNA synthetases (aaRSs) carry out the first step of protein biosynthesis. Several aaRSs are multimeric, and coordination between the dynamics of active sites present in each monomer is a prerequisite for the fast and accurate aminoacylation. However, important lacunae of understanding exist concerning the conformational dynamics of multimeric aaRSs. Questions remained unanswered pertaining to the dynamics of the active site. Little is known concerning the conformational dynamics of the active sites in response to the substrate binding, reorganization of the catalytic residues around reactants, time-dependent changes at the reaction center, which are essential for facilitating the nucleophilic attack, and interactions at the interface of neighboring monomers. In the present work, we carried out all-atom molecular dynamics simulation of dimeric (mk)SerRS from Methanopyrus kandleri bound with tRNA using an explicit solvent system. Two dimeric states of seryl tRNA synthetase (open, substrate bound, and adenylate bound) and two monomeric states (open and substrate bound) are simulated with bound tRNA. The aim is to understand the conformational dynamics of (mk)SerRS during its reaction cycle. While the present results provide a clear dynamical perspective of the active sites of (mk)SerRS, they corroborate with the results from the time-averaged experimental data such as crystallographic and mutation analysis of methanogenic SerRS from M. kandleri and M. barkeri. It is observed from the present simulation that the motif 2 loop gates the active site and its Glu351 and Arg360 stabilizes ATP in a bent state favorable for nucleophilic attack. The flexibility of the walls of the active site gradually reduces near reaction center, which is a more organized region compared to the lid region. The motif 2 loop anchors Ser and ATP using Arg349 in a hydrogen bonded geometry crucial for nucleophilic attack and favorably influences the electrostatic potential at the

  20. In silico detection of tRNA sequence features characteristic to aminoacyl-tRNA synthetase class membership

    PubMed Central

    Jakó, Éena; Ittzés, Péter; Szenes, Áron; Kun, Ádám; Szathmáry, Eörs; Pál, Gábor

    2007-01-01

    Aminoacyl tRNA synthetases (aaRS) are grouped into Class I and II based on primary and tertiary structure and enzyme properties suggesting two independent phylogenetic lineages. Analogously, tRNA molecules can also form two respective classes, based on the class membership of their corresponding aaRS. Although some aaRS–tRNA interactions are not extremely specific and require editing mechanisms to avoid misaminoacylation, most aaRS–tRNA interactions are rather stereospecific. Thus, class-specific aaRS features could be mirrored by class-specific tRNA features. However, previous investigations failed to detect conserved class-specific nucleotides. Here we introduce a discrete mathematical approach that evaluates not only class-specific ‘strictly present’, but also ‘strictly absent’ nucleotides. The disjoint subsets of these elements compose a unique partition, named extended consensus partition (ECP). By analyzing the ECP for both Class I and II tDNA sets from 50 (13 archaeal, 30 bacterial and 7 eukaryotic) species, we could demonstrate that class-specific tRNA sequence features do exist, although not in terms of strictly conserved nucleotides as it had previously been anticipated. This finding demonstrates that important information was hidden in tRNA sequences inaccessible for traditional statistical methods. The ECP analysis might contribute to the understanding of tRNA evolution and could enrich the sequence analysis tool repertoire. PMID:17704131

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

    PubMed Central

    Li, Rongzhong; Macnamara, Lindsay M.; Leuchter, Jessica D.; Alexander, Rebecca W.; Cho, Samuel S.

    2015-01-01

    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. PMID:26184179

  2. No rosetta stone for a sense-antisense origin of aminoacyl tRNA synthetase classes.

    PubMed

    Williams, Tom A; Wolfe, Kenneth H; Fares, Mario A

    2009-02-01

    Aminoacyl tRNA synthetases (aaRS) are crucial enzymes that join amino acids to their cognate tRNAs, thereby implementing the genetic code. These enzymes fall into two unrelated structural classes whose evolution has not been explained. The leading hypothesis, proposed by Rodin and Ohno, is that the two classes originated as a pair of sense-antisense genes encoded on opposite strands of a single DNA molecule. This unusual idea obtained its main support from reports of a "Rosetta stone": a locus where genes for heat shock protein 70 (HSP70) and an Nicotinamide adenine dinulecotide-specific glutamate dehydrogenase (NAD-GDH), which are structurally homologous to the two classes of aaRS, overlap extensively on complementary DNA strands. This remarkable locus was first characterized in the oomycete Achlya klebsiana and has since been reported in many other species. Here we present evidence that the open reading frames on the antisense strand of HSP70 genes are spurious, and we identify a more probable candidate for the gene encoding the oomycete NAD-GDH enzyme. These results cast extensive doubt on the Rosetta Stone argument.

  3. The RNA sequence context defines the mechanistic routes by which yeast arginyl-tRNA synthetase charges tRNA.

    PubMed Central

    Sissler, M; Giegé, R; Florentz, C

    1998-01-01

    Arginylation of tRNA transcripts by yeast arginyl-tRNA synthetase can be triggered by two alternate recognition sets in anticodon loops: C35 and U36 or G36 in tRNA(Arg) and C36 and G37 in tRNA(Asp) (Sissler M, Giegé R, Florentz C, 1996, EMBO J 15:5069-5076). Kinetic studies on tRNA variants were done to explore the mechanisms by which these sets are expressed. Although the synthetase interacts in a similar manner with tRNA(Arg) and tRNA(Asp), the details of the interaction patterns are idiosyncratic, especially in anticodon loops (Sissler M, Eriani G, Martin F, Giegé R, Florentz C, 1997, Nucleic Acids Res 25:4899-4906). Exchange of individual recognition elements between arginine and aspartate tRNA frameworks strongly blocks arginylation of the mutated tRNAs, whereas full exchange of the recognition sets leads to efficient arginine acceptance of the transplanted tRNAs. Unpredictably, the similar catalytic efficiencies of native and transplanted tRNAs originate from different k(cat) and Km combinations. A closer analysis reveals that efficient arginylation results from strong anticooperative effects between individual recognition elements. Nonrecognition nucleotides as well as the tRNA architecture are additional factors that tune efficiency. Altogether, arginyl-tRNA synthetase is able to utilize different context-dependent mechanistic routes to be activated. This confers biological advantages to the arginine aminoacylation system and sheds light on its evolutionary relationship with the aspartate system. PMID:9622124

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

    PubMed Central

    Fang, Pengfei; Guo, Min

    2015-01-01

    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. PMID:26670257

  5. A case of anti-aminoacyl tRNA synthetase antibody syndrome complicated by hemophagocytic syndrome.

    PubMed

    Azuma, Kota; Tamura, Masao; Kurajoh, Masafumi; Hosono, Yuji; Nakajima, Ran; Tsuboi, Kazuyuki; Abe, Takeo; Ogita, Chie; Yokoyama, Yuichi; Furukawa, Tetsuya; Yoshikawa, Takahiro; Saito, Atsushi; Nishioka, Aki; Sekiguchi, Masahiro; Azuma, Naoto; Kitano, Masayasu; Tsunoda, Shinichiro; Omura, Koichiro; Koyama, Hidenori; Matsui, Kiyoshi; Mimori, Tsuneyo; Sano, Hajime

    2016-01-01

      A 48-year-old woman had suffered from a fever and general fatigue, and visited the other hospital for fever elevation in November 2013, at which time interstitial lung disease was revealed. In January 2014, she experienced an eruption in the hand and developed peripheral blood flow damage. Under a diagnosis of adult Still's disease, the patient was administered 0.5 mg of betamethasone as well as cyclosporin at 75 mg/day. In November 2014, general fatigue, fever, and headache were noted, while MRI revealed an enlarged hypophysis and laboratory findings were positive for the anti-pituitary cell antibody, thus a diagnosis of autoimmune hypophysitis was made. Although disease activity was low, she requested hospitalization and was admitted by the Division of Endocrinology and Metabolism at our hospital in May 2015, though only observed. Fever developed again, along with interstitial lung disease, Raynaud's phenomenon, and pain in the crural area again, and we considered the possibility of another disease. After stopping administration of betamethasone and cyclosporin, we made a diagnosis of anti-aminoacyl tRNA synthetase antibody syndrome, and administered methylprednisolone at 500 mg for 3 days as well as prednisolone at 35 mg/day following steroid pulse therapy. Although her condition soon improved, fever, muscle pain, and pancytopenia returned after 3 days. Bone marrow findings revealed the existence of hemophagocytosis, for which we again gave methylprednisolone at 500 mg for 3 days and cyclosporin at 125 mg/day. Thereafter, the patient recovered and was discharged from the hospital.

  6. Radioimmune assay of human platelet prostaglandin synthetase

    SciTech Connect

    Roth, G.J.; Machuga, E.T.

    1982-02-01

    Normal platelet function depends, in part, on platelet PG synthesis. PG synthetase (cyclo-oxygenase) catalyzes the first step in PG synthesis, the formation of PGH/sub 2/ 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 (/sup 125/I)-labelled sheep PG synthetase as antigen. The human platelet enzyme is assayed by its ability to inhibit precipitation of the (/sup 125/I)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 10/sup 9/ 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.

  7. Introduction of a leucine half-zipper engenders multiple high-quality crystals of a recalcitrant tRNA synthetase

    SciTech Connect

    Guo, Min; Shapiro, Ryan; Schimmel, Paul; Yang, Xiang-Lei

    2010-03-01

    E. coli alanyl-tRNA synthetase is recalcitrant to crystallization. A group of leucine substitutions has transformed the protein. Although Escherichia coli alanyl-tRNA synthetase was among the first tRNA synthetases to be sequenced and extensively studied by functional analysis, it has proved to be recalcitrant to crystallization. This challenge remained even for crystallization of the catalytic fragment. By mutationally introducing three stacked leucines onto the solvent-exposed side of an α-helix, an engineered catalytic fragment of the synthetase was obtained that yielded multiple high-quality crystals and cocrystals with different ligands. The engineered α-helix did not form a leucine zipper that interlocked with the same α-helix from another molecule. Instead, using the created hydrophobic spine, it interacted with other surfaces of the protein as a leucine half-zipper (LHZ) to enhance the crystal lattice interactions. The LHZ made crystal lattice contacts in all crystals of different space groups. These results illustrate the power of introducing an LHZ into helices to facilitate crystallization. The authors propose that the method can be unified with surface-entropy reduction and can be broadly used for protein-surface optimization in crystallization.

  8. Modeling of tRNA-assisted mechanism of Arg activation based on a structure of Arg-tRNA synthetase, tRNA, and an ATP analog (ANP).

    PubMed

    Konno, Michiko; Sumida, Tomomi; Uchikawa, Emiko; Mori, Yukie; Yanagisawa, Tatsuo; Sekine, Shun-ichi; Yokoyama, Shigeyuki; Yokoyama, Shigeuki

    2009-09-01

    The ATP-pyrophosphate exchange reaction catalyzed by Arg-tRNA, Gln-tRNA and Glu-tRNA synthetases requires the assistance of the cognate tRNA. tRNA also assists Arg-tRNA synthetase in catalyzing the pyrophosphorolysis of synthetic Arg-AMP at low pH. The mechanism by which the 3'-end A76, and in particular its hydroxyl group, of the cognate tRNA is involved with the exchange reaction catalyzed by those enzymes has yet to be established. We determined a crystal structure of a complex of Arg-tRNA synthetase from Pyrococcus horikoshii, tRNA(Arg)(CCU) and an ATP analog with Rfactor = 0.213 (Rfree = 0.253) at 2.0 A resolution. On the basis of newly obtained structural information about the position of ATP bound on the enzyme, we constructed a structural model for a mechanism in which the formation of a hydrogen bond between the 2'-OH group of A76 of tRNA and the carboxyl group of Arg induces both formation of Arg-AMP (Arg + ATP --> Arg-AMP + pyrophosphate) and pyrophosphorolysis of Arg-AMP (Arg-AMP + pyrophosphate --> Arg + ATP) at low pH. Furthermore, we obtained a structural model of the molecular mechanism for the Arg-tRNA synthetase-catalyzed deacylation of Arg-tRNA (Arg-tRNA + AMP --> Arg-AMP + tRNA at high pH), in which the deacylation of aminoacyl-tRNA bound on Arg-tRNA synthetase and Glu-tRNA synthetase is catalyzed by a quite similar mechanism, whereby the proton-donating group (-NH-C+(NH2)2 or -COOH) of Arg and Glu assists the aminoacyl transfer from the 2'-OH group of tRNA to the phosphate group of AMP at high pH.

  9. Origin and evolution of glutamyl-prolyl tRNA synthetase WHEP domains reveal evolutionary relationships within Holozoa.

    PubMed

    Ray, Partho Sarothi; Fox, Paul L

    2014-01-01

    Repeated domains in proteins that have undergone duplication or loss, and sequence divergence, are especially informative about phylogenetic relationships. We have exploited divergent repeats of the highly structured, 50-amino acid WHEP domains that join the catalytic subunits of bifunctional glutamyl-prolyl tRNA synthetase (EPRS) as a sequence-informed repeat (SIR) to trace the origin and evolution of EPRS in holozoa. EPRS is the only fused tRNA synthetase, with two distinct aminoacylation activities, and a non-canonical translation regulatory function mediated by the WHEP domains in the linker. Investigating the duplications, deletions and divergence of WHEP domains, we traced the bifunctional EPRS to choanozoans and identified the fusion event leading to its origin at the divergence of ichthyosporea and emergence of filozoa nearly a billion years ago. Distribution of WHEP domains from a single species in two or more distinct clades suggested common descent, allowing the identification of linking organisms. The discrete assortment of choanoflagellate WHEP domains with choanozoan domains as well as with those in metazoans supported the phylogenetic position of choanoflagellates as the closest sister group to metazoans. Analysis of clustering and assortment of WHEP domains provided unexpected insights into phylogenetic relationships amongst holozoan taxa. Furthermore, observed gaps in the transition between WHEP domain groupings in distant taxa allowed the prediction of undiscovered or extinct evolutionary intermediates. Analysis based on SIR domains can provide a phylogenetic counterpart to palaentological approaches of discovering "missing links" in the tree of life.

  10. Origin and Evolution of Glutamyl-prolyl tRNA Synthetase WHEP Domains Reveal Evolutionary Relationships within Holozoa

    PubMed Central

    Ray, Partho Sarothi; Fox, Paul L.

    2014-01-01

    Repeated domains in proteins that have undergone duplication or loss, and sequence divergence, are especially informative about phylogenetic relationships. We have exploited divergent repeats of the highly structured, 50-amino acid WHEP domains that join the catalytic subunits of bifunctional glutamyl-prolyl tRNA synthetase (EPRS) as a sequence-informed repeat (SIR) to trace the origin and evolution of EPRS in holozoa. EPRS is the only fused tRNA synthetase, with two distinct aminoacylation activities, and a non-canonical translation regulatory function mediated by the WHEP domains in the linker. Investigating the duplications, deletions and divergence of WHEP domains, we traced the bifunctional EPRS to choanozoans and identified the fusion event leading to its origin at the divergence of ichthyosporea and emergence of filozoa nearly a billion years ago. Distribution of WHEP domains from a single species in two or more distinct clades suggested common descent, allowing the identification of linking organisms. The discrete assortment of choanoflagellate WHEP domains with choanozoan domains as well as with those in metazoans supported the phylogenetic position of choanoflagellates as the closest sister group to metazoans. Analysis of clustering and assortment of WHEP domains provided unexpected insights into phylogenetic relationships amongst holozoan taxa. Furthermore, observed gaps in the transition between WHEP domain groupings in distant taxa allowed the prediction of undiscovered or extinct evolutionary intermediates. Analysis based on SIR domains can provide a phylogenetic counterpart to palaentological approaches of discovering “missing links” in the tree of life. PMID:24968216

  11. TFAM detects co-evolution of tRNA identity rules with lateral transfer of histidyl-tRNA synthetase

    PubMed Central

    Ardell, David H.; Andersson, Siv G. E.

    2006-01-01

    We present TFAM, an automated, statistical method to classify the identity of tRNAs. TFAM, currently optimized for bacteria, classifies initiator tRNAs and predicts the charging identity of both typical and atypical tRNAs such as suppressors with high confidence. We show statistical evidence for extensive variation in tRNA identity determinants among bacterial genomes due to variation in overall tDNA base content. With TFAM we have detected the first case of eukaryotic-like tRNA identity rules in bacteria. An α-proteobacterial clade encompassing Rhizobiales, Caulobacter crescentus and Silicibacter pomeroyi, unlike a sister clade containing the Rickettsiales, Zymomonas mobilis and Gluconobacter oxydans, uses the eukaryotic identity element A73 instead of the highly conserved prokaryotic element C73. We confirm divergence of bacterial histidylation rules by demonstrating perfect covariation of α-proteobacterial tRNAHis acceptor stems and residues in the motif IIb tRNA-binding pocket of their histidyl-tRNA synthetases (HisRS). Phylogenomic analysis supports lateral transfer of a eukaryotic-like HisRS into the α-proteobacteria followed by in situ adaptation of the bacterial tDNAHis and identity rule divergence. Our results demonstrate that TFAM is an effective tool for the bioinformatics, comparative genomics and evolutionary study of tRNA identity. PMID:16473847

  12. Nucleotide sequence of a human tRNA gene heterocluster

    SciTech Connect

    Chang, Y.N.; Pirtle, I.L.; Pirtle, R.M.

    1986-05-01

    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'-/sup 32/P)-labeled bovine liver leucine tRNA and total tRNA as hybridization probes. An 8-kb Hind III fragment of one of these ..gamma..-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.

  13. Rational protein engineering in action: The first crystal structure of a phenylalanine tRNA synthetase from Staphylococcus haemolyticus

    SciTech Connect

    Evdokimov, Artem G.; Mekel, Marlene; Hutchings, Kim; Narasimhan, Lakshmi; Holler, Tod; McGrath, Teresa; Beattie, Bryan; Fauman, Eric; Yan, Chunhong; Heaslet, Holly; Walter, Richard; Finzel, Barry; Ohren, Jeffrey; McConnell, Patrick; Braden, Timothy; Sun, Fang; Spessard, Cindy; Banotai, Craig; Al-Kassim, Loola; Ma, Weijun; Wengender, Paul; Kole, Denis; Garceau, Norman; Toogood, Peter; Liu, Jia

    2008-07-08

    In this article, we describe for the first time the high-resolution crystal structure of a phenylalanine tRNA synthetase from the pathogenic bacterium Staphylococcus haemolyticus. We demonstrate the subtle yet important structural differences between this enzyme and the previously described Thermus thermophilus ortholog. We also explain the structure-activity relationship of several recently reported inhibitors. The native enzyme crystals were of poor quality -- they only diffracted X-rays to 3--5 {angstrom} resolution. Therefore, we have executed a rational surface mutagenesis strategy that has yielded crystals of this 2300-amino acid multidomain protein, diffracting to 2 {angstrom} or better. This methodology is discussed and contrasted with the more traditional domain truncation approach.

  14. tRNA fragments in human health and disease.

    PubMed

    Anderson, Paul; Ivanov, Pavel

    2014-11-28

    Transfer RNA (tRNA) is traditionally considered to be an adaptor molecule that helps ribosomes to decode messenger RNA (mRNA) and synthesize protein. Recent studies have demonstrated that tRNAs also serve as a major source of small non-coding RNAs that possess distinct and varied functions. These tRNA fragments are heterogeneous in size, nucleotide composition, biogenesis and function. Here we describe multiple roles that tRNA fragments play in cell physiology and discuss their relevance to human health and disease.

  15. Identification and functional characterization of a novel bacterial type asparagine synthetase A: a tRNA synthetase paralog from Leishmania donovani.

    PubMed

    Manhas, Reetika; Tripathi, Pankaj; Khan, Sameena; Sethu Lakshmi, Bhavana; Lal, Shambhu Krishan; Gowri, Venkatraman Subramanian; Sharma, Amit; Madhubala, Rentala

    2014-04-25

    Asparagine is formed by two structurally distinct asparagine synthetases in prokaryotes. One is the ammonia-utilizing asparagine synthetase A (AsnA), and the other is asparagine synthetase B (AsnB) that uses glutamine or ammonia as a nitrogen source. In a previous investigation using sequence-based analysis, we had shown that Leishmania spp. possess asparagine-tRNA synthetase paralog asparagine synthetase A (LdASNA) that is ammonia-dependent. Here, we report the cloning, expression, and kinetic analysis of ASNA from Leishmania donovani. Interestingly, LdASNA was both ammonia- and glutamine-dependent. To study the physiological role of ASNA in Leishmania, gene deletion mutations were attempted via targeted gene replacement. Gene deletion of LdASNA showed a growth delay in mutants. However, chromosomal null mutants of LdASNA could not be obtained as the double transfectant mutants showed aneuploidy. These data suggest that LdASNA is essential for survival of the Leishmania parasite. LdASNA enzyme was recalcitrant toward crystallization so we instead crystallized and solved the atomic structure of its close homolog from Trypanosoma brucei (TbASNA) at 2.2 Å. A very significant conservation in active site residues is observed between TbASNA and Escherichia coli AsnA. It is evident that the absence of an LdASNA homolog from humans and its essentiality for the parasites make LdASNA a novel drug target.

  16. Brugia malayi Asparaginyl - tRNA Synthetase Stimulates Endothelial Cell Proliferation, Vasodilation and Angiogenesis

    PubMed Central

    D, Jeeva Jothi; Dhanraj, Muthu; Solaiappan, Shanmugam; Sivanesan, Sanjana; Kron, Michael; Dhanasekaran, Anuradha

    2016-01-01

    A hallmark of chronic infection with lymphatic filarial parasites is the development of lymphatic disease which often results in permanent vasodilation and lymphedema, but all of the mechanisms by which filarial parasites induce pathology are not known. Prior work showed that the asparaginyl-tRNA synthetase (BmAsnRS) of Brugia malayi, an etiological agent of lymphatic filariasis, acts as a physiocrine that binds specifically to interleukin-8 (IL-8) chemokine receptors. Endothelial cells are one of the many cell types that express IL-8 receptors. IL-8 also has been reported previously to induce angiogenesis and vasodilation, however, the effect of BmAsnRS on endothelial cells has not been reported. Therefore, we tested the hypothesis that BmAsnRS might produce physiological changes in endothelial by studying the in vitro effects of BmAsnRS using a human umbilical vein cell line EA.hy926 and six different endothelial cell assays. Our results demonstrated that BmAsnRS produces consistent and statistically significant effects on endothelial cells that are identical to the effects of VEGF, vascular endothelial growth factor. This study supports the idea that new drugs or immunotherapies that counteract the adverse effects of parasite-derived physiocrines may prevent or ameliorate the vascular pathology observed in patients with lymphatic filariasis. PMID:26751209

  17. The Enzymatic Paradox of Yeast Arginyl-tRNA Synthetase: Exclusive Arginine Transfer Controlled by a Flexible Mechanism of tRNA Recognition

    PubMed Central

    Eriani, Gilbert; Geslain, Renaud

    2016-01-01

    Identity determinants are essential for the accurate recognition of transfer RNAs by aminoacyl-tRNA synthetases. To date, arginine determinants in the yeast Saccharomyces cerevisiae have been identified exclusively in vitro and only on a limited number of tRNA Arginine isoacceptors. In the current study, we favor a full cellular approach and expand the investigation of arginine determinants to all four tRNA Arg isoacceptors. More precisely, this work scrutinizes the relevance of the tRNA nucleotides at position 20, 35 and 36 in the yeast arginylation reaction. We built 21 mutants by site-directed mutagenesis and tested their functionality in YAL5, a previously engineered yeast knockout deficient for the expression of tRNA Arg CCG. Arginylation levels were also monitored using Northern blot. Our data collected in vivo correlate with previous observations. C35 is the prominent arginine determinant followed by G36 or U36 (G/U36). In addition, although there is no major arginine determinant in the D loop, the recognition of tRNA Arg ICG relies to some extent on the nucleotide at position 20. This work refines the existing model for tRNA Arg recognition. Our observations indicate that yeast Arginyl-tRNA synthetase (yArgRS) relies on distinct mechanisms to aminoacylate the four isoacceptors. Finally, according to our refined model, yArgRS is able to accommodate tRNA Arg scaffolds presenting N34, C/G35 and G/A/U36 anticodons while maintaining specificity. We discuss the mechanistic and potential physiological implications of these findings. PMID:26844776

  18. The Enzymatic Paradox of Yeast Arginyl-tRNA Synthetase: Exclusive Arginine Transfer Controlled by a Flexible Mechanism of tRNA Recognition.

    PubMed

    McShane, Ariel; Hok, Eveline; Tomberlin, Jensen; Eriani, Gilbert; Geslain, Renaud

    2016-01-01

    Identity determinants are essential for the accurate recognition of transfer RNAs by aminoacyl-tRNA synthetases. To date, arginine determinants in the yeast Saccharomyces cerevisiae have been identified exclusively in vitro and only on a limited number of tRNA Arginine isoacceptors. In the current study, we favor a full cellular approach and expand the investigation of arginine determinants to all four tRNA Arg isoacceptors. More precisely, this work scrutinizes the relevance of the tRNA nucleotides at position 20, 35 and 36 in the yeast arginylation reaction. We built 21 mutants by site-directed mutagenesis and tested their functionality in YAL5, a previously engineered yeast knockout deficient for the expression of tRNA Arg CCG. Arginylation levels were also monitored using Northern blot. Our data collected in vivo correlate with previous observations. C35 is the prominent arginine determinant followed by G36 or U36 (G/U36). In addition, although there is no major arginine determinant in the D loop, the recognition of tRNA Arg ICG relies to some extent on the nucleotide at position 20. This work refines the existing model for tRNA Arg recognition. Our observations indicate that yeast Arginyl-tRNA synthetase (yArgRS) relies on distinct mechanisms to aminoacylate the four isoacceptors. Finally, according to our refined model, yArgRS is able to accommodate tRNA Arg scaffolds presenting N34, C/G35 and G/A/U36 anticodons while maintaining specificity. We discuss the mechanistic and potential physiological implications of these findings.

  19. Adenosine conformations of nucleotides bound to methionyl tRNA synthetase by transferred nuclear Overhauser effect spectroscopy.

    PubMed Central

    Murali, N; Lin, Y; Mechulam, Y; Plateau, P; Rao, B D

    1997-01-01

    The conformations of MgATP and AMP bound to a monomeric tryptic fragment of methionyl tRNA synthetase have been investigated by two-dimensional proton transferred nuclear Overhauser effect spectroscopy (TRNOESY). The sample protocol was chosen to minimize contributions from adventitious binding of the nucleotides to the observed NOE. The experiments were performed at 500 MHz on three different complexes, E.MgATP, E.MgATP.L-methioninol, and E.AMP.L-methioninol. A starter set of distances obtained by fitting NOE build-up curves (not involving H5' and H5") were used to determine a CHARMm energy-minimized structure. The positioning of the H5' and H5" protons was determined on the basis of a conformational search of the torsion angle to obtain the best fit with the observed NOEs for their superposed resonance. Using this structure, a relaxation matrix was set up to calculate theoretical build-up curves for all of the NOEs and compare them with the observed curves. The final structures deduced for the adenosine moieties in the three complexes are very similar, and are described by a glycosidic torsion angle (chi) of 56 degrees +/- 5 degrees and a phase angle of pseudorotation (P) in the range of 47 degrees to 52 degrees, describing a 3(4)T-4E sugar pucker. The glycosidic torsion angle, chi, deduced here for this adenylyl transfer enzyme and those determined previously for three phosphoryl transfer enzymes (creatine kinase, arginine kinase, and pyruvate kinase), and one pyrophosphoryl enzyme (PRibPP synthetase), are all in the range 52 degrees +/- 8 degrees. The narrow range of values suggests a possible common motif for the recognition and binding of the adenosine moiety at the active sites of ATP-utilizing enzymes, irrespective of the point of cleavage on the phosphate chain. Images FIGURE 6 PMID:9129831

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

    SciTech Connect

    Ling, Jiqiang; Peterson, Kaitlyn M.; Simonovic, Ivana; Cho, Chris; Soll, Dieter; Simonovic, Miljan

    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.

  1. Cytoplasmic Leucyl-Trna Synthetase of Neurospora Crassa Is Not Specified by the Leu-5 Locus

    PubMed Central

    Benarous, R.; Chow, C. M.; RajBhandary, U. L.

    1988-01-01

    We generated a λgt11 Neurospora crassa cDNA library and screened the library for the cytoplasmic leucyl-tRNA synthetase (cyto LeuRS) clones using cyto LeuRS specific antibody. Two clones, λNCLRSC1 and λNCLRSC2, were obtained which have inserts of ~2 kbp and ~1.3 kbp, and which overlap by about 0.6 kbp. The following lines of evidence indicate that λNCLRSC1 and λNCLRSC2 encode parts of cyto LeuRS. (1) Antibodies affinity purified using either of the fusion proteins encoded by λNCLRSC1 or λNCLRSC2 inhibit cyto LeuRS activity. Thus, the fusion protein and cyto LeuRS share immunological determinants. (2) The same antibodies also react with an ~115-kDa protein, which comigrates with purified cyto LeuRS, in immunoblots of total N. crassa proteins. We used the cDNA clones to probe a N. crassa genomic DNA library and isolated two genomic DNA clones. Partial sequence analysis of cDNA and genomic DNA clones shows a methionine initiated open reading frame, which includes a stretch of amino acid residues that are highly conserved and that are at the ATP binding site in aminoacyl-tRNA synthetases. Using the cloned DNA as probe, we show that the cyto LeuRS mRNA is ~3900 nucleotides long. Finally, we have used restriction fragment length polymorphism mapping to show that the cyto LeuRS gene resides on the far right of linkage group II and not on linkage group V where the leu-5 mutation, which was previously reported to specify cyto LeuRS, is located. PMID:2842224

  2. Assignment of the human MARS gene, encoding methioninyl-tRNA synthetase, to chromosome 12 using human X Chinese hamster cell hybrids.

    PubMed

    Cirullo, R E; Wasmuth, J J

    1984-05-01

    We have isolated interspecific somatic cell hybrids between a temperature-sensitive Chinese hamster ovary (CHO) cell methioninyl -tRNA synthetase mutant and human peripheral leukocytes. The hybrids were selected at 39 degrees C which requires the retention and expression of the human gene, MARS , which complements the defective CHO gene. In vitro heat-inactivation experiments on the methioninyl -tRNA synthetase activity in cell-free extracts from heat-resistant hybrids indicate that the human form of this enzyme and, therefore, the human MARS gene is present in hybrid cells. Cytogenetic analysis of three independent temperature-resistant hybrids revealed the presence of a single human chromosome, number 12. Two other independent hybrids examined contained human chromosome 12 as well as a second human chromosome. Electrophoretic analysis of extracts from hybrid cell lines for a human chromosome 12 marker isozyme, LDH-B, showed a pattern of heterotetrameric bands consistent with the presence of the human form of this enzyme in these cells. The correlation between the presence of the human form of methioninyl -tRNA synthetase and human chromosome 12 in temperature-resistant hybrids indicates that the human MARS locus is located on this chromosome.

  3. The yeast protein Arc1p binds to tRNA and functions as a cofactor for the methionyl- and glutamyl-tRNA synthetases.

    PubMed Central

    Simos, G; Segref, A; Fasiolo, F; Hellmuth, K; Shevchenko, A; Mann, M; Hurt, E C

    1996-01-01

    Arc1p was found in a screen for components that interact genetically with Los1p, a nuclear pore-associated yeast protein involved in tRNA biogenesis. Arc1p is associated with two proteins which were identified as methionyl-tRNA and glutamyl-tRNA synthetase (MetRS and GluRS) by a new mass spectrometry method. ARC1 gene disruption leads to slow growth and reduced MetRS activity, and synthetically lethal arc1- mutants are complemented by the genes for MetRS and GluRS. Recombinant Arc1p binds in vitro to purified monomeric yeast MetRS, but not to an N-terminal truncated form, and strongly increases its apparent affinity for tRNAMet. Furthermore, Arc1p, which is allelic to the quadruplex nucleic acid binding protein G4p1, exhibits specific binding to tRNA as determined by gel retardation and UV-cross-linking. Arc1p is, therefore, a yeast protein with dual specificity: it associates with tRNA and aminoacyl-tRNA synthetases. This functional interaction may be required for efficient aminoacylation in vivo. Images PMID:8895587

  4. Oxidative stress diverts tRNA synthetase to nucleus for protection against DNA damage.

    PubMed

    Wei, Na; Shi, Yi; Truong, Lan N; Fisch, Kathleen M; Xu, Tao; Gardiner, Elisabeth; Fu, Guangsen; Hsu, Yun-Shiuan Olivia; Kishi, Shuji; Su, Andrew I; Wu, Xiaohua; Yang, Xiang-Lei

    2014-10-23

    Tyrosyl-tRNA synthetase (TyrRS) is known for its essential aminoacylation function in protein synthesis. Here we report a function for TyrRS in DNA damage protection. We found that oxidative stress, which often downregulates protein synthesis, induces TyrRS to rapidly translocate from the cytosol to the nucleus. We also found that angiogenin mediates or potentiates this stress-induced translocalization. The nuclear-localized TyrRS activates transcription factor E2F1 to upregulate the expression of DNA damage repair genes such as BRCA1 and RAD51. The activation is achieved through direct interaction of TyrRS with TRIM28 to sequester this vertebrate-specific epigenetic repressor and its associated HDAC1 from deacetylating and suppressing E2F1. Remarkably, overexpression of TyrRS strongly protects against UV-induced DNA double-strand breaks in zebrafish, whereas restricting TyrRS nuclear entry completely abolishes the protection. Therefore, oxidative stress triggers an essential cytoplasmic enzyme used for protein synthesis to translocate to the nucleus to protect against DNA damage.

  5. Structural characterization of antibiotic self-immunity tRNA synthetase in plant tumour biocontrol agent

    PubMed Central

    Chopra, Shaileja; Palencia, Andrés; Virus, Cornelia; Schulwitz, Sarah; Temple, Brenda R.; Cusack, Stephen; Reader, John

    2016-01-01

    Antibiotic-producing microbes evolved self-resistance mechanisms to avoid suicide. The biocontrol Agrobacterium radiobacter K84 secretes the Trojan Horse antibiotic agrocin 84 that is selectively transported into the plant pathogen A. tumefaciens and processed into the toxin TM84. We previously showed that TM84 employs a unique tRNA-dependent mechanism to inhibit leucyl-tRNA synthetase (LeuRS), while the TM84-producer prevents self-poisoning by expressing a resistant LeuRS AgnB2. We now identify a mechanism by which the antibiotic-producing microbe resists its own toxin. Using a combination of structural, biochemical and biophysical approaches, we show that AgnB2 evolved structural changes so as to resist the antibiotic by eliminating the tRNA-dependence of TM84 binding. Mutagenesis of key resistance determinants results in mutants adopting an antibiotic-sensitive phenotype. This study illuminates the evolution of resistance in self-immunity genes and provides mechanistic insights into a fascinating tRNA-dependent antibiotic with applications for the development of anti-infectives and the prevention of biocontrol emasculation. PMID:27713402

  6. Molecular evolution of aminoacyl tRNA synthetase proteins in the early history of life.

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  8. Diversity of human tRNA genes from the 1000-genomes project.

    PubMed

    Parisien, Marc; Wang, Xiaoyun; Pan, Tao

    2013-12-01

    The sequence diversity of individual human genomes has been extensively analyzed for variations and phenotypic implications for mRNA, miRNA, and long non-coding RNA genes. TRNA (tRNA) also exhibits large sequence diversity in the human genome, but tRNA gene sequence variation and potential functional implications in individual human genomes have not been investigated. Here we capitalize on the sequencing data from the 1000-genomes project to examine the diversity of tRNA genes in the human population. Previous analysis of the reference human genome indicated an unexpected large number of diverse tRNA genes beyond the necessity of translation, suggesting that some tRNA transcripts may perform non-canonical functions. We found 24 new tRNA sequences in>1% and 76 new tRNA sequences in>0.2% of all individuals, indicating that tRNA genes are also subject to evolutionary changes in the human population. Unexpectedly, two abundant new tRNA genes contain base-pair mismatches in the anticodon stem. We experimentally determined that these two new tRNAs have altered structures in vitro; however, one new tRNA is not aminoacylated but extremely stable in HeLa cells, suggesting that this new tRNA can be used for non-canonical function. Our results show that at the scale of human population, tRNA genes are more diverse than conventionally understood, and some new tRNAs may perform non-canonical, extra-translational functions that may be linked to human health and disease.

  9. Structure of the acceptor stem of Escherichia coli tRNA Ala: role of the G3.U70 base pair in synthetase recognition.

    PubMed Central

    Ramos, A; Varani, G

    1997-01-01

    The fidelity of translation of the genetic code depends on accurate tRNA aminoacylation by cognate aminoacyl-tRNA synthetases. Thus, each tRNA has specificity not only for codon recognition, but also for amino acid identity; this aminoacylation specificity is referred to as tRNA identity. The primary determinant of the acceptor identity of Escherichia coli tRNAAlais a wobble G3.U70 pair within the acceptor stem. Despite extensive biochemical and genetic data, the mechanism by which the G3.U70 pair marks the acceptor end of tRNAAla for aminoacylation with alanine has not been clarified at the molecular level. The solution structure of a microhelix derived from the tRNAAla acceptor end has been determined at high precision using a very extensive set of experimental constraints (approximately 32 per nt) obtained by heteronuclear multidimensional NMR methods. The tRNAAla acceptor end is overall similar to A-form RNA, but important differences are observed. The G3.U70 wobble pair distorts the conformation of the phosphodiester backbone and presents the functional groups of U70 in an unusual spatial location. The discriminator base A73 has extensive stacking overlap with G1 within the G1.C72 base pair at the end of the double helical stem and the -CCA end is significantly less ordered than the rest of the molecule. PMID:9153306

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

    DOEpatents

    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.

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

    SciTech Connect

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

    2006-08-01

    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.

  12. Variations in clique and community patterns in protein structures during allosteric communication: investigation of dynamically equilibrated structures of methionyl tRNA synthetase complexes.

    PubMed

    Ghosh, Amit; Vishveshwara, Saraswathi

    2008-11-04

    The allosteric concept has played a key role in understanding the biological functions of proteins. The rigidity or plasticity and the conformational population are the two important ideas invoked in explaining the allosteric effect. Although molecular insights have been gained from a large number of structures, a precise assessment of the ligand-induced conformational changes in proteins at different levels, ranging from gross topology to intricate details, remains a challenge. In this study, we have explored the conformational changes in the complexes of methionyl tRNA synthetase (MetRS) through novel network parameters such as cliques and communities, which identify the rigid regions in the protein structure networks (PSNs) constructed from the noncovalent interactions of amino acid side chains. MetRS belongs to the aminoacyl tRNA synthetase (aaRS) family that plays a crucial role in the translation of genetic code. These enzymes are modular with distinct domains from which extensive genetic, kinetic, and structural data are available, highlighting the role of interdomain communication. The network parameters evaluated here on the conformational ensembles of MetRS complexes, generated from molecular dynamics simulations, have enabled us to understand the interdomain communication in detail. Additionally, the characterization of conformational changes in terms of cliques and communities has also become possible, which had eluded conventional analyses. Furthermore, we find that most of the residues participating in cliques and communities are strikingly different from those that take part in long-range communication. The cliques and communities evaluated here for the first time on PSNs have beautifully captured the local geometries in detail within the framework of global topology. Here the allosteric effect is revealed at the residue level via identification of the important residues specific for structural rigidity and functional flexibility in MetRS. This ought

  13. Comparison of histidine recognition in human and trypanosomatid histidyl-tRNA synthetases.

    PubMed

    Koh, Cho Yeow; Wetzel, Allan B; de van der Schueren, Will J; Hol, Wim G J

    2014-11-01

    As part of a project aimed at obtaining selective inhibitors and drug-like compounds targeting tRNA synthetases from trypanosomatids, we have elucidated the crystal structure of human cytosolic histidyl-tRNA synthetase (Hs-cHisRS) in complex with histidine in order to be able to compare human and parasite enzymes. The resultant structure of Hs-cHisRS•His represents the substrate-bound state (H-state) of the enzyme. It provides an interesting opportunity to compare with ligand-free and imidazole-bound structures Hs-cHisRS published recently, both of which represent the ligand-free state (F-state) of the enzyme. The H-state Hs-cHisRS undergoes conformational changes in active site residues and several conserved motif of HisRS, compared to F-state structures. The histidine forms eight hydrogen bonds with HisRS of which six engage the amino and carboxylate groups of this amino acid. The availability of published imidazole-bound structure provides a unique opportunity to dissect the structural roles of individual chemical groups of histidine. The analysis revealed the importance of the amino and carboxylate groups, of the histidine in leading to these dramatic conformational changes of the H-state. Further, comparison with previously published trypanosomatid HisRS structures reveals a pocket in the F-state of the parasite enzyme that may provide opportunities for developing specific inhibitors of Trypanosoma brucei HisRS.

  14. Large Conformational Changes of Insertion 3 in Human Glycyl-tRNA Synthetase (hGlyRS) during Catalysis*

    PubMed Central

    Deng, Xiangyu; Qin, Xiangjing; Chen, Lei; Jia, Qian; Zhang, Yonghui; Zhang, Zhiyong; Lei, Dongsheng; Ren, Gang; Zhou, Zhihong; Wang, Zhong; Li, Qing; Xie, Wei

    2016-01-01

    Glycyl-tRNA synthetase (GlyRS) is the enzyme that covalently links glycine to cognate tRNA for translation. It is of great research interest because of its nonconserved quaternary structures, unique species-specific aminoacylation properties, and noncanonical functions in neurological diseases, but none of these is fully understood. We report two crystal structures of human GlyRS variants, in the free form and in complex with tRNAGly respectively, and reveal new aspects of the glycylation mechanism. We discover that insertion 3 differs considerably in conformation in catalysis and that it acts like a “switch” and fully opens to allow tRNA to bind in a cross-subunit fashion. The flexibility of the protein is supported by molecular dynamics simulation, as well as enzymatic activity assays. The biophysical and biochemical studies suggest that human GlyRS may utilize its flexibility for both the traditional function (regulate tRNA binding) and alternative functions (roles in diseases). PMID:26797133

  15. Two complementary enzymes for threonylation of tRNA in crenarchaeota: crystal structure of Aeropyrum pernix threonyl-tRNA synthetase lacking a cis-editing domain.

    PubMed

    Shimizu, Satoru; Juan, Ella Czarina Magat; Sato, Yoshiteru; Miyashita, Yu-Ichiro; Hoque, Md Mominul; Suzuki, Kaoru; Sagara, Tsubasa; Tsunoda, Masaru; Sekiguchi, Takeshi; Dock-Bregeon, Anne-Catherine; Moras, Dino; Takénaka, Akio

    2009-11-27

    In protein synthesis, threonyl-tRNA synthetase (ThrRS) must recognize threonine (Thr) from the 20 kinds of amino acids and the cognate tRNA(Thr) from different tRNAs in order to generate Thr-tRNA(Thr). In general, an organism possesses one kind of gene corresponding to ThrRS. However, it has been recently found that some organisms have two different genes for ThrRS in the genome, suggesting that their proteins ThrRS-1 and ThrRS-2 function separately and complement each other in the threonylation of tRNA(Thr), one for catalysis and the other for trans-editing of misacylated Ser-tRNA(Thr). In order to clarify their three-dimensional structures, we performed X-ray analyses of two putatively assigned ThrRSs from Aeropyrum pernix (ApThrRS-1 and ApThrRS-2). These proteins were overexpressed in Escherichia coli, purified, and crystallized. The crystal structure of ApThrRS-1 has been successfully determined at 2.3 A resolution. ApThrRS-1 is a dimeric enzyme composed of two identical subunits, each containing two domains for the catalytic reaction and for anticodon binding. The essential editing domain is completely missing as expected. These structural features reveal that ThrRS-1 catalyzes only the aminoacylation of the cognate tRNA, suggesting the necessity of the second enzyme ThrRS-2 for trans-editing. Since the N-terminal sequence of ApThrRS-2 is similar to the sequence of the editing domain of ThrRS from Pyrococcus abyssi, ApThrRS-2 has been expected to catalyze deaminoacylation of a misacylated serine moiety at the CCA terminus.

  16. Characterization of cDNAs and genomic DNAs for human threonyl- and cysteinyl-tRNA synthetases

    SciTech Connect

    Cruzen, M.E.

    1993-01-01

    Techniques of molecular biology were used to clone, sequence and map two human aminoacyl-tRNA synthetase (aaRS) cDNAs: threonyl-tRNA synthetase (ThrRS) a class II enzyme and cysteinyl-tRNA synthetase (CysRS) a class I enzyme. The predicted protein sequence of human ThrRS is highly homologous to that of lower eukaryotic and prokaryotic ThRSs, particularly in the regions containing the three structural motifs common to all class II synthetases. Signature regions 1 and 2, which characterize the class IIa subgroup (SerRS, ThrRS and HisRS) are highly conserved from bacteria to human. Structural predictions for human ThrRS based on the known structure of the closely related SerRS from E.coli implicate strongly conserved residues in the signature sequences to be important in substrate binding. The amino terminal 100 residues of the deduced amino acid sequence of ThrRS shares structural similarity to SerRS consistent with forming an antiparallel helix implicated in tRNA binding. The 5' untranslated sequence of the human ThrRS gene shares short stretches of common sequence with the gene for hamster HisRS including a binding site for the promoter specific transcription factor sp-1. The deduced amino acid sequence of human CysRS has a high degree of sequence identify to E. coli CysRS. Human CysRS possesses the classic characteristics of a class I synthetase and is most closely related to the MetRS subgroup. The amino terminal half of human CysRS can be modeled as a nucleotide binding fold and shares significant sequence and structural similarity to the other enzymes in this subgroup. The CysRS structural gene (CARS) was mapped to human chromosome 11p15.5 by fluorescent in situ hybridization. CARS is the first aaRS gene to be mapped to chromosome 11. The steady state of both CysRS and ThrRs mRNA were quantitated in several human tissues. Message levels for these enzymes appear to be subjected to differential regulation in different cell types.

  17. Purification, crystallization and preliminary X-ray characterization of a human mitochondrial phenylalanyl-tRNA synthetase

    SciTech Connect

    Levin, Inna; Kessler, Naama; Moor, Nina; Klipcan, Liron; Koc, Emine; Templeton, Paul; Spremulli, Linda; Safro, Mark

    2007-09-01

    The expression, purification and crystallization of recombinant human mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) are reported. Diffraction data were collected to 2.2 Å resolution and the mitPheRS structure was solved using the molecular-replacement method. Human monomeric mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) is an enzyme that catalyzes the charging of tRNA with the cognate amino acid phenylalanine. Human mitPheRS is a chimera of the bacterial α-subunit of PheRS and the B8 domain of its β-subunit. Together, the α-subunit and the ‘RNP-domain’ (B8 domain) at the C-terminus form the minimal structural set to construct an enzyme with phenylalanylation activity. The recombinant human mitPheRS was purified to homogeneity and crystallized in complex with phenylalanine and ATP. The crystals diffracted to 2.2 Å resolution and belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 55, b = 90, c = 96 Å.

  18. Mutation of the human mitochondrial phenylalanine-tRNA synthetase causes infantile-onset epilepsy and cytochrome c oxidase deficiency.

    PubMed

    Almalki, Abdulraheem; Alston, Charlotte L; Parker, Alasdair; Simonic, Ingrid; Mehta, Sarju G; He, Langping; Reza, Mojgan; Oliveira, Jorge M A; Lightowlers, Robert N; McFarland, Robert; Taylor, Robert W; Chrzanowska-Lightowlers, Zofia M A

    2014-01-01

    Mitochondrial aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein synthesis since they charge tRNAs with their cognate amino acids. Mutations in the genes encoding mitochondrial aaRSs have been associated with a wide spectrum of human mitochondrial diseases. Here we report the identification of pathogenic mutations (a partial genomic deletion and a highly conserved p. Asp325Tyr missense variant) in FARS2, the gene encoding mitochondrial phenylalanyl-tRNA synthetase, in a patient with early-onset epilepsy and isolated complex IV deficiency in muscle. The biochemical defect was expressed in myoblasts but not in fibroblasts and associated with decreased steady state levels of COXI and COXII protein and reduced steady state levels of the mt-tRNA(Phe) transcript. Functional analysis of the recombinant mutant p. Asp325Tyr FARS2 protein showed an inability to bind ATP and consequently undetectable aminoacylation activity using either bacterial tRNA or human mt-tRNA(Phe) as substrates. Lentiviral transduction of cells with wildtype FARS2 restored complex IV protein levels, confirming that the p.Asp325Tyr mutation is pathogenic, causing respiratory chain deficiency and neurological deficits on account of defective aminoacylation of mt-tRNA(Phe).

  19. Tyrosyl-tRNA synthetase: the first crystallization of a human mitochondrial aminoacyl-tRNA synthetase

    SciTech Connect

    Bonnefond, Luc; Frugier, Magali; Touzé, Elodie; Lorber, Bernard; Florentz, Catherine; Giegé, Richard Rudinger-Thirion, Joëlle; Sauter, Claude

    2007-04-01

    Crystals of human mitochondrial tyrosyl-tRNA synthetase lacking the C-terminal S4-like domain diffract to 2.7 Å resolution and are suitable for structure determination. Human mitochondrial tyrosyl-tRNA synthetase and a truncated version with its C-terminal S4-like domain deleted were purified and crystallized. Only the truncated version, which is active in tyrosine activation and Escherichia coli tRNA{sup Tyr} charging, yielded crystals suitable for structure determination. These tetragonal crystals, belonging to space group P4{sub 3}2{sub 1}2, were obtained in the presence of PEG 4000 as a crystallizing agent and diffracted X-rays to 2.7 Å resolution. Complete data sets could be collected and led to structure solution by molecular replacement.

  20. Molecular structure of the human argininosuccinate synthetase gene: Occurrence of alternative mRNA splicing

    SciTech Connect

    Freytag, S.O.; Beaudet, A.L.; Bock, H.G.O.; O'Brien, W.E.

    1984-10-01

    The human genome contains one expressed argininosuccinate synthetase gene and ca. 14 pseudogenes that are dispersed to at least 11 human chromosomes. Eleven clones isolated from a human genomic DNA library were characterized extensively by restriction mapping, Southern blotting, and nucleotide sequencing. These 11 clones represent the entire expressed argininosuccinate synthetase gene that spans 63 kilobases and contains at least 13 exons. The expressed gene codes for two mRNAs that differ in their 5' untranslated sequences and arise by alternative splicing involving the inclusion or deletion of an entire exon. In normal human liver and cultured fibroblasts, the predominant mature argininosuccinate synthetase mRNA lacks sequences encoded by exon 2 in the expressed gene. In contrast, the predominant argininosuccinate synthetase mRNA in baboon liver contains exon 2 sequences. A transformed canavanine-resistant human cell line in which argininosuccinate synthetase activity is 180-fold higher than that in wild-type cells contains abundant amounts of both forms of the argininosuccinate synthetase mRNA. The mRNA lacking exon 2 sequences is the more abundant mRNA species in the canavanine-resistant cells. These observations show that splicing of the argininosuccinate synthetase mRNA is species specific in primates and varies among different human cell types.

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

    PubMed

    Castro de Moura, Manuel; Miro, Francesc; Han, Jung Min; Kim, Sunghoon; Celada, Antonio; Ribas de Pouplana, Lluís

    2011-11-01

    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.

  2. Molecular mimicry of human tRNALys anti-codon domain by HIV-1 RNA genome facilitates tRNA primer annealing.

    PubMed

    Jones, Christopher P; Saadatmand, Jenan; Kleiman, Lawrence; Musier-Forsyth, Karin

    2013-02-01

    The primer for initiating reverse transcription in human immunodeficiency virus type 1 (HIV-1) is tRNA(Lys3). Host cell tRNA(Lys) is selectively packaged into HIV-1 through a specific interaction between the major tRNA(Lys)-binding protein, human lysyl-tRNA synthetase (hLysRS), and the viral proteins Gag and GagPol. Annealing of the tRNA primer onto the complementary primer-binding site (PBS) in viral RNA is mediated by the nucleocapsid domain of Gag. The mechanism by which tRNA(Lys3) is targeted to the PBS and released from hLysRS prior to annealing is unknown. Here, we show that hLysRS specifically binds to a tRNA anti-codon-like element (TLE) in the HIV-1 genome, which mimics the anti-codon loop of tRNA(Lys) and is located proximal to the PBS. Mutation of the U-rich sequence within the TLE attenuates binding of hLysRS in vitro and reduces the amount of annealed tRNA(Lys3) in virions. Thus, LysRS binds specifically to the TLE, which is part of a larger LysRS binding domain in the viral RNA that includes elements of the Psi packaging signal. Our results suggest that HIV-1 uses molecular mimicry of the anti-codon of tRNA(Lys) to increase the efficiency of tRNA(Lys3) annealing to viral RNA.

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

    SciTech Connect

    Webb, G.C.; Vaska, V.L.; Ford, J.H.

    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.

  4. Mitochondrial aminoacyl-tRNA synthetases in human disease.

    PubMed

    Konovalova, Svetlana; Tyynismaa, Henna

    2013-04-01

    Mitochondrial aminoacyl-tRNA synthetases (mtARSs) are essential in the process of transferring genetic information from mitochondrial DNA to the complexes of the oxidative phosphorylation system. These synthetases perform an integral step in the initiation of mitochondrial protein synthesis by charging tRNAs with their cognate amino acids. All mtARSs are encoded by nuclear genes, nine of which have recently been described as disease genes for mitochondrial disorders. Unexpectedly, the clinical presentations of these diseases are highly specific to the affected synthetase. Encephalopathy is the most common manifestation but again with gene-specific outcomes. Other clinical presentations include myopathy with anemia, cardiomyopathy, tubulopathy and hearing loss with female ovarian dysgenesis. Here we review the described mutation types and the associated patient phenotypes. The identified mutation spectrum suggests that only mutation types that allow some residual tRNA-charging activity can result in the described mtARS diseases but the molecular mechanisms behind the selective tissue involvement are not currently understood.

  5. Monophyly of class I aminoacyl tRNA synthetase, USPA, ETFP, photolyase, and PP-ATPase nucleotide-binding domains: implications for protein evolution in the RNA.

    PubMed

    Aravind, L; Anantharaman, Vivek; Koonin, Eugene V

    2002-07-01

    Protein sequence and structure comparisons show that the catalytic domains of Class I aminoacyl-tRNA synthetases, a related family of nucleotidyltransferases involved primarily in coenzyme biosynthesis, nucleotide-binding domains related to the UspA protein (USPA domains), photolyases, electron transport flavoproteins, and PP-loop-containing ATPases together comprise a distinct class of alpha/beta domains designated the HUP domain after HIGH-signature proteins, UspA, and PP-ATPase. Several lines of evidence are presented to support the monophyly of the HUP domains, to the exclusion of other three-layered alpha/beta folds with the generic "Rossmann-like" topology. Cladistic analysis, with patterns of structural and sequence similarity used as discrete characters, identified three major evolutionary lineages within the HUP domain class: the PP-ATPases; the HIGH superfamily, which includes class I aaRS and related nucleotidyltransferases containing the HIGH signature in their nucleotide-binding loop; and a previously unrecognized USPA-like group, which includes USPA domains, electron transport flavoproteins, and photolyases. Examination of the patterns of phyletic distribution of distinct families within these three major lineages suggests that the Last Universal Common Ancestor of all modern life forms encoded 15-18 distinct alpha/beta ATPases and nucleotide-binding proteins of the HUP class. This points to an extensive radiation of HUP domains before the last universal common ancestor (LUCA), during which the multiple class I aminoacyl-tRNA synthetases emerged only at a late stage. Thus, substantial evolutionary diversification of protein domains occurred well before the modern version of the protein-dependent translation machinery was established, i.e., still in the RNA world.

  6. Hts1 Encodes Both the Cytoplasmic and Mitochondrial Histidyl-Trna Synthetase of Saccharomyces Cerevisiae: Mutations Alter the Specificity of Compartmentation

    PubMed Central

    Chiu, M. I.; Mason, T. L.; Fink, G. R.

    1992-01-01

    Genetic and biochemical evidence shows that a single nuclear gene HTS1 encodes both the mitochondrial and cytoplasmic histidyl-tRNA synthetases (Hts). The gene specifies two messages, one with two in-frame ATGs (-60 and +1) and another with only the downstream ATG (+1). We have made a new set of mutations that enables us to express only the mitochondrial or the cytoplasmic form and compared the subcellular distribution of the Hts1 protein in these mutants and wild type, using an antibody that interacts with both the mitochondrial and cytoplasmic Hts1 as well as Hts1::LacZ fusions. Mutations in the upstream ATG (-60) or frameshift mutations in the presequence affect only the mitochondrial enzyme and not the cytoplasmic enzyme. Mutations in the downstream ATG (+1 ATG to ATC) destroy the function of the cytosolic enzyme, but do not affect the function of the mitochondrial enzyme. Overexpression of this construct restores cytoplasmic function. Cells expressing a truncated form of Hts containing a deletion of the first 20 amino-terminal residues (Htsc) produce a functional cytoplasmic enzyme, which does not provide mitochondrial function. Overexpression of this truncated cytoplasmic protein provides mitochondrial function and produces detectable levels of the synthetase in the mitochondrion. These experiments suggest that Hts1 contains two domains that together allow efficient localization of Htsm to the mitochondrion: an amino-terminal presequence in the mitochondrial precursor that is likely cleaved upon delivery to the mitochondrion and a second amino-terminal sequence (residues 21-53) present in both the precursor and the cytoplasmic form. Neither one by itself is sufficient to act as an efficient mitochondrial targeting signal. Using our antibody we have been able to detect a protein of increased molecular mass that corresponds to that of the predicted precursor. Taken together these studies show that the specificity of compartmentation of the Hts protein depends

  7. Formation of tRNA granules in the nucleus of heat-induced human cells

    SciTech Connect

    Miyagawa, Ryu; Mizuno, Rie; Watanabe, Kazunori; Ijiri, Kenichi

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer tRNAs are tranlocated into the nucleus in heat-induced HeLa cells. Black-Right-Pointing-Pointer tRNAs form the unique granules in the nucleus. Black-Right-Pointing-Pointer tRNA ganules overlap with nuclear stress granules. -- Abstract: The stress response, which can trigger various physiological phenomena, is important for living organisms. For instance, a number of stress-induced granules such as P-body and stress granule have been identified. These granules are formed in the cytoplasm under stress conditions and are associated with translational inhibition and mRNA decay. In the nucleus, there is a focus named nuclear stress body (nSB) that distinguishes these structures from cytoplasmic stress granules. Many splicing factors and long non-coding RNA species localize in nSBs as a result of stress. Indeed, tRNAs respond to several kinds of stress such as heat, oxidation or starvation. Although nuclear accumulation of tRNAs occurs in starved Saccharomyces cerevisiae, this phenomenon is not found in mammalian cells. We observed that initiator tRNA{sup Met} (Meti) is actively translocated into the nucleus of human cells under heat stress. During this study, we identified unique granules of Meti that overlapped with nSBs. Similarly, elongator tRNA{sup Met} was translocated into the nucleus and formed granules during heat stress. Formation of tRNA granules is closely related to the translocation ratio. Then, all tRNAs may form the specific granules.

  8. Mapping the tRNA binding site on the surface of human DNMT2 methyltransferase.

    PubMed

    Jurkowski, Tomasz P; Shanmugam, Raghuvaran; Helm, Mark; Jeltsch, Albert

    2012-06-05

    The DNMT2 enzyme methylates tRNA-Asp at position C38. Because there is no tRNA-Dnmt2 cocrystal structure available, we have mapped the tRNA binding site of DNMT2 by systematically mutating surface-exposed lysine and arginine residues to alanine and studying the tRNA methylation activity and binding of the corresponding variants. After mutating 20 lysine and arginine residues, we identified eight of them that caused large (>4-fold) decreases in catalytic activity. These residues cluster within and next to a surface cleft in the protein, which is large enough to accommodate the tRNA anticodon loop and stem. This cleft is located next to the binding pocket for the cofactor S-adenosyl-L-methionine, and the catalytic residues of DNMT2 are positioned at its walls or bottom. Many of the variants with strongly reduced catalytic activity showed only a weak loss of tRNA binding or even bound better to tRNA than wild-type DNMT2, which suggests that the enzyme induces some conformational changes in the tRNA in the transition state of the methyl group transfer reaction. Manual placement of tRNA into the structure suggests that DNMT2 mainly interacts with the anticodon stem and loop.

  9. Rational design of an orthogonal tryptophanyl nonsense suppressor tRNA

    PubMed Central

    Hughes, Randall A.; Ellington, Andrew D.

    2010-01-01

    While a number of aminoacyl tRNA synthetase (aaRS):tRNA pairs have been engineered to alter or expand the genetic code, only the Methanococcus jannaschii tyrosyl tRNA synthetase and tRNA have been used extensively in bacteria, limiting the types and numbers of unnatural amino acids that can be utilized at any one time to expand the genetic code. In order to expand the number and type of aaRS/tRNA pairs available for engineering bacterial genetic codes, we have developed an orthogonal tryptophanyl tRNA synthetase and tRNA pair, derived from Saccharomyces cerevisiae. In the process of developing an amber suppressor tRNA, we discovered that the Escherichia coli lysyl tRNA synthetase was responsible for misacylating the initial amber suppressor version of the yeast tryptophanyl tRNA. It was discovered that modification of the G:C content of the anticodon stem and therefore reducing the structural flexibility of this stem eliminated misacylation by the E. coli lysyl tRNA synthetase, and led to the development of a functional, orthogonal suppressor pair that should prove useful for the incorporation of bulky, unnatural amino acids into the genetic code. Our results provide insight into the role of tRNA flexibility in molecular recognition and the engineering and evolution of tRNA specificity. PMID:20571084

  10. Site-specific crosslinking of 4-thiouridine-modified human tRNA(3Lys) to reverse transcriptase from human immunodeficiency virus type I.

    PubMed Central

    Mishima, Y; Steitz, J A

    1995-01-01

    We have mapped specific RNA-protein contacts between human immunodeficiency virus (HIV) type I reverse transcriptase (RT) and its natural primer, human tRNA(3Lys), using a site-specific crosslinking strategy. Four different tRNA(3Lys) constructs with a single 32P-labeled 4-thiouridine (4-thioU) residue at positions -1, 16, 36 or 41 were synthesized. After incubation with RT followed by irradiation, crosslinks were localized to either the p66 or p51 subunit of RT by digestion with nuclease and SDS gel fractionation. 4-thioU at position -1 or 16 transferred label to the p66 subunit almost exclusively (> 90%), whereas position 36 labeled both p66 and p51 (3:1). Position 41 yielded no detectable crosslinks. The region of p66 contacted by position -1 of tRNA(3Lys) was localized to the 203 C-terminal amino acids of RT by CNBr cleavage, whereas a 127 amino acid-CNBr peptide (residues 230-357) from both p66 and p51 was labeled by position 36. Functionality of the 4-thioU-modified tRNA(3Lys)(-1) crosslinked to RT in the presence of an RNA but not a DNA template was demonstrated by the ability of the tRNA to be extended. These results localize the 5' half of the tRNA on the interface between the two RT subunits, closer to the RNase H domain than to the polymerase active site, in accord with previous suggestions. They argue further that a specific binding site for the 5' end of the primer tRNA(3Lys) may exist within the C-terminal portion of the p66 subunit, which could be important for the initiation of reverse transcription. Images PMID:7540137

  11. Chemical Carcinogen-Induced Changes in tRNA Metabolism in Human Cells.

    DTIC Science & Technology

    1983-11-30

    8b. OFFICE SYMBOL 9. PROCUREMENT INSTRUMENT IDENTIFICATION NUMBER ORGANIZATION (If oppl l/.et - Air Force Ofc. of Scientific Res. f/ I Be. ADDRESS...phenotype. Recent studies have also led to the identification of an enzyme which incorporates hypoxanthine into mature tRNA macromolecules. The...observation consistent with our original hypothesis. Investigations into tRNA modifications within the anticodon region have led to the identification of an

  12. Human lysyl-tRNA synthetase is secreted to trigger proinflammatory response

    PubMed Central

    Park, Sang Gyu; Kim, Hye Jin; Min, You Hong; Choi, Eung-Chil; Shin, Young Kee; Park, Bum-Joon; Lee, Sang Won; Kim, Sunghoon

    2005-01-01

    Although aminoacyl-tRNA synthetases (ARSs) are essential for protein synthesis, they also function as regulators and signaling molecules in diverse biological processes. Here, we screened 11 different human ARSs to identify the enzyme that is secreted as a signaling molecule. Among them, we found that lysyl-tRNA synthetase (KRS) was secreted from intact human cells, and its secretion was induced by TNF-α. The secreted KRS bound to macrophages and peripheral blood mononuclear cells to enhance the TNF-α production and their migration. The mitogen-activated protein kinases, extracellular signal-regulated kinase and p38 mitogen-activated protein kinase, and Gαi were determined to be involved in the signal transduction triggered by KRS. All of these activities demonstrate that human KRS may work as a previously uncharacterized signaling molecule, inducing immune response through the activation of monocyte/macrophages. PMID:15851690

  13. Comparison of effects of aspirin and indomethacin on human platelet prostaglandin synthetase.

    PubMed Central

    Crook, D; Collins, A J

    1977-01-01

    Human platelets were incubated in vitro with either aspirin or indomethacin and the prostaglandin synthetase activity of the resultant microsomal fraction from each incubation measured using a radiometric technique. Whereas aspirin produced a dose-related inhibition of the enzyme, indomethacin produced little or no inhibition over the same concentration range (10(-6) mol/l--10(-3) mol/l). Furthermore, administration of aspirin (600 mg) to volunteers produced a highly significant, prolonged inhibition of platelet microsomal prostaglandin synthetase whereas no inhibition was found with indomethacin (50 mg). As indomethacin is considerably more potent than aspirin as an inhibitor of human platelet prostaglandin synthetase in vitro, the results suggest a fundamental difference in the nature of the inhibition produced by each drug, aspirin being an essentially irreversible inhibitor whereas the inhibition produced by indomethacin is reversible. Studies with [3H-acetyl] aspirin have confirmed previous findings (Roth and Majerus, 1975) that aspirin produces an irreversible acetylation of a particulate fraction protein from human platelets. PMID:411427

  14. Structure of Human Phosphopantothenoylcysteine Synthetase at 2.3 Å Resolution

    SciTech Connect

    Manoj, N.; Strauss, E.; Begley, T.P.; Ealick, S.E.

    2010-12-01

    The structure of human phosphopantothenoylcysteine (PPC) synthetase was determined at 2.3 {angstrom} resolution. PPC synthetase is a dimer with identical monomers. Some features of the monomer fold resemble a group of NAD-dependent enzymes, while other features resemble the ribokinase fold. The ATP, phosphopantothenate, and cysteine binding sites were deduced from modeling studies. Highly conserved ATP binding residues include Gly43, Ser61, Gly63, Gly66, Phe230, and Asn258. Highly conserved phosphopantothenate binding residues include Asn59, Ala179, Ala180, and Asp183 from one monomer and Arg55 from the adjacent monomer. The structure predicts a ping pong mechanism with initial formation of an acyladenylate intermediate, followed by release of pyrophosphate and attack by cysteine to form the final products PPC and AMP.

  15. A fluorescence-based coupling reaction for monitoring the activity of recombinant human NAD synthetase.

    PubMed

    Bembenek, Michael E; Kuhn, Eric; Mallender, William D; Pullen, Lester; Li, Ping; Parsons, Thomas

    2005-10-01

    NAD synthetase is responsible for the conversion of nicotinic acid adenine dinucleotide to nicotinamide adenine dinucleotide. This reaction provides a biosynthetic route of the coenzyme and, thus, a source of cellular reducing equivalents. Alterations in the oxidative reductive potential of the cell have been implicated as a contributing factor in many disease states. Thus, this enzyme represents a new class of potential drug targets, and, hence, our efforts were focused upon developing a robust assay for utilization in a high throughput screen. Toward that end, we describe a coupled enzyme assay format for the measurement of recombinant human NAD synthetase by employing lactate dehydrogenase in a cycling/amplification reaction linked ultimately to the fluorescence generation of resorufin from resazurin via diaphorase. We present kinetics of the reaction of NAD synthetase in the coupled assay format, optimization conditions, and inhibition of the reaction by gossypol [1,1',6,6',7,7'-hexahydroxy-3,3'-dimethyl-5,5'-bis(1-methylethyl)-[2,2'- binaphthalene]-8,8'-dicarboxaldehyde] and illustrate the robustness of the assay by demonstrating 384-well microtiter plate uniformity statistics. Collectively, our results show that the assay method is both robust and well suited for this class of enzymes involved in the NAD+ biosynthetic pathway.

  16. Localization of two human autoantigen genes by PCR screening and in situ hybridization-glycyl-tRNA synthetase locates to 7p15 and Alanyl-tRNA synthetase locates to 16q22

    SciTech Connect

    Nichols, R.C.; Pai, S.I.; Liu, P.; Ge, Q.; Targoff, I.N.

    1995-11-01

    Aminoacyl-tRNA synthetases (aminoacyl-RS) catalyze the attachment of an amino acid to its cognate tRNA. Five of 20 human aminoacyl-RS (histidyl-RS, threonyl-RS, isoleucyl-RS, glycyl-RS, and alanyl-RS) have been identified as targets of autoantibodies in the autoimmune disease polymyositis/dermatomyositis (PM/DM; 9). A sixth autoantigenic amino-acyl-RS, lysyl-RS, was recently reported. The genes for histidyl-RS and threonyl-RS have been assigned to chromosome 5, as have the genes for leucyl-RS and arginyl-RS. Six other aminoacyl-RS (glutamyl-prolyl-RS, valyl-RS, cysteinyl-RS, methionyl-RS, tryptophanyl-RS, and asparaginyl-RS) were assigned to chromosomes 1, 6, 11, 12, 14, and 18, respectively. The reason for a preponderance of aminoacyl-RS genes on chromosome 5 is unknown, but it has been suggested that regulatory relatedness might be a factor. Recently the entire or partial cDNA sequences for two autoantigenic aminoacyl-RS genes, glycyl-RS (gene symbol GARS; 4) and alanyl-RS (gene symbol AARS; 1), were reported. To understand further the genesis of autoimmune responses to aminoacyl-RS and to determine whether genes for autoantigenic aminoacyl-RS colocalize to chromosome 5, we have determined the chromosomal site of the GARS and AARS genes by PCR-based screening of somatic cell hybrid panels and by fluorescence in situ hybridization (FISH) analysis. 10 refs., 1 fig.

  17. Inhibition of human estrogen synthetase (aromatase) by flavones.

    PubMed

    Kellis, J T; Vickery, L E

    1984-09-07

    Several naturally occurring and synthetic flavones were found to inhibit the aromatization of androstenedione and testosterone to estrogens catalyzed by human placental and ovarian microsomes. These flavones include (in order of decreasing potency) 7,8-benzoflavone, chrysin, apigenin, flavone, flavanone, and quercetin; 5,6-benzoflavone was not inhibitory. 7,8-Benzoflavone and chrysin were potent competitive inhibitors and induced spectral changes in the aromatase cytochrome P-450 indicative of substrate displacement. Flavones may thus compete with steroids in their interaction with certain monooxygenases and thereby alter steroid hormone metabolism.

  18. Inhibition of human glutamine synthetase by L-methionine-S,R-sulfoximine-relevance to the treatment of neurological diseases.

    PubMed

    Jeitner, Thomas M; Cooper, Arthur J L

    2014-12-01

    At high concentrations, the glutamine synthetase inhibitor L-methionine-S,R-sulfoximine (MSO) is a convulsant, especially in dogs. Nevertheless, sub-convulsive doses of MSO are neuroprotective in rodent models of hyperammonemia, acute liver disease, and amyotrophic lateral sclerosis and suggest MSO may be clinically useful. Previous work has also shown that much lower doses of MSO are required to produce convulsions in dogs than in primates. Evidence from the mid-20th century suggests that humans are also less sensitive. In the present work, the inhibition of recombinant human glutamine synthetase by MSO is shown to be biphasic-an initial reversible competitive inhibition (K i 1.19 mM) is followed by rapid irreversible inactivation. This K i value for the human enzyme accounts, in part, for relative insensitivity of primates to MSO and suggests that this inhibitor could be used to safely inhibit glutamine synthetase activity in humans.

  19. Exploring the Catalytic Mechanism of Human Glutamine Synthetase by Computer Simulations.

    PubMed

    Issoglio, Federico M; Campolo, Nicolas; Zeida, Ari; Grune, Tilman; Radi, Rafael; Estrin, Dario A; Bartesaghi, Silvina

    2016-10-13

    Glutamine synthetase is an important enzyme that catalyzes the ATP-dependent formation of glutamine from glutamate and ammonia. In mammals, it plays a key role in preventing excitotoxicity in the brain and detoxifying ammonia in the liver. In plants and bacteria, it is fundamental for nitrogen metabolism, being critical for the survival of the organism. In this work, we show how the use of classical molecular dynamics simulations and multiscale quantum mechanics/molecular mechanics simulations allowed us to examine the structural properties and dynamics of human glutamine synthetase (HsGS), as well as the reaction mechanisms involved in the catalytic process with atomic level detail. Our results suggest that glutamine formation proceeds through a two-step mechanism that includes a first step in which the γ-glutamyl phosphate intermediate forms, with a 5 kcal/mol free energy barrier and a -8 kcal/mol reaction free energy, and then a second rate-limiting step involving the ammonia nucleophilic attack, with a free energy barrier of 19 kcal/mol and a reaction free energy of almost zero. A detailed analysis of structural features within each step exposed the relevance of the acid-base equilibrium related to protein residues and substrates in the thermodynamics and kinetics of the reactions. These results provide a comprehensive study of HsGS dynamics and establish the groundwork for further analysis regarding changes in HsGS activity, as occur in natural variants and post-translational modifications.

  20. The mRNA of human cytoplasmic arginyl-tRNA synthetase recruits prokaryotic ribosomes independently.

    PubMed

    Yang, Fang; Ji, Quan-Quan; Ruan, Liang-Liang; Ye, Qing; Wang, En-Duo

    2014-07-25

    There are two isoforms of cytoplasmic arginyl-tRNA synthetase (hcArgRS) in human cells. The long form is a component of the multiple aminoacyl-tRNA synthetase complex, and the other is an N-terminal truncated form (NhcArgRS), free in the cytoplasm. It has been shown that the two forms of ArgRS arise from alternative translational initiation in a single mRNA. The short form is produced from the initiation at a downstream, in-frame AUG start codon. Interestingly, our data suggest that the alternative translational initiation of hcArgRS mRNA also takes place in Escherichia coli transformants. When the gene encoding full-length hcArgRS was overexpressed in E. coli, two forms of hcArgRS were observed. The N-terminal sequencing experiment identified that the short form was identical to the NhcArgRS in human cytoplasm. By constructing a bicistronic system, our data support that the mRNA encoding the N-terminal extension of hcArgRS has the capacity of independently recruiting E. coli ribosomes. Furthermore, two critical elements for recruiting prokaryotic ribosomes were identified, the “AGGA” core of the Shine-Dalgarno sequence and the “A-rich” sequence located just proximal to the alternative in-frame initiation site. Although the mechanisms of prokaryotic and eukaryotic translational initiation are distinct, they share some common features. The ability of the hcArgRS mRNA to recruit the prokaryotic ribosome may provide clues for shedding light on the mechanism of alternative translational initiation of hcArgRS mRNA in eukaryotic cells.

  1. Distribution of immunoreactive glutamine synthetase in the adult human and mouse brain. Qualitative and quantitative observations with special emphasis on extra-astroglial protein localization.

    PubMed

    Bernstein, Hans-Gert; Bannier, Jana; Meyer-Lotz, Gabriela; Steiner, Johann; Keilhoff, Gerburg; Dobrowolny, Henrik; Walter, Martin; Bogerts, Bernhard

    2014-11-01

    Glutamine synthetase catalyzes the ATP-dependent condensation of ammonia and glutamate to form glutamine, thus playing a pivotal role in glutamate and glutamine homoeostasis. Despite a plethora of studies on this enzyme, knowledge about the regional and cellular distribution of this enzyme in human brain is still fragmentary. Therefore, we mapped fourteen post-mortem brains of psychically healthy individuals for the distribution of the glutamine synthetase immunoreactive protein. It was found that glutamine synthetase immunoreactivity is expressed in multiple gray and white matter astrocytes, but also in oligodendrocytes, ependymal cells and certain neurons. Since a possible extra-astrocytic expression of glutamine synthetase is highly controversial, we paid special attention to its appearance in oligodendrocytes and neurons. By double immunolabeling of mouse brain slices and cultured mouse brain cells for glutamine synthetase and cell-type-specific markers we provide evidence that besides astrocytes subpopulations of oligodendrocytes, microglial cells and neurons express glutamine synthetase. Moreover, we show that glutamine synthetase-immunopositive neurons are not randomly distributed throughout human and mouse brain, but represent a subpopulation of nitrergic (i.e. neuronal nitric oxide synthase expressing) neurons. Possible functional implications of an extra-astrocytic localization of glutamine synthetase are discussed.

  2. An exposed cysteine residue of human angiostatic mini tryptophanyl-tRNA synthetase.

    PubMed

    Wakasugi, Keisuke

    2010-04-13

    Human tryptophanyl-tRNA synthetase (TrpRS) catalyzes the aminoacylation of tRNA(Trp). Human TrpRS exists in two forms: a major form that is the full-length protein and a truncated form (mini TrpRS) in which most of the N-terminal extension is absent. Human mini, but not full-length, TrpRS has angiostatic activity. Because the full-length protein, which lacks angiostatic activity, has all of the amino acid determinants of the mini form, which has activity, I searched for conformational differences between the two proteins. Using a disulfide cross-linking assay, I showed that the molecular environment around Cys62 is significantly different between the two proteins. This difference can be explained by inspection of the three-dimensional structure of the full-length protein. These results give a clear demonstration of a significant difference, around a specific residue (Cys62), between a potent angiostatic and nonangiostatic version of human TrpRS.

  3. Congenital Visual Impairment and Progressive Microcephaly Due to Lysyl-Transfer Ribonucleic Acid (RNA) Synthetase (KARS) Mutations: The Expanding Phenotype of Aminoacyl-Transfer RNA Synthetase Mutations in Human Disease.

    PubMed

    McMillan, Hugh J; Humphreys, Peter; Smith, Amanda; Schwartzentruber, Jeremy; Chakraborty, Pranesh; Bulman, Dennis E; Beaulieu, Chandree L; Majewski, Jacek; Boycott, Kym M; Geraghty, Michael T

    2015-07-01

    Aminoacyl-transfer ribonucleic acid (RNA) synthetases (ARSs) are a group of enzymes required for the first step of protein translation. Each aminoacyl-transfer RNA synthetase links a specific amino acid to its corresponding transfer RNA component within the cytoplasm, mitochondria, or both. Mutations in ARSs have been linked to a growing number of diseases. Lysyl-transfer RNA synthetase (KARS) links the amino acid lysine to its cognate transfer RNA. We report 2 siblings with severe infantile visual loss, progressive microcephaly, developmental delay, seizures, and abnormal subcortical white matter. Exome sequencing identified mutations within the KARS gene (NM_005548.2):c.1312C>T; p.Arg438Trp and c.1573G>A; p.Glu525Lys occurring within a highly conserved region of the catalytic domain. Our patients' phenotype is remarkably similar to a phenotype recently reported in glutaminyl-transfer RNA synthetase (QARS), another bifunctional ARS gene. This finding expands the phenotypic spectrum associated with mutations in KARS and draws attention to aminoacyl-transfer RNA synthetase as a group of enzymes that are increasingly being implicated in human disease.

  4. Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase

    SciTech Connect

    Brizio, Carmen; Galluccio, Michele; Wait, Robin; Torchetti, Enza Maria; Bafunno, Valeria; Accardi, Rosita; Gianazza, Elisabetta; Indiveri, Cesare; Barile, Maria . E-mail: m.barile@biologia.uniba.it

    2006-06-09

    FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63 kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60 kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl{sub 2}, as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8 {+-} 1.3 nmol of FAD synthesized/min/mg protein and exhibited a K {sub M} value for FMN of 1.5 {+-} 0.3 {mu}M. This is First report on characterization of human FADS, and First cloning and over-expression of FADS from an organism higher than yeast.

  5. Molecular cloning and chromosomal localization of human holocarboxylase synthetase, a gene responsible for biotin dependency

    SciTech Connect

    Suzuki, Y.; Aoki, Y.; Ishida, Y.

    1994-09-01

    Holocarboxylase synthetase (HCS) catalyzes biotin incorporation into various carboxylases that require biotin as a prosthetic group. They are acetyl-CoA carboxylase, a rate-limiting enzyme of fatty acid synthesis; pyruvate carboxylase, a key enzyme of gluconeogenesis; propionyl-CoA carboxylase and 3-methylcrotonyl-CoA carboxylase, enzymes involved in amino acid catabolism. HCS is therefore involved in various metabolic processes and is a key enzyme for biotin utilization by mammalian cells. Deficiency of HCS in man is known to cause biotin-responsive multiple carboxylase deficiency. Isolation of cDNA clones for the enzyme is essential to understand HCS and its deficiency at the molecular level. We purified bovine liver HCS and sequenced its proteolytic peptides. Degenerative oligonucleotide primers were synthesized from the two peptide sequences and used to amplify a putative HCS cDNA fragment from human liver by PCR. Using the amplified DNA fragment as a probe, we screened {lambda}gt10 human liver cDNA library and isolated 12 positive clones. The isolated cDNAs encoded a protein of 726 amino acids with molecular mass of 80,759. The protein contained several sequences identical or similar to those of peptides derived from the bovine liver HCS. The predicted protein had a homologous region with BirA which acts as both a biotin-[acetyl-CoA-carboxylase] ligase and a biotin repressor in E. coli, suggesting a functional relationship between the two proteins. We expressed the protein using pET3 a vector in E. coli (BL21 strain) and raised antiserum against the expressed protein. The antiserum immunoprecipitated HCS activities of human lymphoblasts and bovine liver. A one-base deletion and a missense mutation were found in cells from siblings with HCS deficiency. The human HCS gene was assigned to chromosome 21, region 21q22.1 by fluorescence in situ hybridization analysis.

  6. Chemical validation of trypanothione synthetase: a potential drug target for human trypanosomiasis.

    PubMed

    Torrie, Leah S; Wyllie, Susan; Spinks, Daniel; Oza, Sandra L; Thompson, Stephen; Harrison, Justin R; Gilbert, Ian H; Wyatt, Paul G; Fairlamb, Alan H; Frearson, Julie A

    2009-12-25

    In the search for new therapeutics for the treatment of human African trypanosomiasis, many potential drug targets in Trypanosoma brucei have been validated by genetic means, but very few have been chemically validated. Trypanothione synthetase (TryS; EC 6.3.1.9; spermidine/glutathionylspermidine:glutathione ligase (ADP-forming)) is one such target. To identify novel inhibitors of T. brucei TryS, we developed an in vitro enzyme assay, which was amenable to high throughput screening. The subsequent screen of a diverse compound library resulted in the identification of three novel series of TryS inhibitors. Further chemical exploration resulted in leads with nanomolar potency, which displayed mixed, uncompetitive, and allosteric-type inhibition with respect to spermidine, ATP, and glutathione, respectively. Representatives of all three series inhibited growth of bloodstream T. brucei in vitro. Exposure to one of our lead compounds (DDD86243; 2 x EC(50) for 72 h) decreased intracellular trypanothione levels to <10% of wild type. In addition, there was a corresponding 5-fold increase in the precursor metabolite, glutathione, providing strong evidence that DDD86243 was acting on target to inhibit TryS. This was confirmed with wild-type, TryS single knock-out, and TryS-overexpressing cell lines showing expected changes in potency to DDD86243. Taken together, these data provide initial chemical validation of TryS as a drug target in T. brucei.

  7. Human selenophosphate synthetase 1 has five splice variants with unique interactions, subcellular localizations and expression patterns

    SciTech Connect

    Kim, Jin Young; Lee, Kwang Hee; Shim, Myoung Sup; Shin, Hyein; Xu, Xue-Ming; Carlson, Bradley A.; Hatfield, Dolph L.; Lee, Byeong Jae

    2010-06-18

    Selenophosphate synthetase 1 (SPS1) is an essential cellular gene in higher eukaryotes. Five alternative splice variants of human SPS1 (major type, {Delta}E2, {Delta}E8, +E9, +E9a) were identified wherein +E9 and +E9a make the same protein. The major type was localized in both the nuclear and plasma membranes, and the others in the cytoplasm. All variants form homodimers, and in addition, the major type forms a heterodimer with {Delta}E2, and {Delta}E8 with +E9. The level of expression of each splice variant was different in various cell lines. The expression of each alternative splice variant was regulated during the cell cycle. The levels of the major type and {Delta}E8 were gradually increased until G2/M phase and then gradually decreased. {Delta}E2 expression peaked at mid-S phase and then gradually decreased. However, +E9/+E9a expression decreased gradually after cell cycle arrest. The possible involvement of SPS1 splice variants in cell cycle regulation is discussed.

  8. Resveratrol compounds inhibit human holocarboxylase synthetase and cause a lean phenotype in Drosophila melanogaster

    PubMed Central

    Cordonier, Elizabeth L.; Adjam, Riem; Camara Teixeira, Daniel; Onur, Simone; Zbasnik, Richard; Read, Paul E.; Döring, Frank; Schlegel, Vicki L.; Zempleni, Janos

    2015-01-01

    Holocarboxylase synthetase (HLCS) is the sole protein-biotin ligase in the human proteome. HLCS has key regulatory functions in intermediary metabolism, including fatty acid metabolism, and in gene repression through epigenetic mechanisms. The objective of this study was to identify foodborne inhibitors of HLCS that alter HLCS-dependent pathways in metabolism and gene regulation. When libraries of extracts from natural products and chemically pure compounds were screened for HLCS inhibitor activity, resveratrol compounds in grape materials caused an HLCS inhibition of >98% in vitro. The potency of these compounds was piceatannol > resveratrol > piceid. Grape-borne compounds other than resveratrol metabolites also contributed toward HLCS inhibition, e.g., p-coumaric acid and cyanidin chloride. HLCS inhibitors had meaningful effects on body fat mass. When Drosophila melanogaster brummer mutants, which are genetically predisposed to storing excess amounts of lipids, were fed diets enriched with grape leaf extracts and piceid, body fat mass decreased by more than 30% in males and females. However, Drosophila responded to inhibitor treatment with an increase in the expression of HLCS, which elicited an increase in the abundance of biotinylated carboxylases in vivo. We conclude that mechanisms other than inhibition of HLCS cause body fat loss in flies. We propose that the primary candidate is the inhibition of the insulin receptor/Akt signaling pathway. PMID:26303405

  9. Neurodegenerative disease-associated mutants of a human mitochondrial aminoacyl-tRNA synthetase present individual molecular signatures

    PubMed Central

    Sauter, Claude; Lorber, Bernard; Gaudry, Agnès; Karim, Loukmane; Schwenzer, Hagen; Wien, Frank; Roblin, Pierre; Florentz, Catherine; Sissler, Marie

    2015-01-01

    Mutations in human mitochondrial aminoacyl-tRNA synthetases are associated with a variety of neurodegenerative disorders. The effects of these mutations on the structure and function of the enzymes remain to be established. Here, we investigate six mutants of the aspartyl-tRNA synthetase correlated with leukoencephalopathies. Our integrated strategy, combining an ensemble of biochemical and biophysical approaches, reveals that mutants are diversely affected with respect to their solubility in cellular extracts and stability in solution, but not in architecture. Mutations with mild effects on solubility occur in patients as allelic combinations whereas those with strong effects on solubility or on aminoacylation are necessarily associated with a partially functional allele. The fact that all mutations show individual molecular and cellular signatures and affect amino acids only conserved in mammals, points towards an alternative function besides aminoacylation. PMID:26620921

  10. Effects of single-nucleotide polymorphisms in the human holocarboxylase synthetase gene on enzyme catalysis.

    PubMed

    Esaki, Shingo; Malkaram, Sridhar A; Zempleni, Janos

    2012-04-01

    Holocarboxylase synthetase (HLCS) is a biotin protein ligase, which has a pivotal role in biotin-dependent metabolic pathways and epigenetic phenomena in humans. Knockdown of HLCS produces phenotypes such as heat susceptibility and decreased life span in Drosophila melanogaster, whereas knockout of HLCS appears to be embryonic lethal. HLCS comprises 726 amino acids in four domains. More than 2500 single-nucleotide polymorphisms (SNPs) have been identified in human HLCS. Here, we tested the hypotheses that HLCS SNPs impair enzyme activity, and that biotin supplementation restores the activities of HLCS variants to wild-type levels. We used an in silico approach to identify five SNPs that alter the amino acid sequence in the N-terminal, central, and C-terminal domains in human HLCS. Recombinant HLCS was used for enzyme kinetics analyses of HLCS variants, wild-type HLCS, and the L216R mutant, which has a biotin ligase activity near zero. The biotin affinity of variant Q699R is lower than that of the wild-type control, but the maximal activity was restored to that of wild-type HLCS when assay mixtures were supplemented with biotin. In contrast, the biotin affinities of HLCS variants V96F and G510R are not significantly different from the wild-type control, but their maximal activities remained moderately lower than that of wild-type HLCS even when assay mixtures were supplemented with biotin. The V96 L SNP did not alter enzyme kinetics. Our findings suggest that individuals with HLCS SNPs may benefit from supplemental biotin, yet to different extents depending on the genotype.

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

    PubMed

    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.

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

    PubMed Central

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

    2013-01-01

    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. Crystal structure of tetrameric form of human lysyl-tRNA synthetase: Implications for multisynthetase complex formation

    SciTech Connect

    Guo, Min; Ignatov, Michael; Musier-Forsyth, Karin; Schimmel, Paul; Yang, Xiang-Lei

    2008-09-17

    In mammals, many aminoacyl-tRNA synthetases are bound together in a multisynthetase complex (MSC) as a reservoir of procytokines and regulation molecules for functions beyond aminoacylation. The {alpha}{sub 2} homodimeric lysyl-tRNA synthetase (LysRS) is tightly bound in the MSC and, under specific conditions, is secreted to trigger a proinflammatory response. Results by others suggest that {alpha}{sub 2} LysRS is tightly bound into the core of the MSC with homodimeric {beta}{sub 2} p38, a scaffolding protein that itself is multifunctional. Not understood is how the two dimeric proteins combine to make a presumptive {alpha}{sub 2}{beta}{sub 2} heterotetramer and, in particular, the location of the surfaces on LysRS that would accommodate the p38 interactions. Here we present a 2.3-{angstrom} crystal structure of a tetrameric form of human LysRS. The relatively loose (as seen in solution) tetramer interface is assembled from two eukaryote-specific sequences, one in the catalytic- and another in the anticodon-binding domain. This same interface is predicted to provide unique determinants for interaction with p38. The analyses suggest how the core of the MSC is assembled and, more generally, that interactions and functions of synthetases can be built and regulated through dynamic protein-protein interfaces. These interfaces are created from small adaptations to what is otherwise a highly conserved (through evolution) polypeptide sequence.

  14. Increased expression of argininosuccinate synthetase protein predicts poor prognosis in human gastric cancer.

    PubMed

    Shan, Yan-Shen; Hsu, Hui-Ping; Lai, Ming-Derg; Yen, Meng-Chi; Luo, Yi-Pey; Chen, Yi-Ling

    2015-01-01

    Aberrant expression of argininosuccinate synthetase (ASS1, also known as ASS) has been found in cancer cells and is involved in the carcinogenesis of gastric cancer. The aim of the present study was to investigate the level of ASS expression in human gastric cancer and to determine the possible correlations between ASS expression and clinicopathological findings. Immunohistochemistry was performed on paraffin‑embedded tissues to determine whether ASS was expressed in 11 of 11 specimens from patients with gastric cancer. The protein was localized primarily to the cytoplasm of cancer cells and normal epithelium. In the Oncomine cancer microarray database, expression of the ASS gene was significantly increased in gastric cancer tissues. To investigate the clinicopathological and prognostic roles of ASS expression, we performed western blot analysis of 35 matched specimens of gastric adenocarcinomas and normal tissue obtained from patients treated at the National Cheng Kung University Hospital. The ratio of relative ASS expression (expressed as the ASS/β-actin ratio) in tumor tissues to that in normal tissues was correlated with large tumor size (P=0.007) and with the tumor, node, metastasis (TNM) stage of the American Joint Committee on Cancer staging system (P=0.031). Patients whose cancer had increased the relative expression of ASS were positive for perineural invasion and had poor recurrence-free survival. In summary, ASS expression in gastric cancer was associated with a poor prognosis. Further study of mechanisms to silence the ASS gene or decrease the enzymatic activity of ASS protein has the potential to provide new treatments for patients with gastric cancer.

  15. Crystal structure of human phosphoribosylpyrophosphate synthetase 1 reveals a novel allosteric site.

    PubMed

    Li, Sheng; Lu, Yongcheng; Peng, Baozhen; Ding, Jianping

    2007-01-01

    PRPP (phosphoribosylpyrophosphate) is an important metabolite essential for nucleotide synthesis and PRS (PRPP synthetase) catalyses synthesis of PRPP from R5P (ribose 5-phosphate) and ATP. The enzymatic activity of PRS is regulated by phosphate ions, divalent metal cations and ADP. In the present study we report the crystal structures of recombinant human PRS1 in complexes with SO4(2-) ions alone and with ATP, Cd2+ and SO4(2-) ions respectively. The AMP moiety of ATP binds at the ATP-binding site, and a Cd2+ ion binds at the active site and in a position to interact with the beta- and gamma-phosphates of ATP. A SO4(2-) ion, an analogue of the activator phosphate, was found to bind at both the R5P-binding site and the allosteric site defined previously. In addi-tion, an extra SO4(2-) binds at a site at the dimer interface between the ATP-binding site and the allosteric site. Binding of this SO4(2-) stabilizes the conformation of the flexible loop at the active site, leading to the formation of the active, open conformation which is essential for binding of ATP and initiation of the catalytic reaction. This is the first time that structural stabilization at the active site caused by binding of an activator has been observed. Structural and biochemical data show that mutations of some residues at this site influence the binding of SO4(2-) and affect the enzymatic activity. The results in the present paper suggest that this new SO4(2-)-binding site is a second allosteric site to regulate the enzymatic activity which might also exist in other eukaryotic PRSs (except plant PRSs of class II), but not in bacterial PRSs.

  16. N114S mutation causes loss of ATP-induced aggregation of human phosphoribosylpyrophosphate synthetase 1

    SciTech Connect

    Liu Honglin; Peng, Xiaohui; Zhao Fang; Zhang Guobin; Tao Ye; Luo Zhaofeng; Li Yang; Teng Maikun; Li Xu Wei Shiqiang

    2009-02-20

    This study examined recombinant wild-type human phosphoribosylpyrophosphate synthetase 1 (wt-PRS1, EC 2.7.6.1) and the point mutant Asn114Ser PRS1 (N114S-Mutant) in cells of a patient with primary gout. Dynamic light-scattering and sedimentation velocity experiments indicated that the monomeric wt-PRS1 in solution was assembled into hexamers after adding the substrate ATP. However, this ATP-induced aggregation effect was not observed with N114S-Mutant, which has a 50% higher enzymatic activity than that of wt-PRS1. Synchrotron radiation circular dichroism spectroscopy revealed that the point mutation causes an increase of {alpha}-helix content and a decrease of turn content. Examination of the crystal structure of wt-PRS1 indicated that 12 hydrogen bonds formed by 6 pairs of N114 and D139 have an important role in stabilizing the hexamer. We suggest that the substitution of S114 for N114 in N114S-Mutant leads to the rupture of 12 hydrogen bonds and breakage of the PO{sub 4}{sup 3-} allosteric site where PO{sub 4}{sup 3-} functions as a fixer of the ATP-binding loop. Therefore, we consider that formation of the hexamer as the structural basis of the ADP allosteric inhibition is greatly weakened by the N114S mutation, and that alteration of the ATP-binding loop conformation is the key factor in the increased activity of N114S-Mutant. These two factors could be responsible for the high level of activity of N114S-Mutant in this patient.

  17. Chemical Carcinogen-Induced Changes in tRNA Metabolism in Human Cells.

    DTIC Science & Technology

    1981-11-01

    Comparison of nucleic acid catabolism by normal human fibroblasts and fibroblasts transformed with methylazoxymethyl alcohol ( MAMA ),an activated...catabolite in long-term, pulse-chase experiments. However, the kinetics of catabolism differed, in that only the MAMA -transformed cells had generated...cells, while the free base 7-methylguanine was secreted preferentially by the MAMA -transformed cells. The transformed cells also produced small amounts

  18. Chromosomal localization of the gene for the human trifunctional enzyme, methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase.

    PubMed Central

    Rozen, R; Barton, D; Du, J; Hum, D W; MacKenzie, R E; Francke, U

    1989-01-01

    A trifunctional protein in man, 5,10-methylenetetrahydrofolate dehydrogenase-5,10-methenyltetrahydrofolate cyclohydrolase-10-formyltetrahydrofolate synthetase, catalyzes three consecutive steps in the interconversion of tetrahydrofolate derivatives; these derivatives supply one-carbon units for intermediary metabolism. Somatic cell hybridization and in situ hybridization were used to localize the functional gene coding for this protein--to human chromosome 14q24, near the c-fos and TGF-beta 3 loci. A second hybridizing sequence, possibly a pseudogene, was identified near the centromere of the X chromosome, at Xp11. Images Figure 1 PMID:2786332

  19. Spectrophotometric assays for monitoring tRNA aminoacylation and aminoacyl-tRNA hydrolysis reactions.

    PubMed

    First, Eric A; Richardson, Charles J

    2017-01-15

    Aminoacyl-tRNA synthetases play a central role in protein synthesis, catalyzing the attachment of amino acids to their cognate tRNAs. Here, we describe a spectrophotometric assay for tyrosyl-tRNA synthetase in which the Tyr-tRNA product is cleaved, regenerating the tRNA substrate. As tRNA is the limiting substrate in the assay, recycling it substantially increases the sensitivity of the assay while simultaneously reducing its cost. The tRNA aminoacylation reaction is monitored spectrophotometrically by coupling the production of AMP to the conversion of NAD(+) to NADH. We have adapted the tyrosyl-tRNA synthetase assay to monitor: (1) aminoacylation of tRNA by l- or d-tyrosine, (2) cyclodipeptide formation by cyclodipeptide synthases, (3) hydrolysis of d-aminoacyl-tRNAs by d-tyrosyl-tRNA deacylase, and (4) post-transfer editing by aminoacyl-tRNA synthetases. All of these assays are continuous and homogenous, making them amenable for use in high-throughput screens of chemical libraries. In the case of the cyclodipeptide synthase, d-tyrosyl-tRNA deacylase, and post-transfer editing assays, the aminoacyl-tRNAs are generated in situ, avoiding the need to synthesize and purify aminoacyl-tRNA substrates prior to performing the assays. Lastly, we describe how the tyrosyl-tRNA synthetase assay can be adapted to monitor the activity of other aminoacyl-tRNA synthetases and how the approach to regenerating the tRNA substrate can be used to increase the sensitivity and decrease the cost of commercially available aminoacyl-tRNA synthetase assays.

  20. Multiple adaptive mechanisms affect asparagine synthetase substrate availability in asparaginase-resistant MOLT-4 human leukaemia cells.

    PubMed Central

    Aslanian, A M; Kilberg, M S

    2001-01-01

    Childhood acute lymphoblastic leukaemia is treated by combination chemotherapy with a number of drugs, almost always including the enzyme L-asparaginase (ASNase). Although the initial remission rate is quite high, relapse and associated drug resistance remain a problem. In vitro studies have demonstrated an adaptive increase in asparagine synthetase (AS) expression in ASNase-resistant cells, which is believed to permit ASNase-resistant human leukaemia cells to survive in vivo. The present results, obtained with ASNase-sensitive and -resistant human MOLT-4 leukaemia cell lines, illustrate that several other adaptive processes occur to provide sufficient amounts of the AS substrates, aspartate and glutamine, required to support this increased enzymic activity. In both cell populations, aspartate is derived almost exclusively from intracellular sources, whereas the necessary glutamine arises from both intracellular and extracellular sources. Transport of glutamine into ASNase-resistant cells is significantly enhanced compared with the parental cells, whereas amino acid efflux (e.g. asparagine) is reduced. Most of the adaptive change for the amino acid transporters, Systems A, ASC and L, is rapidly (12 h) reversed following ASNase removal. The enzymic activity of glutamine synthetase is also enhanced in ASNase-resistant cells by a post-transcriptional mechanism. The results demonstrate that there are several sites of metabolic adaptation in ASNase-treated leukaemia cells that serve to promote the replenishment of both glutamine and asparagine. PMID:11485552

  1. Elaborate uORF/IRES features control expression and localization of human glycyl-tRNA synthetase

    PubMed Central

    Alexandrova, Jana; Paulus, Caroline; Rudinger-Thirion, Joëlle; Jossinet, Fabrice; Frugier, Magali

    2015-01-01

    The canonical activity of glycyl-tRNA synthetase (GARS) is to charge glycine onto its cognate tRNAs. However, outside translation, GARS also participates in many other functions. A single gene encodes both the cytosolic and mitochondrial forms of GARS but 2 mRNA isoforms were identified. Using immunolocalization assays, in vitro translation assays and bicistronic constructs we provide experimental evidence that one of these mRNAs tightly controls expression and localization of human GARS. An intricate regulatory domain was found in its 5′-UTR which displays a functional Internal Ribosome Entry Site and an upstream Open Reading Frame. Together, these elements hinder the synthesis of the mitochondrial GARS and target the translation of the cytosolic enzyme to ER-bound ribosomes. This finding reveals a complex picture of GARS translation and localization in mammals. In this context, we discuss how human GARS expression could influence its moonlighting activities and its involvement in diseases. PMID:26327585

  2. A role for [Fe4S4] clusters in tRNA recognition—a theoretical study

    PubMed Central

    Stiebritz, Martin T.

    2014-01-01

    Over the past several years, structural studies have led to the unexpected discovery of iron–sulfur clusters in enzymes that are involved in DNA replication/repair and protein biosynthesis. Although these clusters are generally well-studied cofactors, their significance in the new contexts often remains elusive. One fascinating example is a tryptophanyl-tRNA synthetase from the thermophilic bacterium Thermotoga maritima, TmTrpRS, that has recently been structurally characterized. It represents an unprecedented connection among a primordial iron–sulfur cofactor, RNA and protein biosynthesis. Here, a possible role of the [Fe4S4] cluster in tRNA anticodon-loop recognition is investigated by means of density functional theory and comparison with the structure of a human tryptophanyl-tRNA synthetase/tRNA complex. It turns out that a cluster-coordinating cysteine residue, R224, and polar main chain atoms form a characteristic structural motif for recognizing a putative 5′ cytosine or 5′ 2-thiocytosine moiety in the anticodon loop of the tRNA molecule. This motif provides not only affinity but also specificity by creating a structural and energetical penalty for the binding of other bases, such as uracil. PMID:24753428

  3. Domain movements during CCA-addition: A new function for motif C in the catalytic core of the human tRNA nucleotidyltransferases

    PubMed Central

    Ernst, Felix G M; Rickert, Christian; Bluschke, Alexander; Betat, Heike; Steinhoff, Heinz-Jürgen; Mörl, Mario

    2015-01-01

    CCA-adding enzymes are highly specific RNA polymerases that synthesize and maintain the sequence CCA at the tRNA 3′-end. This nucleotide triplet is a prerequisite for tRNAs to be aminoacylated and to participate in protein biosynthesis. During CCA-addition, a set of highly conserved motifs in the catalytic core of these enzymes is responsible for accurate sequential nucleotide incorporation. In the nucleotide binding pocket, three amino acid residues form Watson-Crick-like base pairs to the incoming CTP and ATP. A reorientation of these templating amino acids switches the enzyme's specificity from CTP to ATP recognition. However, the mechanism underlying this essential structural rearrangement is not understood. Here, we show that motif C, whose actual function has not been identified yet, contributes to the switch in nucleotide specificity during polymerization. Biochemical characterization as well as EPR spectroscopy measurements of the human enzyme reveal that mutating the highly conserved amino acid position D139 in this motif interferes with AMP incorporation and affects interdomain movements in the enzyme. We propose a model of action, where motif C forms a flexible spring element modulating the relative orientation of the enzyme's head and body domains to accommodate the growing 3′-end of the tRNA. Furthermore, these conformational transitions initiate the rearranging of the templating amino acids to switch the specificity of the nucleotide binding pocket from CTP to ATP during CCA-synthesis. PMID:25849199

  4. Domain movements during CCA-addition: a new function for motif C in the catalytic core of the human tRNA nucleotidyltransferases.

    PubMed

    Ernst, Felix G M; Rickert, Christian; Bluschke, Alexander; Betat, Heike; Steinhoff, Heinz-Jürgen; Mörl, Mario

    2015-01-01

    CCA-adding enzymes are highly specific RNA polymerases that synthesize and maintain the sequence CCA at the tRNA 3'-end. This nucleotide triplet is a prerequisite for tRNAs to be aminoacylated and to participate in protein biosynthesis. During CCA-addition, a set of highly conserved motifs in the catalytic core of these enzymes is responsible for accurate sequential nucleotide incorporation. In the nucleotide binding pocket, three amino acid residues form Watson-Crick-like base pairs to the incoming CTP and ATP. A reorientation of these templating amino acids switches the enzyme's specificity from CTP to ATP recognition. However, the mechanism underlying this essential structural rearrangement is not understood. Here, we show that motif C, whose actual function has not been identified yet, contributes to the switch in nucleotide specificity during polymerization. Biochemical characterization as well as EPR spectroscopy measurements of the human enzyme reveal that mutating the highly conserved amino acid position D139 in this motif interferes with AMP incorporation and affects interdomain movements in the enzyme. We propose a model of action, where motif C forms a flexible spring element modulating the relative orientation of the enzyme's head and body domains to accommodate the growing 3'-end of the tRNA. Furthermore, these conformational transitions initiate the rearranging of the templating amino acids to switch the specificity of the nucleotide binding pocket from CTP to ATP during CCA-synthesis.

  5. tRNA genes and the genetic code.

    PubMed

    Foltan, Jaromir S

    2008-08-07

    The genetic code describes translational assignments between codons and amino acids. tRNAs and aminoacyl-tRNA synthetases (aaRSs) are those molecules by means of which these assignments are established. Any aaRS recognizes its tRNAs according to some of their nucleotides called identity elements (IEs). Let a 1Mut-similarity Sim (1Mut) be the average similarity between such tRNA genes whose codons differ by one point mutation. We showed that: (1) a global maximum of Sim (1Mut) is reached at the standard genetic code 27 times for 4 sets of IEs of tRNA genes of eukaryotic species, while it is so only 5 times for similarities Sim (C&R) between all tRNA genes whose codons lie in the same column or row of the code. Therefore, point mutations of anticodons were tested by nature to recruit tRNAs from one isoaccepting group to another, (2) because plain similarities Sim (all) between tRNA genes of species within any of the three domains of life are higher than between tRNA genes of species belonging to different domains, tRNA genes retained information about early evolution of cells, (3) we searched the order of tRNAs in which they were most probably assigned to their codons and amino acids. The beginning Ala, (Val), Pro, Ile, Lys, Arg, Trp, Met, Asp, Cys, (Ser) of our resulting chronology lies under a plateau on a graph of Sim (1Mut,IE)(univ.ancestors) plotted over this chronology for a set S(IE) of all IEs of tRNA genes, whose universal ancestors were separately computed for each codon. This plateau has remained preserved along the whole line of evolution of the code and is consistent with observations of Ribas de Pouplana and Schimmel [2001. Aminoacy1-tRNA synthetases: potential markers of genetic code development. Trends Biochem. Sci. 26, 591-598] that specific pairs of aaRSs-one from each of their two classes-can be docked simultaneously onto the acceptor stem of tRNA and hence an interaction existed between their ancestors using a reduced code, (4) sharpness of a

  6. Expression cloning of a human cDNA encoding folylpoly(gamma-glutamate) synthetase and determination of its primary structure.

    PubMed Central

    Garrow, T A; Admon, A; Shane, B

    1992-01-01

    A human cDNA for folypoly(gamma-glutamate) synthetase [FPGS; tetrahydrofolate:L-glutamate gamma-ligase (ADP forming), EC 6.3.2.17] has been cloned by functional complementation of an Escherichia coli folC mutant. The cDNA encodes a 545-residue protein of M(r) 60,128. The deduced sequence has regions that are highly homologous to peptide sequences obtained from purified pig liver FPGS and shows limited homology to the E. coli and Lactobacillus casei FPGSs. Expression of the cDNA in E. coli results in elevated expression of an enzyme with characteristics of mammalian FPGS. Expression of the cDNA in AUXB1, a mammalian cell lacking FPGS activity, overcomes the cell's requirement for thymidine and purines but does not overcome the cell's glycine auxotrophy, consistent with expression of the protein in the cytosol but not the mitochondria. PMID:1409616

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

    PubMed Central

    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 in two other G1 ts mutants at the restrictive temperature. Cells trapped in S phase by a thymidine block also contained decreased levels of tRNA4Lys when raised to 39 degrees. Both tRNA4Lys levels and cell division increased when the cells were returned to the permissive temperature. An in vitro assay was established for the modification of tRNA5Lys to tRNA4Lys with tRNA6Lys and tRNA2Lys as intermediates. The first reaction is the synthesis of tRNA6Lys which involves the introduction of a modified uridine at the third position of the anticodon. Extracts of 694 cells grown at 33 degrees were able to modify rat liver [3H] tRNA5Lys to tRNA6Lys and tRNA4Lys in vitro when assayed at 25 degrees but not at 39 degrees. Extracts of Balb/c 3T3 cells, however, were more active at 39 degrees than at 25 degrees showing that the normal enzyme is not temperature sensitive. Ts-694 cell tRNA, isolated from cells grown at 33 degrees was aminoacylated at both 25 degrees and 39 degrees with rat liver synthetases. tRNA4Lys was present at both temperatures indicating that ts-694 cells do not contain a temperature sensitive tRNA4Lys. Images PMID:6569451

  8. A Human Disease-causing Point Mutation in Mitochondrial Threonyl-tRNA Synthetase Induces Both Structural and Functional Defects*

    PubMed Central

    Wang, Yong; Zhou, Xiao-Long; Ruan, Zhi-Rong; Liu, Ru-Juan; Eriani, Gilbert; Wang, En-Duo

    2016-01-01

    Mitochondria require all translational components, including aminoacyl-tRNA synthetases (aaRSs), to complete organelle protein synthesis. Some aaRS mutations cause mitochondrial disorders, including human mitochondrial threonyl-tRNA synthetase (hmtThrRS) (encoded by TARS2), the P282L mutation of which causes mitochondrial encephalomyopathies. However, its catalytic and structural consequences remain unclear. Herein, we cloned TARS2 and purified the wild-type and P282L mutant hmtThrRS. hmtThrRS misactivates non-cognate Ser and uses post-transfer editing to clear erroneously synthesized products. In vitro and in vivo analyses revealed that the mutation induces a decrease in Thr activation, aminoacylation, and proofreading activities and a change in the protein structure and/or stability, which might cause reduced catalytic efficiency. We also identified a splicing variant of TARS2 mRNA lacking exons 8 and 9, the protein product of which is targeted into mitochondria. In HEK293T cells, the variant does not dimerize and cannot complement the ThrRS knock-out strain in yeast, suggesting that the truncated protein is inactive and might have a non-canonical function, as observed for other aaRS fragments. The present study describes the aminoacylation and editing properties of hmtThrRS, clarifies the molecular consequences of the P282L mutation, and shows that the yeast ThrRS-deletion model is suitable to test pathology-associated point mutations or alternative splicing variants of mammalian aaRS mRNAs. PMID:26811336

  9. Mitochondrial leucine tRNA level and PTCD1 are regulated in response to leucine starvation.

    PubMed

    Schild, Christof; Hahn, Dagmar; Schaller, André; Jackson, Christopher Benjamin; Rothen-Rutishauser, Barbara; Mirkovitch, Jelena; Nuoffer, Jean-Marc

    2014-07-01

    Pentatricopeptide repeat domain protein 1 (PTCD1) is a novel human protein that was recently shown to decrease the levels of mitochondrial leucine tRNAs. The physiological role of this regulation, however, remains unclear. Here we show that amino acid starvation by leucine deprivation significantly increased the mRNA steady-state levels of PTCD1 in human hepatocarcinoma (HepG2) cells. Amino acid starvation also increased the mitochondrially encoded leucine tRNA (tRNA(Leu(CUN))) and the mRNA for the mitochondrial leucyl-tRNA synthetase (LARS2). Despite increased PTCD1 mRNA steady-state levels, amino acid starvation decreased PTCD1 on the protein level. Decreasing PTCD1 protein concentration increases the stability of the mitochondrial leucine tRNAs, tRNA(Leu(CUN)) and tRNA(Leu(UUR)) as could be shown by RNAi experiments against PTCD1. Therefore, it is likely that decreased PTCD1 protein contributes to the increased tRNA(Leu(CUN)) levels in amino acid-starved cells. The stabilisation of the mitochondrial leucine tRNAs and the upregulation of the mitochondrial leucyl-tRNA synthetase LARS2 might play a role in adaptation of mitochondria to amino acid starvation.

  10. Structure of a tryptophanyl-tRNA synthetase containing an iron–sulfur cluster

    PubMed Central

    Han, Gye Won; Yang, Xiang-Lei; McMullan, Daniel; Chong, Yeeting E.; Krishna, S. Sri; Rife, Christopher L.; Weekes, Dana; Brittain, Scott M.; Abdubek, Polat; Ambing, Eileen; Astakhova, Tamara; Axelrod, Herbert L.; Carlton, Dennis; Caruthers, Jonathan; Chiu, Hsiu-Ju; Clayton, Thomas; Duan, Lian; Feuerhelm, Julie; Grant, Joanna C.; Grzechnik, Slawomir K.; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kumar, Abhinav; Marciano, David; Miller, Mitchell D.; Morse, Andrew T.; Nigoghossian, Edward; Okach, Linda; Paulsen, Jessica; Reyes, Ron; van den Bedem, Henry; White, Aprilfawn; Wolf, Guenter; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Elsliger, Marc-André; Schimmel, Paul; Wilson, Ian A.

    2010-01-01

    A novel aminoacyl-tRNA synthetase that contains an iron–sulfur cluster in the tRNA anticodon-binding region and efficiently charges tRNA with tryptophan has been found in Thermotoga maritima. The crystal structure of TmTrpRS (tryptophanyl-tRNA synthetase; TrpRS; EC 6.1.1.2) reveals an iron–sulfur [4Fe–­4S] cluster bound to the tRNA anticodon-binding (TAB) domain and an l-­tryptophan ligand in the active site. None of the other T. maritima aminoacyl-tRNA synthetases (AARSs) contain this [4Fe–4S] cluster-binding motif (C-x 22-C-x 6-C-x 2-C). It is speculated that the iron–sulfur cluster contributes to the stability of TmTrpRS and could play a role in the recognition of the anticodon. PMID:20944229

  11. Shaping tRNA

    ERIC Educational Resources Information Center

    Priano, Christine

    2013-01-01

    This model-building activity provides a quick, visual, hands-on tool that allows students to examine more carefully the cloverleaf structure of a typical tRNA molecule. When used as a supplement to lessons that involve gene expression, this exercise reinforces several concepts in molecular genetics, including nucleotide base-pairing rules, the…

  12. Secreted human glycyl-tRNA synthetase implicated in defense against ERK-activated tumorigenesis.

    PubMed

    Park, Min Chul; Kang, Taehee; Jin, Da; Han, Jung Min; Kim, Sang Bum; Park, Yun Jung; Cho, Kiwon; Park, Young Woo; Guo, Min; He, Weiwei; Yang, Xiang-Lei; Schimmel, Paul; Kim, Sunghoon

    2012-03-13

    Although adaptive systems of immunity against tumor initiation and destruction are well investigated, less understood is the role, if any, of endogenous factors that have conventional functions. Here we show that glycyl-tRNA synthetase (GRS), an essential component of the translation apparatus, circulates in serum and can be secreted from macrophages in response to Fas ligand that is released from tumor cells. Through cadherin (CDH)6 (K-cadherin), GRS bound to different ERK-activated tumor cells, and released phosphatase 2A (PP2A) from CDH6. The activated PP2A then suppressed ERK signaling through dephosphorylation of ERK and induced apoptosis. These activities were inhibited by blocking GRS with a soluble fragment of CDH6. With in vivo administration of GRS, growth of tumors with a high level of CDH6 and ERK activation were strongly suppressed. Our results implicate a conventional cytoplasmic enzyme in translation as an intrinsic component of the defense against ERK-activated tumor formation.

  13. Identification of holocarboxylase synthetase chromatin binding sites in human mammary cell lines using the DNA adenine methyltransferase identification technology.

    PubMed

    Singh, Dipika; Pannier, Angela K; Zempleni, Janos

    2011-06-01

    Holocarboxylase synthetase (HCS) is a chromatin protein that is essential for mediating the covalent binding of biotin to histones. Biotinylation of histones plays crucial roles in the repression of genes and repeats in the human genome. We tested the feasibility of DNA adenine methyltransferase identification (DamID) technology to map HCS binding sites in human mammary cell lines. Full-length HCS was fused to DNA adenine methyltransferase (Dam) for subsequent transfection into breast cancer (MCF-7) and normal breast (MCF-10A) cells. HCS docking sites in chromatin were identified by using the unique adenine methylation sites established by Dam in the fusion construct; docking sites were unambiguously identified using methylation-sensitive digestion, cloning, and sequencing. In total, 15 novel HCS binding sites were identified in the two cell lines, and the following 4 of the 15 overlapped between MCF-7 and MCF-10A cells: inositol polyphosphate-5-phosphatase A, corticotropin hormone precursor, ribosome biogenesis regulatory protein, and leptin precursor. We conclude that DamID is a useful technology to map HCS binding sites in human chromatin and propose that the entire set of HCS binding sites could be mapped by combining DamID with microarray technology.

  14. Assignment of two human autoantigen genes-isoleucyl-tRNA synthetase locates to 9q21 and lysyl-tRNA synthetase locates to 16q23-q24

    SciTech Connect

    Nichols, R.C.; Blinder, J.; Pai, S.I.

    1996-08-15

    Protein synthesis is initiated by the attachment of amino acids to cognate tRNAs by aminoacyl-tRNA synthetases (aaRS). Five of twenty human aaRS (histidyl-RS, threonyl-RS, alanyl-RS, glycyl-RS, and isoleucyl-RS) have been identified as targets of autoantibodies in the autoimmune disease polymyositis/dermatomyositis. Autoantibodies to human lysyl-RS, a sixth autoantigenic aminoacyl-RS, were recently identified. The genes for histidyl-RS and threonyl-RS have been localized to chromosome 5, and we recently reported that the genes for alanyl-RS and glycyl-RS localize to chromosomes 16 and 7, respectively. To understand the genesis of autoimmune responses to aaRS better, we have used PCR-based screening of somatic cell hybrid panels and fluorescence in situ hybridization (FISH) to assign the genes for isoleucyl-RS and lysyl-RS. 19 refs., 1 fig.

  15. Asparagine synthetase expression alone is sufficient to induce l-asparaginase resistance in MOLT-4 human leukaemia cells.

    PubMed Central

    Aslanian, A M; Fletcher, B S; Kilberg, M S

    2001-01-01

    Childhood acute lymphoblastic leukaemia (ALL) is treated by combination chemotherapy with a number of drugs, always including the enzyme L-asparaginase (ASNase). Although the initial remission rate is quite high, relapse and associated drug resistance are a significant problem. In vitro studies have demonstrated increased asparagine synthetase (AS) expression in ASNase-resistant cells, which has led to the hypothesis that elevated AS activity permits drug-resistant survival. The data presented show that not only is elevated AS expression a property of ASNase-resistant MOLT-4 human leukaemia cells, but that short-term (12 h) treatment of the cells with ASNase causes a relatively rapid induction of AS expression. The results also document that the elevated expression of AS in ASNase-resistant cells is not fully reversible, even 6 weeks after ASNase removal from the culture medium. Furthermore, ASNase resistance, assessed as both drug-insensitive cell growth rates and decreased drug-induced apoptosis, parallels this irreversible AS expression. Mimicking the elevated AS activity in ASNase-resistant cells by overexpression of the human AS protein by stable retroviral transformation of parental MOLT4 cells is sufficient to induce the ASNase-resistance phenotype. These data document that ASNase resistance in ALL cells is a consequence of elevated AS expression and that although other drug-induced metabolic changes occur, they are secondary to the increased asparagine biosynthetic rate. PMID:11415466

  16. Knockdown of asparagine synthetase by RNAi suppresses cell growth in human melanoma cells and epidermoid carcinoma cells.

    PubMed

    Li, Hui; Zhou, Fusheng; Du, Wenhui; Dou, Jinfa; Xu, Yu; Gao, Wanwan; Chen, Gang; Zuo, Xianbo; Sun, Liangdan; Zhang, Xuejun; Yang, Sen

    2016-05-01

    Melanoma, the most aggressive form of skin cancer, causes more than 40,000 deaths each year worldwide. And epidermoid carcinoma is another major form of skin cancer, which could be studied together with melanoma in several aspects. Asparagine synthetase (ASNS) gene encodes an enzyme that catalyzes the glutamine- and ATP-dependent conversion of aspartic acid to asparagine, and its expression is associated with the chemotherapy resistance and prognosis in several human cancers. The present study aims to explore the potential role of ASNS in melanoma cells A375 and human epidermoid carcinoma cell line A431. We applied a lentivirus-mediated RNA interference (RNAi) system to study its function in cell growth of both cells. The results revealed that inhibition of ASNS expression by RNAi significantly suppressed the growth of melanoma cells and epidermoid carcinoma cells, and induced a G0/G1 cell cycle arrest in melanoma cells. Knockdown of ASNS in A375 cells remarkably downregulated the expression levels of CDK4, CDK6, and Cyclin D1, and upregulated the expression of p21. Therefore, our study provides evidence that ASNS may represent a potential therapeutic target for the treatment of melanoma.

  17. The T box mechanism: tRNA as a regulatory molecule

    PubMed Central

    Green, Nicholas J.; Grundy, Frank J.; Henkin, Tina M.

    2009-01-01

    The T box mechanism is widely used in Gram-positive bacteria to regulate expression of aminoacyl-tRNA synthetase genes and genes involved in amino acid biosynthesis and uptake. Binding of a specific uncharged tRNA to a riboswitch element in the nascent transcript causes a structural change in the transcript that promotes expression of the downstream coding sequence. In most cases, this occurs by stabilization of an antiterminator element that competes with formation of a terminator helix. Specific tRNA recognition by the nascent transcript results in increased expression of genes important for tRNA aminoacylation in response to decreased pools of charged tRNA. PMID:19932103

  18. Activation of LXR increases acyl-CoA synthetase activity through direct regulation of ACSL3 in human placental trophoblast cells.

    PubMed

    Weedon-Fekjaer, M Susanne; Dalen, Knut Tomas; Solaas, Karianne; Staff, Anne Cathrine; Duttaroy, Asim K; Nebb, Hilde Irene

    2010-07-01

    Placental fatty acid transport and metabolism are important for proper growth and development of the feto-placental unit. The nuclear receptors, liver X receptors alpha and beta (LXRalpha and LXRbeta), are key regulators of lipid metabolism in many tissues, but little is known about their role in fatty acid transport and metabolism in placenta. The current study investigates the LXR-mediated regulation of long-chain acyl-CoA synthetase 3 (ACSL3) and its functions in human placental trophoblast cells. We demonstrate that activation of LXR increases ACSL3 expression, acyl-CoA synthetase activity, and fatty acid uptake in human tropholast cells. Silencing of ACSL3 in these cells attenuates the LXR-mediated increase in acyl-CoA synthetase activity. Furthermore, we show that ACSL3 is directly regulated by LXR through a conserved LXR responsive element in the ACSL3 promoter. Our results suggest that LXR plays a regulatory role in fatty acid metabolism by direct regulation of ACSL3 in human placental trophoblast cells.

  19. Interactions between avian myeloblastosis reverse transcriptase and tRNATrp. Mapping of complexed tRNA with chemicals and nucleases.

    PubMed

    Garret, M; Romby, P; Giegé, R; Litvak, S

    1984-03-12

    The interactions between beef tRNATrp with avian myeloblastosis reverse transcriptase have been studied by statistical chemical modifications of phosphate (ethylnitrosourea) and cytidine (dimethyl sulfate) residues, as well as by digestion of complexed tRNA by Cobra venom nuclease and Neurospora crassa endonuclease. Results with nucleases and chemicals show that reverse transcriptase interacts preferentially with the D arm, the anticodon stem and the T psi stem. All these regions are located in the outside of the L-shaped structure of tRNA. This domain of interaction is different to that reported previously in the complex of beef tRNA with the cognate aminoacyl-tRNA synthetase (M. Garret et al.; Eur. J. Biochem. In press). Avian reverse transcriptase destabilizes the region of tRNA where most of the tertiary interactions maintaining the structure of tRNA are located.

  20. Interactions between avian myeloblastosis reverse transcriptase and tRNATrp. Mapping of complexed tRNA with chemicals and nucleases.

    PubMed Central

    Garret, M; Romby, P; Giegé, R; Litvak, S

    1984-01-01

    The interactions between beef tRNATrp with avian myeloblastosis reverse transcriptase have been studied by statistical chemical modifications of phosphate (ethylnitrosourea) and cytidine (dimethyl sulfate) residues, as well as by digestion of complexed tRNA by Cobra venom nuclease and Neurospora crassa endonuclease. Results with nucleases and chemicals show that reverse transcriptase interacts preferentially with the D arm, the anticodon stem and the T psi stem. All these regions are located in the outside of the L-shaped structure of tRNA. This domain of interaction is different to that reported previously in the complex of beef tRNA with the cognate aminoacyl-tRNA synthetase (M. Garret et al.; Eur. J. Biochem. In press). Avian reverse transcriptase destabilizes the region of tRNA where most of the tertiary interactions maintaining the structure of tRNA are located. Images PMID:6200830

  1. Dual Role of the Molybdenum Cofactor Biosynthesis Protein MOCS3 in tRNA Thiolation and Molybdenum Cofactor Biosynthesis in Humans*

    PubMed Central

    Chowdhury, Mita Mullick; Dosche, Carsten; Löhmannsröben, Hans-Gerd; Leimkühler, Silke

    2012-01-01

    We studied two pathways that involve the transfer of persulfide sulfur in humans, molybdenum cofactor biosynthesis and tRNA thiolation. Investigations using human cells showed that the two-domain protein MOCS3 is shared between both pathways. MOCS3 has an N-terminal adenylation domain and a C-terminal rhodanese-like domain. We showed that MOCS3 activates both MOCS2A and URM1 by adenylation and a subsequent sulfur transfer step for the formation of the thiocarboxylate group at the C terminus of each protein. MOCS2A and URM1 are β-grasp fold proteins that contain a highly conserved C-terminal double glycine motif. The role of the terminal glycine of MOCS2A and URM1 was examined for the interaction and the cellular localization with MOCS3. Deletion of the C-terminal glycine of either MOCS2A or URM1 resulted in a loss of interaction with MOCS3. Enhanced cyan fluorescent protein and enhanced yellow fluorescent protein fusions of the proteins were constructed, and the fluorescence resonance energy transfer efficiency was determined by the decrease in the donor lifetime. The cellular localization results showed that extension of the C terminus with an additional glycine of MOCS2A and URM1 altered the localization of MOCS3 from the cytosol to the nucleus. PMID:22453920

  2. A glycolytic enzyme, enolase, is recruited as a cofactor of tRNA targeting toward mitochondria in Saccharomyces cerevisiae

    PubMed Central

    Entelis, Nina; Brandina, Irina; Kamenski, Piotr; Krasheninnikov, Igor A.; Martin, Robert P.; Tarassov, Ivan

    2006-01-01

    In many organisms, mitochondria import nuclear DNA-encoded small RNAs. In yeast Saccharomyces cerevisiae, one out of two cytoplasmic isoacceptor tRNAsLys is partially addressed into the organelle. Mitochondrial targeting of this tRNA was shown to depend on interaction with the precursor of mitochondrial lysyl–tRNA synthetase, preMsk1p. However, preMsk1p alone was unable to direct tRNA targeting, suggesting the existence of additional protein factor(s). Here, we identify the glycolytic enzyme, enolase, as such a factor. We demonstrate that recombinant enolase and preMSK1p are sufficient to direct tRNA import in vitro and that depletion of enolase inhibits tRNA import in vivo. Enzymatic and tRNA targeting functions of enolase appear to be independent. Three newly characterized properties of the enolase can be related to its novel function: (1) specific affinity to the imported tRNA, (2) the ability to facilitate formation of the complex between preMsk1p and the imported tRNA, and (3) partial targeting toward the mitochondrial outer membrane. We propose a model suggesting that the cell exploits mitochondrial targeting of the enolase in order to address the tRNA toward peri-mitochondrially synthesized preMsk1p. Our results indicate an alternative molecular chaperone function of glycolytic enzyme enolase in tRNA mitochondrial targeting. PMID:16738406

  3. tRNA acceptor-stem and anticodon bases embed separate features of amino acid chemistry

    PubMed Central

    Carter, Charles W.; Wolfenden, Richard

    2016-01-01

    abstract The universal genetic code is a translation table by which nucleic acid sequences can be interpreted as polypeptides with a wide range of biological functions. That information is used by aminoacyl-tRNA synthetases to translate the code. Moreover, amino acid properties dictate protein folding. We recently reported that digital correlation techniques could identify patterns in tRNA identity elements that govern recognition by synthetases. Our analysis, and the functionality of truncated synthetases that cannot recognize the tRNA anticodon, support the conclusion that the tRNA acceptor stem houses an independent code for the same 20 amino acids that likely functioned earlier in the emergence of genetics. The acceptor-stem code, related to amino acid size, is distinct from a code in the anticodon that is related to amino acid polarity. Details of the acceptor-stem code suggest that it was useful in preserving key properties of stereochemically-encoded peptides that had developed the capacity to interact catalytically with RNA. The quantitative embedding of the chemical properties of amino acids into tRNA bases has implications for the origins of molecular biology. PMID:26595350

  4. Cloning and characterization of a putative human holocytochrome c-type synthetase gene (HCCS) isolated from the critical region for microphthalmia with linear skin defects (MLS)

    SciTech Connect

    Schaefer, L.; Ballabio, A.; Zoghbi, H.Y.

    1996-06-01

    Microphthalmia with linear skin defects syndrome (MLS) is an X-linked male-lethal disorder associated with X chromosomal rearrangements resulting in monosomy from Xpter to Xp22. Features include microphthalmia, sclerocornea, linear skin defects, and agenesis of the corpus callosum. Using a cross-species conservation strategy, an expressed sequence from the 450- to the 550-kb MLS critical region on Xp22 was identified by screening a human embryo cDNA library. Northern analysis revealed a transcript of {approx}2.6 kb in all tissues examined, with weaker expression of {approx}1.2- and {approx}5.2-kb transcripts. The strongest expression was observed in heart and skeletal muscle. Sequence analysis of a 3-kb cDNA contig revealed an 807-bp open reading frame encoding a putative 268-amino-acid-protein. Comparison of the sequence with sequences in the databases revealed homology with holocytochrome c-type synthetases, which catalyze the covalent addition of a heme group onto c-type cytochromes in the mitochondria. The c-type cytochromes are required for proper functioning of the electron transport pathway. The human gene (HGMW-approved symbol HCCS) and the corresponding murine gene characterized in this paper are the first mammalian holocytochrome c-type synthetases to be described in the literature. Because of the lack of a neuromuscular phenotype in MLS, it is uncertain whether the deletion of a mitochondrial holocytochrome synthetase would contribute to the phenotype seen in MLS. The expression pattern of this gene and knowledge about the function of holocytochrome synthetases, however, suggest that it is a good candidate for X-linked encephalomyopathies typically associated with mitochondrial dysfunction. 25 refs., 4 figs.

  5. Piecemeal Buildup of the Genetic Code, Ribosomes, and Genomes from Primordial tRNA Building Blocks

    PubMed Central

    Caetano-Anollés, Derek; Caetano-Anollés, Gustavo

    2016-01-01

    The origin of biomolecular machinery likely centered around an ancient and central molecule capable of interacting with emergent macromolecular complexity. tRNA is the oldest and most central nucleic acid molecule of the cell. Its co-evolutionary interactions with aminoacyl-tRNA synthetase protein enzymes define the specificities of the genetic code and those with the ribosome their accurate biosynthetic interpretation. Phylogenetic approaches that focus on molecular structure allow reconstruction of evolutionary timelines that describe the history of RNA and protein structural domains. Here we review phylogenomic analyses that reconstruct the early history of the synthetase enzymes and the ribosome, their interactions with RNA, and the inception of amino acid charging and codon specificities in tRNA that are responsible for the genetic code. We also trace the age of domains and tRNA onto ancient tRNA homologies that were recently identified in rRNA. Our findings reveal a timeline of recruitment of tRNA building blocks for the formation of a functional ribosome, which holds both the biocatalytic functions of protein biosynthesis and the ability to store genetic memory in primordial RNA genomic templates. PMID:27918435

  6. Mutations of Human NARS2, Encoding the Mitochondrial Asparaginyl-tRNA Synthetase, Cause Nonsyndromic Deafness and Leigh Syndrome

    PubMed Central

    Shahzad, Mohsin; Huang, Vincent H.; Qaiser, Tanveer A.; Potluri, Prasanth; Mahl, Sarah E.; Davila, Antonio; Nazli, Sabiha; Hancock, Saege; Yu, Margret; Gargus, Jay; Chang, Richard; Al-sheqaih, Nada; Newman, William G.; Abdenur, Jose; Starr, Arnold; Hegde, Rashmi; Dorn, Thomas; Busch, Anke; Park, Eddie; Wu, Jie; Schwenzer, Hagen; Flierl, Adrian; Florentz, Catherine; Sissler, Marie; Khan, Shaheen N.; Li, Ronghua; Guan, Min-Xin; Friedman, Thomas B.; Wu, Doris K.; Procaccio, Vincent; Riazuddin, Sheikh; Wallace, Douglas C.; Ahmed, Zubair M.; Huang, Taosheng; Riazuddin, Saima

    2015-01-01

    Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome. PMID:25807530

  7. Mutations of human NARS2, encoding the mitochondrial asparaginyl-tRNA synthetase, cause nonsyndromic deafness and Leigh syndrome.

    PubMed

    Simon, Mariella; Richard, Elodie M; Wang, Xinjian; Shahzad, Mohsin; Huang, Vincent H; Qaiser, Tanveer A; Potluri, Prasanth; Mahl, Sarah E; Davila, Antonio; Nazli, Sabiha; Hancock, Saege; Yu, Margret; Gargus, Jay; Chang, Richard; Al-Sheqaih, Nada; Newman, William G; Abdenur, Jose; Starr, Arnold; Hegde, Rashmi; Dorn, Thomas; Busch, Anke; Park, Eddie; Wu, Jie; Schwenzer, Hagen; Flierl, Adrian; Florentz, Catherine; Sissler, Marie; Khan, Shaheen N; Li, Ronghua; Guan, Min-Xin; Friedman, Thomas B; Wu, Doris K; Procaccio, Vincent; Riazuddin, Sheikh; Wallace, Douglas C; Ahmed, Zubair M; Huang, Taosheng; Riazuddin, Saima

    2015-03-01

    Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome.

  8. Human endomembrane H sup + pump strongly resembles the ATP-synthetase of Archaebacteria

    SciTech Connect

    Suedhof, T.C.; Stone, D.K.; Johnston, P.A.; Xie, Xiaosong ); Fried, V.A. )

    1989-08-01

    Preparations of mammalian H{sup +} pumps that acidify intracellular vesicles contain eight or nine polypeptides, ranging in size from 116 to 17 kDa. Biochemical analysis indicates that the 70- and 58-kDa polypeptides are subunits critical for ATP hydrolysis. The amino acid sequences of the major catalytic subunits (58 and 70 kDa) of the endomembrane H{sup +} pump are unknown from animal cells. The authors report here the complete sequence of the 58-kDa subunit derived from a human kidney cDNA clone and partial sequences of the 70- and 58-kDa subunits purified from clathrin-coated vesicles of bovine brain. The amino acid sequences of both proteins strongly resemble the sequences of the corresponding subunits of the vacuolar H{sup +} pumps of Archaebacteria, plants, and fungi. The archaebacterial enzyme is believed to use a H{sup +} gradient to synthesize ATP. Thus, a common ancestral protein has given rise to a H{sup +} pump that synthesizes ATP in one organism and hydrolyzes it in another and is highly conserved from prokaryotes to humans. The same pump appears to mediate the acidification of intracellular organelles, including coated vesicles, lysosomes, and secretory granules, as well as extracellular fluids such as urine.

  9. Discovery and characterization of a novel class of pyrazolopyrimidinedione tRNA synthesis inhibitors.

    PubMed

    Montgomery, Justin I; Smith, James F; Tomaras, Andrew P; Zaniewski, Richard; McPherson, Craig J; McAllister, Laura A; Hartman-Neumann, Sandra; Arcari, Joel T; Lescoe, Marykay; Gutierrez, Jemy; Yuan, Ying; Limberakis, Chris; Miller, Alita A

    2015-06-01

    A high-throughput phenotypic screen for novel antibacterial agents led to the discovery of a novel pyrazolopyrimidinedione, PPD-1, with preferential activity against methicillin-resistant Staphylococcus aureus (MRSA). Resistance mapping revealed the likely target of inhibition to be lysyl tRNA synthetase (LysRS). Preliminary structure-activity relationship (SAR) studies led to an analog, PPD-2, which gained Gram-negative antibacterial activity at the expense of MRSA activity and resistance to this compound mapped to prolyl tRNA synthetase (ProRS). These targets of inhibition were confirmed in vitro, with PPD-1 showing IC₅₀s of 21.7 and 35 μM in purified LysRS and ProRS enzyme assays, and PPD-2, 151 and 0.04 μM, respectively. The highly attractive chemical properties of these compounds combined with intriguing preliminary SAR suggest that further exploration of this compelling novel series is warranted.

  10. Multiple erythroid isoforms of human long-chain acyl-CoA synthetases are produced by switch of the fatty acid gate domains

    PubMed Central

    Soupene, Eric; Kuypers, Frans A

    2006-01-01

    Background The formation of acyl-CoA by the action of acyl-CoA synthetases plays a crucial role in membrane lipid turnover, including the plasma membrane of erythrocytes. In human, five Acyl-CoA Synthetase Long-chain (ACSL) genes have been identified with as many as 3 different transcript variants for each. Results Acyl-CoA Synthetase Long-chain member 6 (ACSL6) is responsible for activation of long-chain fatty acids in erythrocytes. Two additional transcript variants were also isolated from brain and testis. We report the expression in reticulocytes of two new variants and of the one isolated from brain. All three represented different spliced variants of a mutually exclusive exon pair. They encode a slightly different short motif which contains a conserved structural domain, the fatty acid Gate domain. The motifs differ in the presence of either the aromatic residue phenylalanine (Phe) or tyrosine (Tyr). Based on homology, two new isoforms for the closely related ACSL1 were predicted and characterized. One represented a switch of the Phe- to the Tyr-Gate domain motif, the other resulted from the exclusion of both. Swapping of this motif also appears to be common in all mammalian ACSL member 1 and 6 homologs. Conclusion We propose that a Phe to Tyr substitution or deletion of the Gate domain, is the structural reason for the conserved alternative splicing that affects these motifs. Our findings support our hypothesis that this region is structurally important to define the activity of these enzymes. PMID:16834775

  11. Studies on the thiol group of lactose synthetase A protein from human milk and on the binding of uridine diphosphate galactose to the enzyme

    PubMed Central

    Kitchen, B. J.; Andrews, P.

    1974-01-01

    The lactose synthetase activity of A protein from human milk was much decreased but not abolished by reaction with thiol-group reagents. Protection experiments indicated that a free thiol group on the enzyme is situated near the UDP-galactose binding site and inactivation of the enzyme with p-hydroxymercuribenzoate was probably due to prevention of UDP-galactose binding. Affinity chromatography showed that the mercuribenzoate substituent also decreased the affinity of A protein for N-acetylglucosamine but complex-formation between A protein–N-acetylglucosamine and α-lactalbumin was relatively unaffected. UDP-galactose appears to be bound to the enzyme mainly through its pyrophosphate group with Mn2+ ion and through the cis hydroxyls of ribose, whereas its hexose moiety has little if any affinity for the enzyme. Lactose synthetase activity remaining after the reaction with thiol-group reagents indicates that a free thiol group is not an essential part of the A protein active site. PMID:4375968

  12. Identification of a residue crucial for the angiostatic activity of human mini tryptophanyl-tRNA synthetase by focusing on its molecular evolution.

    PubMed

    Nakamoto, Terumasa; Miyanokoshi, Miki; Tanaka, Tomoaki; Wakasugi, Keisuke

    2016-04-20

    Human tryptophanyl-tRNA synthetase (TrpRS) exists in two forms: a full-length TrpRS and a mini TrpRS. We previously found that human mini, but not full-length, TrpRS is an angiostatic factor. Moreover, it was shown that the interaction between mini TrpRS and the extracellular domain of vascular endothelial (VE)-cadherin is crucial for its angiostatic activity. However, the molecular mechanism of the angiostatic activity of human mini TrpRS is only partly understood. In the present study, we investigated the effects of truncated (mini) form of TrpRS proteins from human, bovine, or zebrafish on vascular endothelial growth factor (VEGF)-stimulated chemotaxis of human umbilical vein endothelial cells (HUVECs). We show that both human and bovine mini TrpRSs inhibited VEGF-induced endothelial migration, whereas zebrafish mini TrpRS did not. Next, to identify residues crucial for the angiostatic activity of human mini TrpRS, we prepared several site-directed mutants based on amino acid sequence alignments among TrpRSs from various species and demonstrated that a human mini K153Q TrpRS mutant cannot inhibit VEGF-stimulated HUVEC migration and cannot bind to the extracellular domain of VE-cadherin. Taken together, we conclude that the Lys153 residue of human mini TrpRS is a VE-cadherin binding site and is therefore crucial for its angiostatic activity.

  13. Identification of a residue crucial for the angiostatic activity of human mini tryptophanyl-tRNA synthetase by focusing on its molecular evolution

    PubMed Central

    Nakamoto, Terumasa; Miyanokoshi, Miki; Tanaka, Tomoaki; Wakasugi, Keisuke

    2016-01-01

    Human tryptophanyl-tRNA synthetase (TrpRS) exists in two forms: a full-length TrpRS and a mini TrpRS. We previously found that human mini, but not full-length, TrpRS is an angiostatic factor. Moreover, it was shown that the interaction between mini TrpRS and the extracellular domain of vascular endothelial (VE)-cadherin is crucial for its angiostatic activity. However, the molecular mechanism of the angiostatic activity of human mini TrpRS is only partly understood. In the present study, we investigated the effects of truncated (mini) form of TrpRS proteins from human, bovine, or zebrafish on vascular endothelial growth factor (VEGF)-stimulated chemotaxis of human umbilical vein endothelial cells (HUVECs). We show that both human and bovine mini TrpRSs inhibited VEGF-induced endothelial migration, whereas zebrafish mini TrpRS did not. Next, to identify residues crucial for the angiostatic activity of human mini TrpRS, we prepared several site-directed mutants based on amino acid sequence alignments among TrpRSs from various species and demonstrated that a human mini K153Q TrpRS mutant cannot inhibit VEGF-stimulated HUVEC migration and cannot bind to the extracellular domain of VE-cadherin. Taken together, we conclude that the Lys153 residue of human mini TrpRS is a VE-cadherin binding site and is therefore crucial for its angiostatic activity. PMID:27094087

  14. Monitoring mis-acylated tRNA suppression efficiency in mammalian cells via EGFP fluorescence recovery.

    PubMed

    Ilegems, Erwin; Pick, Horst M; Vogel, Horst

    2002-12-01

    A reporter assay was developed to detect and quantify nonsense codon suppression by chemically aminoacylated tRNAs in mammalian cells. It is based on the cellular expression of the enhanced green fluorescent protein (EGFP) as a reporter for the site-specific amino acid incorporation in its sequence using an orthogonal suppressor tRNA derived from Escherichia coli. Suppression of an engineered amber codon at position 64 in the EGFP run-off transcript could be achieved by the incorporation of a leucine via an in vitro aminoacylated suppressor tRNA. Microinjection of defined amounts of mutagenized EGFP mRNA and suppressor tRNA into individual cells allowed us to accurately determine suppression efficiencies by measuring the EGFP fluorescence intensity in individual cells using laser-scanning confocal microscopy. Control experiments showed the absence of natural suppression or aminoacylation of the synthetic tRNA by endogenous aminoacyl-tRNA synthetases. This reporter assay opens the way for the optimization of essential experimental parameters for expanding the scope of the suppressor tRNA technology to different cell types.

  15. Alternative pathways for editing non-cognate amino acids by aminoacyl-tRNA synthetases.

    PubMed Central

    Jakubowski, H; Fersht, A R

    1981-01-01

    Evidence is presented that the editing mechanisms of aminoacyl-tRNA synthetase operate by two alternative pathways: pre-transfer, by hydrolysis of the non-cognate aminoacyl adenylate; post-transfer, by hydrolysis of the mischarged tRNA. The methionyl-tRNA synthetases from Escherichia coli and Bacillus stearothermophilus and isoleucyl-tRNA synthetase from E. coli, for example, are shown to reject misactivated homocysteine rapidly by the pre-transfer route. A novel feature of this reaction is that homocysteine thiolactone is formed by the facile cyclisation of the homocysteinyl adenylate. Valyl-tRNA synthetases, on the other hand, reject the more readily activated non-cognate amino acids by primarily the post-transfer route. The features governing the choice of pathway are discussed. PMID:7024910

  16. Long chain acyl-CoA synthetase 3-mediated phosphatidylcholine synthesis is required for assembly of very low density lipoproteins in human hepatoma Huh7 cells.

    PubMed

    Yao, Hongbing; Ye, Jin

    2008-01-11

    Hepatocytes play a crucial role in regulating lipid metabolism by exporting cholesterol and triglyceride into plasma through secretion of very low density lipoproteins (VLDL). VLDL production is also required for release of hepatitis C virus (HCV) from infected hepatocytes. Here, we show that long chain acyl-CoA synthetase 3 (ACSL3) plays a crucial role in secretion of VLDL and HCV from hepatocytes. In cultured human hepatoma Huh7 cells, ACSL3 is specifically required for incorporation of fatty acids into phosphatidylcholine. In cells receiving small interfering RNA targeting ACSL3, secretion of apolipoprotein B, the major protein component of VLDL, was inhibited and the lipoprotein was rapidly degraded. This inhibition in secretion was completely eliminated when these cells were treated with phosphatidylcholine. Treatment of cells with small interfering RNA targeting ACSL3 also inhibited secretion of HCV from Huh7-derived cells. These results identify ACSL3 as a new enzymatic target to limit VLDL secretion and HCV infection.

  17. A new mechanism of post-transfer editing by aminoacyl-tRNA synthetases: catalysis of hydrolytic reaction by bacterial-type prolyl-tRNA synthetase.

    PubMed

    Boyarshin, Konstantin S; Priss, Anastasia E; Rayevskiy, Alexsey V; Ilchenko, Mykola M; Dubey, Igor Ya; Kriklivyi, Ivan A; Yaremchuk, Anna D; Tukalo, Michael A

    2017-02-01

    Aminoacyl tRNA synthetases are enzymes that specifically attach amino acids to cognate tRNAs for use in the ribosomal stage of translation. For many aminoacyl tRNA synthetases, the required level of amino acid specificity is achieved either by specific hydrolysis of misactivated aminoacyl-adenylate intermediate (pre-transfer editing) or by hydrolysis of the mischarged aminoacyl-tRNA (post-transfer editing). To investigate the mechanism of post-transfer editing of alanine by prolyl-tRNA synthetase from the pathogenic bacteria Enterococcus faecalis, we used molecular modeling, molecular dynamic simulations, quantum mechanical (QM) calculations, site-directed mutagenesis of the enzyme, and tRNA modification. The results support a new tRNA-assisted mechanism of hydrolysis of misacylated Ala-tRNA(Pro). The most important functional element of this catalytic mechanism is the 2'-OH group of the terminal adenosine 76 of Ala-tRNA(Pro), which forms an intramolecular hydrogen bond with the carbonyl group of the alanine residue, strongly facilitating hydrolysis. Hydrolysis was shown by QM methods to proceed via a general acid-base catalysis mechanism involving two functionally distinct water molecules. The transition state of the reaction was identified. Amino acid residues of the editing active site participate in the coordination of substrate and both attacking and assisting water molecules, performing the proton transfer to the 3'-O atom of A76.

  18. Antiviral activity of human oligoadenylate synthetases-like (OASL) is mediated by enhancing retinoic acid-inducible gene I (RIG-I) signaling

    PubMed Central

    Zhu, Jianzhong; Zhang, Yugen; Ghosh, Arundhati; Cuevas, Rolando A.; Forero, Adriana; Dhar, Jayeeta; Ibsen, Mikkel Søes; Schmid-Burgk, Jonathan Leo; Schmidt, Tobias; Ganapathiraju, Madhavi K.; Fujita, Takashi; Hartmann, Rune; Barik, Sailen; Hornung, Veit; Coyne, Carolyn B.; Sarkar, Saumendra N.

    2014-01-01

    SUMMARY Virus infection is sensed in the cytoplasm by retinoic acid-inducible gene I (RIG-I, also known as DDX58), which requires RNA and polyubiquitin binding to induce type I interferon (IFN), and activate cellular innate immunity. We show that the human IFN-inducible oligoadenylate synthetases-like (OASL) protein had antiviral activity and mediated RIG-I activation by mimicking polyubiquitin. Loss of OASL expression reduced RIG-I signaling and enhanced virus replication in human cells. Conversely, OASL expression suppressed replication of a number of viruses in a RIG-I-dependent manner and enhanced RIG-I-mediated IFN induction. OASL interacted and colocalized with RIG-I, and through its C-terminal ubiquitin-like domain specifically enhanced RIG-I signaling. Bone marrow derived macrophages from mice deficient for Oasl2 showed that among the two mouse orthologs of human OASL; Oasl2 is functionally similar to human OASL. Our findings show a mechanism by which human OASL contributes to host antiviral responses by enhancing RIG-I activation. PMID:24931123

  19. The binding of tyrosinyl-5'-AMP to tyrosyl-tRNA synthetase (E.coli).

    PubMed Central

    Grosse, F; Krauss, G; Kownatzki, R; Maass, G

    1979-01-01

    The binding between tyrosyl-tRNA synthetase (E.coli) and the alkylanalogue of the aminoacyladenylate, tyrosinyl-5'-AMP, has been investigated by fluorescence titrations and rapid mixing experiments. Tyrosyl-tRNA synthetase has two equivalent and independent binding sites for tyrosinyl-5'-AMP. The intrinsic binding constant is 4 x 10(7)M-1. The binding sites for tRNATyr and tyrosinyl-5'-AMP are independent of each other, the anticooperative mode of tRNA binding being preserved in the presence of tyrosinyl-5-AMP. PMID:377229

  20. Human holocarboxylase synthetase with a start site at methionine-58 is the predominant nuclear variant of this protein and has catalytic activity

    SciTech Connect

    Bao, Baolong; Wijeratne, Subhashinee S.K.; Rodriguez-Melendez, Rocio; Zempleni, Janos

    2011-08-19

    Highlights: {yields} Unambiguous evidence is provided that methionine-58 serves as an in-frame alternative translation site for holocarboxylase synthetase (HLCS58). {yields} Full-length HLCS and HLCS58 enter the nucleus, but HLCS58 is the predominant variant. {yields} HLCS58 has biological activity as biotin protein ligase. -- Abstract: Holocarboxylase synthetase (HLCS) catalyzes the covalent binding of biotin to both carboxylases in extranuclear structures and histones in cell nuclei, thereby mediating important roles in intermediary metabolism, gene regulation, and genome stability. HLCS has three putative translational start sites (methionine-1, -7, and -58), but lacks a strong nuclear localization sequence that would explain its participation in epigenetic events in the cell nucleus. Recent evidence suggests that small quantities of HLCS with a start site in methionine-58 (HLCS58) might be able to enter the nuclear compartment. We generated the following novel insights into HLCS biology. First, we generated a novel HLCS fusion protein vector to demonstrate that methionine-58 is a functional translation start site in human cells. Second, we used confocal microscopy and western blots to demonstrate that HLCS58 enters the cell nucleus in meaningful quantities, and that full-length HLCS localizes predominantly in the cytoplasm but may also enter the nucleus. Third, we produced recombinant HLCS58 to demonstrate its biological activity toward catalyzing the biotinylation of both carboxylases and histones. Collectively, these observations are consistent with roles of HLCS58 and full-length HLCS in nuclear events. We conclude this report by proposing a novel role for HLCS in epigenetic events, mediated by physical interactions between HLCS and other chromatin proteins as part of a larger multiprotein complex that mediates gene repression.

  1. [Polymorphism in the human 2'-5'-oligoadenylate synthetase genes (OAS), associated with predisposition to severe forms of tick-borne encephalitis, in populations from North Eurasia].

    PubMed

    Barkhash, A V; Babenko, V N; Kobzev, V F; Romashchenko, A G; Voevoda, M I

    2010-01-01

    2'-5'-oligoadenylate synthetases are a family of interferon-induced enzymes which play an important role in the antiviral defense in mammals. In human genome three genes encoding functional synthetases (OAS1, OAS2 and OAS3) form a cluster. Previously we found that particular genotypes and/or alleles of five single nucleotide polymorphisms (SNPs) located within OAS2 and OAS3 genes are associated with predisposition to severe forms of tick-borne encephalitis (TBE) in Russian population. In current study we investigated the distribution of three of that SNPs (OAS3rs2285932 (C/T Ile438Ile), OAS3rs2072136 (G/A, Ser567Ser) and OAS2 rs15895 (G/A, Trp720Ter relative to p71 isoform)) in seven populations from North Eurasia: Caucasians (Russians and Germans (from Altai region)), Central Asian Mongoloids (Altaians, Khakasses, Tuvinians and Shorians) and Arctic Mongoloids (Chukchi). Differences between populations in genotype, allele and haplotype frequencies and in linkage disequilibrium structure for these SNPs were detected. We found that these frequencies correlate with the ethnicity of the populations and with their supposed differential exposure to TBE virus. Particularly, the lowest frequencies of G/G genotype for OAS3 gene rs2072136 SNP (that according to our previously obtained data is associated with predisposition to severe forms of TBE) were found in Altaians, Khakasses, Tuvinians and Shorians who may highly contact with TBE virus in places of their habitation. Thus, data obtained allow to suppose that TBE virus might act as a selection factor for particular OAS genes variants in Central Asian Mongoloids.

  2. Polyspecific pyrrolysyl-tRNA synthetases from directed evolution

    PubMed Central

    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

    2014-01-01

    Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl 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 Nε-acetyl-Lys (AcK) onto tRNAPyl. Here, we examine an Nε-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

  3. Polyspecific pyrrolysyl-tRNA synthetases from directed evolution.

    PubMed

    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.

  4. Structure of Prolyl-tRNA Synthetase-Halofuginone Complex Provides Basis for Development of Drugs against Malaria and Toxoplasmosis.

    PubMed

    Jain, Vitul; Yogavel, Manickam; Oshima, Yoshiteru; Kikuchi, Haruhisa; Touquet, Bastien; Hakimi, Mohamed-Ali; Sharma, Amit

    2015-05-05

    The Chinese herb Dichroa febrifuga has traditionally treated malaria-associated fever. Its active component febrifugine (FF) and derivatives such as halofuginone (HF) are potent anti-malarials. Here, we show that FF-based derivatives arrest parasite growth by direct interaction with and inhibition of the protein translation enzyme prolyl-tRNA synthetase (PRS). Dual administration of inhibitors that target different tRNA synthetases suggests high utility of these drug targets. We reveal the ternary complex structure of PRS-HF and adenosine 5'-(β,γ-imido)triphosphate where the latter facilitates HF integration into the PRS active site. Structural analyses also highlight spaces within the PRS architecture for HF derivatization of its quinazolinone, but not piperidine, moiety. We also show a remarkable ability of HF to kill the related human parasite Toxoplasma gondii, suggesting wider HF efficacy against parasitic PRSs. Hence, our cell-, enzyme-, and structure-based data on FF-based inhibitors strengthen the case for their inclusion in anti-malarial and anti-toxoplasmosis drug development efforts.

  5. Methionyl-tRNA synthetase from Caenorhabditis elegans: A specific multidomain organization for convergent functional evolution

    PubMed Central

    Havrylenko, Svitlana; Legouis, Renaud; Negrutskii, Boris; Mirande, Marc

    2010-01-01

    Methionyl-tRNA synthetase (MetRS) is a multidomain protein that specifically binds tRNAMet and catalyzes the synthesis of methionyl-tRNAMet. The minimal, core enzyme found in Aquifex aeolicus is made of a catalytic domain, which catalyzes the aminoacylation reaction, and an anticodon-binding domain, which promotes tRNA–protein association. In eukaryotes, additional domains are appended in cis or in trans to the core enzyme and increase the stability of the tRNA–protein complexes. Eventually, as observed for MetRS from Homo sapiens, the C-terminal appended domain causes a slow release of aminoacyl-tRNA and establishes a limiting step in the global aminoacylation reaction. Here, we report that MetRS from the nematode Caenorhabditis elegans displays a new type of structural organization. Its very C-terminal appended domain is related to the oligonucleotide binding-fold-based tRNA-binding domain (tRBD) recovered at the C-terminus of MetRS from plant, but, in the nematode enzyme, this domain is separated from the core enzyme by an insertion domain. Gel retardation and tRNA aminoacylation experiments show that MetRS from nematode is functionally related to human MetRS despite the fact that their appended tRBDs have distinct structural folds, and are not orthologs. Thus, functional convergence of human and nematode MetRS is the result of parallel and convergent evolution that might have been triggered by the selective pressure to invent processivity of tRNA handling in translation in higher eukaryotes. PMID:20954242

  6. tRNA synthase suppression activates de novo cysteine synthesis to compensate for cystine and glutathione deprivation during ferroptosis.

    PubMed

    Shimada, Kenichi; Stockwell, Brent R

    2016-03-01

    Glutathione is a major endogenous reducing agent in cells, and cysteine is a limiting factor in glutathione synthesis. Cysteine is obtained by uptake or biosynthesis, and mammalian cells often rely on either one or the other pathway. Because of the scarcity of glutathione, blockade of cysteine uptake causes oxidative cell death known as ferroptosis. A new study suggests that tRNA synthetase suppression activates the endogenous biosynthesis of cysteine, compensates such cysteine loss, and thus makes cells resistant to ferroptosis.

  7. Cocrystal structure of a T-box riboswitch Stem I domain in complex with its cognate tRNA

    PubMed Central

    Zhang, Jinwei; Ferré-D’Amaré, Adrian R.

    2013-01-01

    In Gram-positive bacteria, T-box riboswitches regulate expression of aminoacyl-tRNA synthetases (ARSs) and other proteins in response to fluctuating tRNA aminoacylation levels under various nutritional states1. T-boxes reside in the 5’-untranslated regions (UTRs) of the mRNAs they regulate, and comprise two conserved domains. Stem I harbors the specifier trinucleotide that base-pairs with the anticodon of cognate tRNA. 3’ to Stem I is the antiterminator domain, which base-pairs with the tRNA acceptor end and evaluates its aminoacylation state2. Despite high phylogenetic conservation and widespread occurrence in pathogens, the structural basis of tRNA recognition3,4 by this riboswitch remains ill-defined. Here, we demonstrate that the ~100-nucleotide T-box Stem I is necessary and sufficient for specific, high-affinity (Kd ~150 nM) tRNA binding, and report its structure in complex with cognate tRNA at 3.2 Å resolution. Stem I recognizes the overall architecture of tRNA in addition to its anticodon, something accomplished by large ribonucleoproteins (RNPs) like the ribosome or proteins such as ARSs5, but unprecedented for a compact mRNA domain. The C-shaped Stem I cradles the L-shaped tRNA forming an extended (1604 Å2) intermolecular interface. In addition to the specifier-anticodon interaction, two interdigitated T-loops near the apex of Stem I stack on the tRNA elbow in a manner analogous to those of the J11/12-J12/11 motif6 of RNase P and the L1 stalk7 of the ribosomal E-site. Since these RNPs and T-boxes are unrelated, this strategy to recognize an universal tRNA feature likely evolved convergently. Mutually induced fit of Stem I and the tRNA exploiting the intrinsic flexibility of tRNA and its conserved post-transcriptional modifications results in high shape complementarity, which in addition to providing specificity and affinity, globally organizes the T-box to orchestrate tRNA-dependent transcription regulation. PMID:23892783

  8. Export and transport of tRNA are coupled to a multi-protein complex.

    PubMed Central

    Kruse, C; Willkomm, D K; Grünweller, A; Vollbrandt, T; Sommer, S; Busch, S; Pfeiffer, T; Brinkmann, J; Hartmann, R K; Müller, P K

    2000-01-01

    Vigilin is a ubiquitous multi heterogeneous nuclear ribonucleoprotein (hnRNP) K homologous (KH)-domain protein. Here we demonstrate that purified recombinant human vigilin binds tRNA molecules with high affinity, although with limited specificity. Nuclear microinjection experiments revealed for the first time that the immuno-affinity-purified nuclear vigilin core complex (VCC(N)) as well as recombinant vigilin accelerate tRNA export from the nucleus in human cells. The nuclear tRNA receptor exportin-t is part of the VCC(N). Elongation factor (EF)-1alpha is enriched in VCC(N) and its cytoplasmic counterpart VCC(C), whereas EF-1beta, EF-1gamma and EF-1delta are basically confined to the VCC(C). Our results suggest further that vigilin and exportin-t might interact during tRNA export, provide evidence that the channeled tRNA cycle is already initiated in the nucleus, and illustrate that intracellular tRNA trafficking is associated with discrete changes in the composition of cellular cytoplasmic multi-protein complexes containing tRNA. PMID:10657246

  9. Pyrrolysyl-tRNA synthetase, an aminoacyl-tRNA synthetase for genetic code expansion

    PubMed Central

    Crnković, Ana; Suzuki, Tateki; Söll, Dieter; Reynolds, Noah M.

    2016-01-01

    Genetic code expansion (GCE) has become a central topic of synthetic biology. GCE relies on engineered aminoacyl-tRNA synthetases (aaRSs) and a cognate tRNA species to allow codon reassignment by co-translational insertion of non-canonical amino acids (ncAAs) into proteins. Introduction of such amino acids increases the chemical diversity of recombinant proteins endowing them with novel properties. Such proteins serve in sophisticated biochemical and biophysical studies both in vitro and in vivo, they may become unique biomaterials or therapeutic agents, and they afford metabolic dependence of genetically modified organisms for biocontainment purposes. In the Methanosarcinaceae the incorporation of the 22nd genetically encoded amino acid, pyrrolysine (Pyl), is facilitated by pyrrolysyl-tRNA synthetase (PylRS) and the cognate UAG-recognizing tRNAPyl. This unique aaRS•tRNA pair functions as an orthogonal translation system (OTS) in most model organisms. The facile directed evolution of the large PylRS active site to accommodate many ncAAs, and the enzyme’s anticodon-blind specific recognition of the cognate tRNAPyl make this system highly amenable for GCE purposes. The remarkable polyspecificity of PylRS has been exploited to incorporate >100 different ncAAs into proteins. Here we review the Pyl-OT system and selected GCE applications to examine the properties of an effective OTS. PMID:28239189

  10. Methylated nucleosides in tRNA and tRNA methyltransferases

    PubMed Central

    Hori, Hiroyuki

    2014-01-01

    To date, more than 90 modified nucleosides have been found in tRNA and the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s). Recent studies of the biosynthetic pathways have demonstrated that the availability of methyl group donors for the methylation in tRNA is important for correct and efficient protein synthesis. In this review, I focus on the methylated nucleosides and tRNA methyltransferases. The primary functions of tRNA methylations are linked to the different steps of protein synthesis, such as the stabilization of tRNA structure, reinforcement of the codon-anticodon interaction, regulation of wobble base pairing, and prevention of frameshift errors. However, beyond these basic functions, recent studies have demonstrated that tRNA methylations are also involved in the RNA quality control system and regulation of tRNA localization in the cell. In a thermophilic eubacterium, tRNA modifications and the modification enzymes form a network that responses to temperature changes. Furthermore, several modifications are involved in genetic diseases, infections, and the immune response. Moreover, structural, biochemical, and bioinformatics studies of tRNA methyltransferases have been clarifying the details of tRNA methyltransferases and have enabled these enzymes to be classified. In the final section, the evolution of modification enzymes is discussed. PMID:24904644

  11. [Anti-synthetase syndrome].

    PubMed

    Novak, Srdan

    2012-01-01

    Antysynthetase syndrome is considered as a group ofidiopathic inflammatory myositis with charcteristic serologic hallmark--antibodies which recognise the aminoacyl-tRNA synthetasses (ARS). Clinical picture of those patients contains myositis and/or intersticial lung disease (ILD) and/or arthritis and/or fever and/or Raynaud phenomenon and sometimes characteristic look of mechanic's hands. Myositis can be overt, sometimes even absent, while IBP is major cause of morbidity and determines the outcome of the disease. Untill now eight different any-synthetase autoantibodies are recognised, and most frequent are findings of anti-histidyl-tRNa synthetase antibodies. Patients with other ARS autoantibodies usually have severe ILD. Drug of choice are steroids in dosage of 1 mg/kg with immunosupresive agent (azatioprin or methotrexate) while in severe IBP cyclophosphamide is needed. Recently succsesful treatment with rituximab in combination with cyclophosphamide is reported.

  12. Structures of Trypanosoma brucei Methionyl-tRNA Synthetase with Urea-Based Inhibitors Provide Guidance for Drug Design against Sleeping Sickness

    PubMed Central

    Koh, Cho Yeow; Kim, Jessica E.; Wetzel, Allan B.; de van der Schueren, Will J.; Shibata, Sayaka; Ranade, Ranae M.; Liu, Jiyun; Zhang, Zhongsheng; Gillespie, J. Robert; Buckner, Frederick S.; Verlinde, Christophe L. M. J.; Fan, Erkang; Hol, Wim G. J.

    2014-01-01

    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. PMID:24743796

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

    PubMed

    Koh, Cho Yeow; Kim, Jessica E; Wetzel, Allan B; de van der Schueren, Will J; Shibata, Sayaka; Ranade, Ranae M; Liu, Jiyun; Zhang, Zhongsheng; Gillespie, J Robert; Buckner, Frederick S; Verlinde, Christophe L M J; Fan, Erkang; Hol, Wim G J

    2014-04-01

    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. tRNA regulation of gene expression: Interactions of an mRNA 5′-UTR with a regulatory tRNA

    PubMed Central

    Nelson, Audrey R.; Henkin, Tina M.; Agris, Paul F.

    2006-01-01

    Many genes encoding aminoacyl-tRNA synthetases and other amino acid–related products in Gram-positive bacteria, including important pathogens, are regulated through interaction of unacylated tRNA with the 5′-untranslated region (5′-UTR) of the mRNA. Each gene regulated by this mechanism responds specifically to the cognate tRNA, and specificity is determined by pairing of the anticodon of the tRNA with a codon sequence in the “Specifier Loop” of the 5′-UTR. For the 5′-UTR to function in gene regulation, the mRNA folding interactions must be sufficiently stable to present the codon sequence for productive binding to the anticodon of the matching tRNA. A model bimolecular system was developed in which the interaction between two half molecules (“Common” and “Specifier”) would reconstitute the Specifier Loop region of the 5′-UTR of the Bacillus subtilis glyQS gene, encoding GlyRS mRNA. Gel mobility shift analysis and fluorescence spectroscopy yielded experimental K ds of 27.6 ± 1.0 μM and 10.5 ± 0.7 μM, respectively, for complex formation between Common and Specifier half molecules. The reconstituted 5′-UTR of the glyQS mRNA bound the anticodon stem and loop of tRNAGly (ASLGlyGCC) specifically and with a significant affinity (K d = 20.2 ± 1.4 μM). Thus, the bimolecular 5′-UTR and ASLGlyGCC models mimic the RNA–RNA interaction required for T box gene regulation in vivo. PMID:16741230

  15. Altered alpha subunits in phenylalanyl-tRNA synthetases from p-fluorophenylalanine-resistant strains of Escherichis coli.

    PubMed

    Hennecke, H; Böck, A

    1975-07-01

    Three different phenylalanyl-tRNA synthetases have been purified to near homogeneity, one from a wild-type strain of Escherichia coli and the others from two independently isolated p-fluorophenyalanine-resistant strains. The mutant enzymes were not able to use p-fluorophenylalanine as a substrate for activation and attachment to tRNA. They proved to be indistinguishable from the wild-type enzyme by several electrophoretic and immunological criteria. The alpha and beta subunits of all three enzymes have been prepared by a method described in this paper. The isolated subunits per se did not reveal any significant enzyme activity, but combined they were able to form active phenylalanyl tRNA synthetase after a defined reconstitution process. Mixed reconstitution experiments between wild-type and mutant subunits indicate that the mutant alpha subunit is responsible for p-fluorophenylalanine resistance and therefore seems to carry the phenylalanine-binding site or to participate in its formation.

  16. Structural diversity and protein engineering of the aminoacyl-tRNA synthetases.

    PubMed

    Perona, John J; Hadd, Andrew

    2012-11-06

    Aminoacyl-tRNA synthetases (aaRS) are the enzymes that ensure faithful transmission of genetic information in all living cells, and are central to the developing technologies for expanding the capacity of the translation apparatus to incorporate nonstandard amino acids into proteins in vivo. The 24 known aaRS families are divided into two classes that exhibit functional evolutionary convergence. Each class features an active site domain with a common fold that binds ATP, the amino acid, and the 3'-terminus of tRNA, embellished by idiosyncratic further domains that bind distal portions of the tRNA and enhance specificity. Fidelity in the expression of the genetic code requires that the aaRS be selective for both amino acids and tRNAs, a substantial challenge given the presence of structurally very similar noncognate substrates of both types. Here we comprehensively review central themes concerning the architectures of the protein structures and the remarkable dual-substrate selectivities, with a view toward discerning the most important issues that still substantially limit our capacity for rational protein engineering. A suggested general approach to rational design is presented, which should yield insight into the identities of the protein-RNA motifs at the heart of the genetic code, while also offering a basis for improving the catalytic properties of engineered tRNA synthetases emerging from genetic selections.

  17. Mitochondrial tRNA sequences as unusual replication origins: pathogenic implications for Homo sapiens.

    PubMed

    Seligmann, Hervé; Krishnan, Neeraja M; Rao, Basuthkar J

    2006-12-07

    The heavy strand of vertebrate mitochondrial genomes accumulates deaminations proportionally to the time it spends single-stranded during replication. A previous study showed that the strength of genome-wide deamination gradients originating from tRNA gene's locations increases with their capacities to form secondary structures resembling mitochondrial origins of light strand replication (OL), suggesting an alternative function for tRNA sequences. We hypothesize that this function is frequently pathogenic for those tRNA genes that normally do not form OL-like structures, because this could cause excess mutations in genome regions unadapted to tolerate them. In human mitochondrial genomes, pathogenic tRNA variants usually form less OL-like structures than non-pathogenic ones in cases where the normal non-pathogenic tRNA variant can function as OL, as evolutionary analyses reveal. For tRNAs lacking the putative OL-like functioning capacity, pathogenic variants form more OL-like secondary structures, particularly structures that might invoke bi-directional replication (true for 14 among 21 tRNA species, p<0.05, sign test; significantly at p<0.05 (1 tailed test) for 7 tRNA species), but not more unidirectional replication invoking structures. Accounting for the functional cloverleaf-like structure-forming capacities of tRNAs yields similar results. Rare, non-pathogenic tRNA mutants tend to form more OL-like structures than the common, non-pathogenic ones, suggesting weak directional selection also among non-pathogenic variants. The duration spent single stranded by a region of the heavy strand (D(ssH)) during replication, estimated by integrating over all regions that can function as OL in Homo sapiens mitochondrial genomes, increases with distance of that region from the Dloop. This suggests convergence of single-strandedness during replication and transcription, and explains conserved locations of tRNA species in mitochondrial genomes and bacterial operons. These

  18. Global tRNA misacylation induced by anaerobiosis and antibiotic exposure broadly increases stress resistance in Escherichia coli

    PubMed Central

    Schwartz, Michael H.; Waldbauer, Jacob R.; Zhang, Lichun; Pan, Tao

    2016-01-01

    High translational fidelity is commonly considered a requirement for optimal cellular health and protein function. However, recent findings have shown that inducible mistranslation specifically with methionine engendered at the tRNA charging level occurs in mammalian cells, yeast and archaea, yet it was unknown whether bacteria were capable of mounting a similar response. Here, we demonstrate that Escherichia coli misacylates non-methionyl-tRNAs with methionine in response to anaerobiosis and antibiotic exposure via the methionyl–tRNA synthetase (MetRS). Two MetRS succinyl-lysine modifications independently confer high tRNA charging fidelity to the otherwise promiscuous, unmodified enzyme. Strains incapable of tRNA mismethionylation are less adept at growth in the presence of antibiotics and stressors. The presence of tRNA mismethionylation and its potential role in mistranslation within the bacterial domain establishes this response as a pervasive biological mechanism and connects it to diverse cellular functions and modes of fitness. PMID:27672035

  19. Different sequence signatures in the upstream regions of plant and animal tRNA genes shape distinct modes of regulation.

    PubMed

    Zhang, Gong; Lukoszek, Radoslaw; Mueller-Roeber, Bernd; Ignatova, Zoya

    2011-04-01

    In eukaryotes, the transcription of tRNA genes is initiated by the concerted action of transcription factors IIIC (TFIIIC) and IIIB (TFIIIB) which direct the recruitment of polymerase III. While TFIIIC recognizes highly conserved, intragenic promoter elements, TFIIIB binds to the non-coding 5'-upstream regions of the tRNA genes. Using a systematic bioinformatic analysis of 11 multicellular eukaryotic genomes we identified a highly conserved TATA motif followed by a CAA-motif in the tRNA upstream regions of all plant genomes. Strikingly, the 5'-flanking tRNA regions of the animal genomes are highly heterogeneous and lack a common conserved sequence signature. Interestingly, in the animal genomes the tRNA species that read the same codon share conserved motifs in their upstream regions. Deep-sequencing analysis of 16 human tissues revealed multiple splicing variants of two of the TFIIIB subunits, Bdp1 and Brf1, with tissue-specific expression patterns. These multiple forms most likely modulate the TFIIIB-DNA interactions and explain the lack of a uniform signature motif in the tRNA upstream regions of animal genomes. The anticodon-dependent 5'-flanking motifs provide a possible mechanism for independent regulation of the tRNA transcription in various human tissues.

  20. Structure of Leishmania major methionyl-tRNA synthetase in complex with intermediate products methionyladenylate and pyrophosphate.

    PubMed

    Larson, Eric T; Kim, Jessica E; Zucker, Frank H; Kelley, Angela; Mueller, Natascha; Napuli, Alberto J; Verlinde, Christophe L M J; Fan, Erkang; Buckner, Frederick S; Van Voorhis, Wesley C; Merritt, Ethan A; Hol, Wim G J

    2011-03-01

    Leishmania parasites cause two million new cases of leishmaniasis each year with several hundreds of millions of people at risk. Due to the paucity and shortcomings of available drugs, we have undertaken the crystal structure determination of a key enzyme from Leishmania major in hopes of creating a platform for the rational design of new therapeutics. Crystals of the catalytic core of methionyl-tRNA synthetase from L. major (LmMetRS) were obtained with the substrates MgATP and methionine present in the crystallization medium. These crystals yielded the 2.0 Å resolution structure of LmMetRS in complex with two products, methionyladenylate and pyrophosphate, along with a Mg(2+) ion that bridges them. This is the first class I aminoacyl-tRNA synthetase (aaRS) structure with pyrophosphate bound. The residues of the class I aaRS signature sequence motifs, KISKS and HIGH, make numerous contacts with the pyrophosphate. Substantial differences between the LmMetRS structure and previously reported complexes of Escherichia coli MetRS (EcMetRS) with analogs of the methionyladenylate intermediate product are observed, even though one of these analogs only differs by one atom from the intermediate. The source of these structural differences is attributed to the presence of the product pyrophosphate in LmMetRS. Analysis of the LmMetRS structure in light of the Aquifex aeolicus MetRS-tRNA(Met) complex shows that major rearrangements of multiple structural elements of enzyme and/or tRNA are required to allow the CCA acceptor triplet to reach the methionyladenylate intermediate in the active site. Comparison with sequences of human cytosolic and mitochondrial MetRS reveals interesting differences near the ATP- and methionine-binding regions of LmMetRS, suggesting that it should be possible to obtain compounds that selectively inhibit the parasite enzyme.

  1. Probing the substrate-binding sites of aminoacyl-tRNA synthetases with the procion dye green HE-4BD.

    PubMed Central

    McArdell, J E; Duffield, M; Atkinson, T

    1989-01-01

    A reactive bis-dichloro derivative of the Procion dye Green HE-4BD was shown to inactivate irreversibly methionyl-tRNA synthetase (MTS) from Escherichia coli and also tryptophyl-tRNA synthetase (WTS) and tyrosyl-tRNA synthetase (YTS) from Bacillus stearothermophilus at pH 8.5 and 37 degrees C. At a 5-fold excess of reactive dye over enzyme subunit concentration MTS was quantitatively inactivated within 20 min in the ATP/pyrophosphate exchange assay, whereas WTS and YTS show an 80% loss of activity over the same time period. The inactivation is affected by the addition of substrates, which either protect (WTS and YTS) or promote (YTS with tyrosine) the dye-mediated enzyme inactivation. Green HE-4BD-OH was shown to be a competitive inhibitor of MTS with respect to MgATP, methionine and tRNA substrates. PMID:2658972

  2. Maf1-mediated regulation of yeast RNA polymerase III is correlated with CCA addition at the 3' end of tRNA precursors.

    PubMed

    Foretek, Dominika; Nuc, Przemysław; Żywicki, Marek; Karlowski, Wojciech M; Kudla, Grzegorz; Boguta, Magdalena

    2016-08-27

    In eukaryotic cells tRNA synthesis is negatively regulated by the protein Maf1, conserved from yeast to humans. Maf1 from yeast Saccharomyces cerevisiae mediates repression of trna transcription when cells are transferred from medium with glucose to medium with glycerol, a non-fermentable carbon source. The strain with deleted gene encoding Maf1 (maf1Δ) is viable but accumulates tRNA precursors. In this study tRNA precursors were analysed by RNA-Seq and Northern hybridization in wild type strain and maf1Δ mutant grown in glucose medium or upon shift to repressive conditions. A negative effect of maf1Δ mutant on the addition of the auxiliary CCA nucleotides to the 3' end of pre-tRNAs was observed in cells shifted to unfavourable growth conditions. This effect was reduced by overexpression of the yeast CCA1 gene encoding ATP(CTP):tRNA nucleotidyltransferase. The CCA sequence at the 3' end is important for export of tRNA precursors from the nucleus and essential for tRNA charging with amino acids. Data presented here indicate that CCA-addition to intron-containing end-processed tRNA precursors is a limiting step in tRNA maturation when there is no Maf1 mediated RNA polymerase III (Pol III) repression. The correlation between CCA synthesis and Pol III regulation by Maf1 could be important in coordination of tRNA transcription, processing and regulation of translation.

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

    NASA Technical Reports Server (NTRS)

    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.

  4. Genetics Home Reference: glutathione synthetase deficiency

    MedlinePlus

    ... Facebook Share on Twitter Your Guide to Understanding Genetic Conditions Search MENU Toggle navigation Home Page Search ... Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Home Health Conditions glutathione synthetase deficiency glutathione synthetase ...

  5. Mitochondrial tRNA mutations and disease.

    PubMed

    Yarham, John W; Elson, Joanna L; Blakely, Emma L; McFarland, Robert; Taylor, Robert W

    2010-01-01

    Mitochondrial (mt-) tRNA (MTT) gene mutations are an important cause of human morbidity and are associated with a wide range of pathology, from isolated organ-specific diseases such as myopathy or hearing loss, through to multisystem disorders with encephalopathy, gastrointestinal dysmotility, and life-threatening cardiomyopathy. Our understanding of how MTT mutations cause disease remains poor and progress has been hampered by the complex interaction of genotype with phenotype that can result in patients who harbor the same mutation exhibiting starkly contrasting phenotypes, whereas other (genetically heterogeneous) patients manifest clinically identical syndromes. A further complexity is the highly polymorphic nature of mitochondrial DNA (mtDNA), which must temper any reflex assumptions of pathogenicity for novel MTT substitutions. Nevertheless significant progress is being made and we shall review the methods employed to identify and characterize MTT mutations as pathogenic. Also important is our understanding of the molecular processes involved and we shall discuss the data available on two of the most studied MTT mutations (m.8344A > G and m.3243A > G) as well as other potential pathogenic mechanisms. Knowledge of factors influencing the inheritance of MTT mutations, and therefore the likelihood of disease transmission, is of particular importance to female patients. At present, the factors determining transmission remain elusive, but we shall examine several possible mechanisms and discuss the evidence for each. Finally, a number of different yeast and mouse models are currently used to investigate mitochondrial disease and we will assess the importance of and difficulties associated with each model as well as the future of possible therapies for patients with mitochondrial disease.

  6. The T-loop region of animal mitochondrial tRNA(Ser)(AGY) is a main recognition site for homologous seryl-tRNA synthetase.

    PubMed Central

    Ueda, T; Yotsumoto, Y; Ikeda, K; Watanabe, K

    1992-01-01

    Recognition sites of bovine mitochondrial serine tRNA specific for condons AGY [tRNA(Ser) (AGY)] by the cognate mitochondrial seryl-tRNA synthetase were studied using a range of tRNA(Ser)(AGY) variants which were obtained by the in vitro transcription of synthetic tRNA genes with T7 RNA polymerase. Base replacements in the anticodon and discriminator sites did not affect serine acceptance. However, deletion and/or replacement in the T-loop region completely deprived the variants of their charging activities. Point mutation experiments in this region also showed that the adenosine residue in the middle of the T-loop (position 58), which is involved in tertiary interaction between the T-loop and the truncated D-arm [de Bruijn and Klug, 1983] played a significant role in the recognition process by the synthetase. PMID:1375735

  7. Emerging roles of tRNA in adaptive translation, signalling dynamics and disease.

    PubMed

    Kirchner, Sebastian; Ignatova, Zoya

    2015-02-01

    tRNAs, nexus molecules between mRNAs and proteins, have a central role in translation. Recent discoveries have revealed unprecedented complexity of tRNA biosynthesis, modification patterns, regulation and function. In this Review, we present emerging concepts regarding how tRNA abundance is dynamically regulated and how tRNAs (and their nucleolytic fragments) are centrally involved in stress signalling and adaptive translation, operating across a wide range of timescales. Mutations in tRNAs or in genes affecting tRNA biogenesis are also linked to complex human diseases with surprising heterogeneity in tissue vulnerability, and we highlight cell-specific aspects that modulate the disease penetrance of tRNA-based pathologies.

  8. Metazoan tRNA introns generate stable circular RNAs in vivo

    PubMed Central

    Lu, Zhipeng; Filonov, Grigory S.; Noto, John J.; Schmidt, Casey A.; Hatkevich, Talia L.; Wen, Ying; Jaffrey, Samie R.; Matera, A. Gregory

    2015-01-01

    We report the discovery of a class of abundant circular noncoding RNAs that are produced during metazoan tRNA splicing. These transcripts, termed tRNA intronic circular (tric)RNAs, are conserved features of animal transcriptomes. Biogenesis of tricRNAs requires anciently conserved tRNA sequence motifs and processing enzymes, and their expression is regulated in an age-dependent and tissue-specific manner. Furthermore, we exploited this biogenesis pathway to develop an in vivo expression system for generating “designer” circular RNAs in human cells. Reporter constructs expressing RNA aptamers such as Spinach and Broccoli can be used to follow the transcription and subcellular localization of tricRNAs in living cells. Owing to the superior stability of circular vs. linear RNA isoforms, this expression system has a wide range of potential applications, from basic research to pharmaceutical science. PMID:26194134

  9. tRNA Biology in Mitochondria

    PubMed Central

    Salinas-Giegé, Thalia; Giegé, Richard; Giegé, Philippe

    2015-01-01

    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. PMID:25734984

  10. Rosiglitazone Inhibits Acyl-CoA Synthetase Activity and Fatty Acid Partitioning to Diacylglycerol and Triacylglycerol via a Peroxisome Proliferator–Activated Receptor-γ–Independent Mechanism in Human Arterial Smooth Muscle Cells and Macrophages

    PubMed Central

    Askari, Bardia; Kanter, Jenny E.; Sherrid, Ashley M.; Golej, Deidre L.; Bender, Andrew T.; Liu, Joey; Hsueh, Willa A.; Beavo, Joseph A.; Coleman, Rosalind A.; Bornfeldt, Karin E.

    2010-01-01

    Rosiglitazone is an insulin-sensitizing agent that has recently been shown to exert beneficial effects on atherosclerosis. In addition to peroxisome proliferator–activated receptor (PPAR)-γ, rosiglitazone can affect other targets, such as directly inhibiting recombinant long-chain acyl-CoA synthetase (ACSL)-4 activity. Because it is unknown if ACSL4 is expressed in vascular cells involved in atherosclerosis, we investigated the ability of rosiglitazone to inhibit ACSL activity and fatty acid partitioning in human and murine arterial smooth muscle cells (SMCs) and macrophages. Human and murine SMCs and human macrophages expressed Acsl4, and rosiglitazone inhibited Acsl activity in these cells. Furthermore, rosiglitazone acutely inhibited partitioning of fatty acids into phospholipids in human SMCs and inhibited fatty acid partitioning into diacylglycerol and triacylglycerol in human SMCs and macrophages through a PPAR-γ–independent mechanism. Conversely, murine macrophages did not express ACSL4, and rosiglitazone did not inhibit ACSL activity in these cells, nor did it affect acute fatty acid partitioning into cellular lipids. Thus, rosiglitazone inhibits ACSL activity and fatty acid partitioning in human and murine SMCs and in human macrophages through a PPAR-γ–independent mechanism likely to be mediated by ACSL4 inhibition. Therefore, rosiglitazone might alter the biological effects of fatty acids in these cells and in atherosclerosis. PMID:17259370

  11. Evolution meets disease: penetrance and functional epistasis of mitochondrial tRNA mutations.

    PubMed

    Moreno-Loshuertos, Raquel; Ferrín, Gustavo; Acín-Pérez, Rebeca; Gallardo, M Esther; Viscomi, Carlo; Pérez-Martos, Acisclo; Zeviani, Massimo; Fernández-Silva, Patricio; Enríquez, José Antonio

    2011-04-01

    About half of the mitochondrial DNA (mtDNA) mutations causing diseases in humans occur in tRNA genes. Particularly intriguing are those pathogenic tRNA mutations than can reach homoplasmy and yet show very different penetrance among patients. These mutations are scarce and, in addition to their obvious interest for understanding human pathology, they can be excellent experimental examples to model evolution and fixation of mitochondrial tRNA mutations. To date, the only source of this type of mutations is human patients. We report here the generation and characterization of the first mitochondrial tRNA pathological mutation in mouse cells, an m.3739G>A transition in the mitochondrial mt-Ti gene. This mutation recapitulates the molecular hallmarks of a disease-causing mutation described in humans, an m.4290T>C transition affecting also the human mt-Ti gene. We could determine that the pathogenic molecular mechanism, induced by both the mouse and the human mutations, is a high frequency of abnormal folding of the tRNA(Ile) that cannot be charged with isoleucine. We demonstrate that the cells harboring the mouse or human mutant tRNA have exacerbated mitochondrial biogenesis triggered by an increase in mitochondrial ROS production as a compensatory response. We propose that both the nature of the pathogenic mechanism combined with the existence of a compensatory mechanism can explain the penetrance pattern of this mutation. This particular behavior can allow a scenario for the evolution of mitochondrial tRNAs in which the fixation of two alleles that are individually deleterious can proceed in two steps and not require the simultaneous mutation of both.

  12. Correction of the consequences of mitochondrial 3243A>G mutation in the MT-TL1 gene causing the MELAS syndrome by tRNA import into mitochondria.

    PubMed

    Karicheva, Olga Z; Kolesnikova, Olga A; Schirtz, Tom; Vysokikh, Mikhail Y; Mager-Heckel, Anne-Marie; Lombès, Anne; Boucheham, Abdeldjalil; Krasheninnikov, Igor A; Martin, Robert P; Entelis, Nina; Tarassov, Ivan

    2011-10-01

    Mutations in human mitochondrial DNA are often associated with incurable human neuromuscular diseases. Among these mutations, an important number have been identified in tRNA genes, including 29 in the gene MT-TL1 coding for the tRNA(Leu(UUR)). The m.3243A>G mutation was described as the major cause of the MELAS syndrome (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes). This mutation was reported to reduce tRNA(Leu(UUR)) aminoacylation and modification of its anti-codon wobble position, which results in a defective mitochondrial protein synthesis and reduced activities of respiratory chain complexes. In the present study, we have tested whether the mitochondrial targeting of recombinant tRNAs bearing the identity elements for human mitochondrial leucyl-tRNA synthetase can rescue the phenotype caused by MELAS mutation in human transmitochondrial cybrid cells. We demonstrate that nuclear expression and mitochondrial targeting of specifically designed transgenic tRNAs results in an improvement of mitochondrial translation, increased levels of mitochondrial DNA-encoded respiratory complexes subunits, and significant rescue of respiration. These findings prove the possibility to direct tRNAs with changed aminoacylation specificities into mitochondria, thus extending the potential therapeutic strategy of allotopic expression to address mitochondrial disorders.

  13. The modular structure of Escherichia coli threonyl-tRNA synthetase as both an enzyme and a regulator of gene expression.

    PubMed

    Caillet, Joël; Nogueira, Teresa; Masquida, Benoît; Winter, Flore; Graffe, Monique; Dock-Brégeon, Anne-Catherine; Torres-Larios, Alfredo; Sankaranarayanan, Rajan; Westhof, Eric; Ehresmann, Bernard; Ehresmann, Chantal; Romby, Pascale; Springer, Mathias

    2003-02-01

    In addition to its role in tRNA aminoacylation, Escherichia coli threonyl-tRNA synthetase is a regulatory protein which binds a site, called the operator, located in the leader of its own mRNA and inhibits translational initiation by competing with ribosome binding. This work shows that the two essential steps of regulation, operator recognition and inhibition of ribosome binding, are performed by different domains of the protein. The catalytic and the C-terminal domain of the protein are involved in binding the two anticodon arm-like structures in the operator whereas the N-terminal domain of the enzyme is responsible for the competition with the ribosome. This is the first demonstration of a modular structure for a translational repressor and is reminiscent of that of transcriptional regulators. The mimicry between the operator and tRNA, suspected on the basis of previous experiments, is further supported by the fact that identical regions of the synthetase recognize both the operator and the tRNA anticodon arm. Based on these results, and recent structural data, we have constructed a computer-derived molecular model for the operator-threonyl-tRNA synthetase complex, which sheds light on several essential aspects of the regulatory mechanism.

  14. Revised nomenclature for the mammalian long-chain acyl-CoA synthetase gene family.

    PubMed

    Mashek, Douglas G; Bornfeldt, Karin E; Coleman, Rosalind A; Berger, Johannes; Bernlohr, David A; Black, Paul; DiRusso, Concetta C; Farber, Steven A; Guo, Wen; Hashimoto, Naohiro; Khodiyar, Varsha; Kuypers, Frans A; Maltais, Lois J; Nebert, Daniel W; Renieri, Alessandra; Schaffer, Jean E; Stahl, Andreas; Watkins, Paul A; Vasiliou, Vasilis; Yamamoto, Tokuo T

    2004-10-01

    By consensus, the acyl-CoA synthetase (ACS) community, with the advice of the human and mouse genome nomenclature committees, has revised the nomenclature for the mammalian long-chain acyl-CoA synthetases. ACS is the family root name, and the human and mouse genes for the long-chain ACSs are termed ACSL1,3-6 and Acsl1,3-6, respectively. Splice variants of ACSL3, -4, -5, and -6 are cataloged. Suggestions for naming other family members and for the nonmammalian acyl-CoA synthetases are made.

  15. Archaeal aminoacyl-tRNA synthetases interact with the ribosome to recycle tRNAs.

    PubMed

    Godinic-Mikulcic, Vlatka; Jaric, Jelena; Greber, Basil J; Franke, Vedran; Hodnik, Vesna; Anderluh, Gregor; Ban, Nenad; Weygand-Durasevic, Ivana

    2014-04-01

    Aminoacyl-tRNA synthetases (aaRS) are essential enzymes catalyzing the formation of aminoacyl-tRNAs, the immediate precursors for encoded peptides in ribosomal protein synthesis. Previous studies have suggested a link between tRNA aminoacylation and high-molecular-weight cellular complexes such as the cytoskeleton or ribosomes. However, the structural basis of these interactions and potential mechanistic implications are not well understood. To biochemically characterize these interactions we have used a system of two interacting archaeal aaRSs: an atypical methanogenic-type seryl-tRNA synthetase and an archaeal ArgRS. More specifically, we have shown by thermophoresis and surface plasmon resonance that these two aaRSs bind to the large ribosomal subunit with micromolar affinities. We have identified the L7/L12 stalk and the proteins located near the stalk base as the main sites for aaRS binding. Finally, we have performed a bioinformatics analysis of synonymous codons in the Methanothermobacter thermautotrophicus genome that supports a mechanism in which the deacylated tRNAs may be recharged by aaRSs bound to the ribosome and reused at the next occurrence of a codon encoding the same amino acid. These results suggest a mechanism of tRNA recycling in which aaRSs associate with the L7/L12 stalk region to recapture the tRNAs released from the preceding ribosome in polysomes.

  16. T box riboswitches in Actinobacteria: Translational regulation via novel tRNA interactions

    PubMed Central

    Sherwood, Anna V.; Grundy, Frank J.; Henkin, Tina M.

    2015-01-01

    The T box riboswitch regulates many amino acid-related genes in Gram-positive bacteria. T box riboswitch-mediated gene regulation was shown previously to occur at the level of transcription attenuation via structural rearrangements in the 5′ untranslated (leader) region of the mRNA in response to binding of a specific uncharged tRNA. In this study, a novel group of isoleucyl-tRNA synthetase gene (ileS) T box leader sequences found in organisms of the phylum Actinobacteria was investigated. The Stem I domains of these RNAs lack several highly conserved elements that are essential for interaction with the tRNA ligand in other T box RNAs. Many of these RNAs were predicted to regulate gene expression at the level of translation initiation through tRNA-dependent stabilization of a helix that sequesters a sequence complementary to the Shine–Dalgarno (SD) sequence, thus freeing the SD sequence for ribosome binding and translation initiation. We demonstrated specific binding to the cognate tRNAIle and tRNAIle-dependent structural rearrangements consistent with regulation at the level of translation initiation, providing the first biochemical demonstration, to our knowledge, of translational regulation in a T box riboswitch. PMID:25583497

  17. The selective tRNA aminoacylation mechanism based on a single G•U pair.

    PubMed

    Naganuma, Masahiro; Sekine, Shun-ichi; Chong, Yeeting Esther; Guo, Min; Yang, Xiang-Lei; Gamper, Howard; Hou, Ya-Ming; Schimmel, Paul; Yokoyama, Shigeyuki

    2014-06-26

    Ligation of tRNAs with their cognate amino acids, by aminoacyl-tRNA synthetases, establishes the genetic code. Throughout evolution, tRNA(Ala) selection by alanyl-tRNA synthetase (AlaRS) has depended predominantly on a single wobble base pair in the acceptor stem, G3•U70, mainly on the kcat level. Here we report the crystal structures of an archaeal AlaRS in complex with tRNA(Ala) with G3•U70 and its A3•U70 variant. AlaRS interacts with both the minor- and the major-groove sides of G3•U70, widening the major groove. The geometry difference between G3•U70 and A3•U70 is transmitted along the acceptor stem to the 3'-CCA region. Thus, the 3'-CCA region of tRNA(Ala) with G3•U70 is oriented to the reactive route that reaches the active site, whereas that of the A3•U70 variant is folded back into the non-reactive route. This novel mechanism enables the single wobble pair to dominantly determine the specificity of tRNA selection, by an approximate 100-fold difference in kcat.

  18. The selective tRNA aminoacylation mechanism based on a single G•U pair

    PubMed Central

    Naganuma, Masahiro; Sekine, Shun-ichi; Chong, Yeeting Esther; Guo, Min; Yang, Xiang-Lei; Gamper, Howard; Hou, Ya-Ming; Schimmel, Paul; Yokoyama, Shigeyuki

    2015-01-01

    Ligation of tRNAs with their cognate amino acids, by aminoacyl-tRNA synthetases, establishes the genetic code. Throughout evolution, tRNAAla selection by alanyl-tRNA synthetase (AlaRS) has depended predominantly on a single wobble base pair in the acceptor stem, G3•U70, mainly on the kcat level. Here we report the crystal structures of an archaeal AlaRS in complex with tRNAAla with G3•U70 and its A3•U70 variant. AlaRS interacts with both the minor- and major-groove sides of G3•U70, widening the major groove. The geometry difference between G3•U70 and A3•U70 is transmitted along the acceptor stem to the 3′-CCA region. Thus, the 3′-CCA region of tRNAAla with G3•U70 is oriented to the reactive route that reaches the active site, whereas that of the A3•U70 variant is folded back into the “non-reactive route”. This novel mechanism enables the single wobble pair to dominantly determine the specificity of tRNA selection, by an approximate 100-fold difference in kcat. PMID:24919148

  19. Essentiality of threonylcarbamoyladenosine (t6A), a universal tRNA modification, in bacteria

    PubMed Central

    Thiaville, Patrick C.; Yacoubi, Basma El; Köhrer, Caroline; Thiaville, Jennifer J.; Deutsch, Chris; Iwata-Reuyl, Dirk; Bacusmo, Jo Marie; Armengaud, Jean; Bessho, Yoshitaka; Wetzel, Collin; Cao, Xiaoyu; Limbach, Patrick A.; RajBhandary, Uttam L.; de Crécy-Lagard, Valérie

    2016-01-01

    Threonylcarbamoyladenosine (t6A) is a modified nucleoside universally conserved in tRNAs in all three kingdoms of life. The recently discovered genes for t6A synthesis, including tsaC and tsaD, are essential in model prokaryotes but not essential in yeast. These genes had been identified as antibacterial targets even before their functions were known. However, the molecular basis for this prokaryotic-specific essentiality has remained a mystery. Here, we show that t6A is a strong positive determinant for aminoacylation of tRNA by bacterial-type but not by eukaryotic-type isoleucyl-tRNA synthetases and might also be a determinant for the essential enzyme tRNAIle-lysidine synthetase. We confirm that t6A is essential in Escherichia coli and a survey of genome-wide essentiality studies shows that genes for t6A synthesis are essential in most prokaryotes. This essentiality phenotype is not universal in Bacteria as t6A is dispensable in Deinococcus radiodurans, Thermus thermophilus, Synechocystis PCC6803 and Streptococcus mutans. Proteomic analysis of t6A- D. radiodurans strains revealed an induction of the proteotoxic stress response and identified genes whose translation is most affected by the absence of t6A in tRNAs. Thus, although t6A is universally conserved in tRNAs, its role in translation might vary greatly between organisms. PMID:26337258

  20. In vivo selection of lethal mutations reveals two functional domains in arginyl-tRNA synthetase.

    PubMed Central

    Geslain, R; Martin, F; Delagoutte, B; Cavarelli, J; Gangloff, J; Eriani, G

    2000-01-01

    Using random mutagenesis and a genetic screening in yeast, we isolated 26 mutations that inactivate Saccharomyces cerevisiae arginyl-tRNA synthetase (ArgRS). The mutations were identified and the kinetic parameters of the corresponding proteins were tested after purification of the expression products in Escherichia coli. The effects were interpreted in the light of the crystal structure of ArgRS. Eighteen functional residues were found around the arginine-binding pocket and eight others in the carboxy-terminal domain of the enzyme. Mutations of these residues all act by strongly impairing the rates of tRNA charging and arginine activation. Thus, ArgRS and tRNA(Arg) can be considered as a kind of ribonucleoprotein, where the tRNA, before being charged, is acting as a cofactor that activates the enzyme. Furthermore, by using different tRNA(Arg) isoacceptors and heterologous tRNA(Asp), we highlighted the crucial role of several residues of the carboxy-terminal domain in tRNA recognition and discrimination. PMID:10744027

  1. Recombinant expression, purification, and crystallization of the glutaminyl-tRNA synthetase from Toxoplasma gondii.

    PubMed

    van Rooyen, Jason M; Hakimi, Mohamed-Ali; Belrhali, Hassan

    2015-06-01

    Aminoacyl tRNA synthetases play a critical role in protein synthesis by providing precursor transfer-RNA molecules correctly charged with their cognate amino-acids. The essential nature of these enzymes make them attractive targets for designing new drugs against important pathogenic protozoans like Toxoplasma. Because no structural data currently exists for a protozoan glutaminyl-tRNA synthetase (QRS), an understanding of its potential as a drug target and its function in the assembly of the Toxoplasma multi-aminoacyl tRNA (MARS) complex is therefore lacking. Here we describe the optimization of expression and purification conditions that permitted the recovery and crystallization of both domains of the Toxoplasma QRS enzyme from a heterologous Escherichia coli expression system. Expression of full-length QRS was only achieved after the addition of an N-terminal histidine affinity tag and the isolated protein was active on both cellular and in vitro produced Toxoplasma tRNA. Taking advantage of the proteolytic susceptibility of QRS to cleavage into component domains, N-terminal glutathione S-transferase (GST) motif-containing domain fragments were isolated and crystallization conditions discovered. Isolation of the C-terminal catalytic domain was accomplished after subcloning the domain and optimizing expression conditions. Purified catalytic domain survived cryogenic storage and yielded large diffraction-quality crystals over-night after optimization of screening conditions. This work will form the basis of future structural studies into structural-functional relationships of both domains including potential targeted drug-design studies and investigations into the assembly of the Toxoplasma MARS complex.

  2. Genome recoding by tRNA modifications

    PubMed Central

    Tuorto, Francesca

    2016-01-01

    RNA modifications are emerging as an additional regulatory layer on top of the primary RNA sequence. These modifications are particularly enriched in tRNAs where they can regulate not only global protein translation, but also protein translation at the codon level. Modifications located in or in the vicinity of tRNA anticodons are highly conserved in eukaryotes and have been identified as potential regulators of mRNA decoding. Recent studies have provided novel insights into how these modifications orchestrate the speed and fidelity of translation to ensure proper protein homeostasis. This review highlights the prominent modifications in the tRNA anticodon loop: queuosine, inosine, 5-methoxycarbonylmethyl-2-thiouridine, wybutosine, threonyl–carbamoyl–adenosine and 5-methylcytosine. We discuss the functional relevance of these modifications in protein translation and their emerging role in eukaryotic genome recoding during cellular adaptation and disease. PMID:27974624

  3. Crystal structure of the N-terminal anticodon-binding domain of the nondiscriminating aspartyl-tRNA synthetase from Helicobacter pylori.

    PubMed

    Songsiriritthigul, Chomphunuch; Suebka, Suwimon; Chen, Chun Jung; Fuengfuloy, Pitchayada; Chuawong, Pitak

    2017-02-01

    The N-terminal anticodon-binding domain of the nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) plays a crucial role in the recognition of both tRNA(Asp) and tRNA(Asn). Here, the first X-ray crystal structure of the N-terminal domain of this enzyme (ND-AspRS1-104) from the human-pathogenic bacterium Helicobacter pylori is reported at 2.0 Å resolution. The apo form of H. pylori ND-AspRS1-104 shares high structural similarity with the N-terminal anticodon-binding domains of the discriminating aspartyl-tRNA synthetase (D-AspRS) from Escherichia coli and ND-AspRS from Pseudomonas aeruginosa, allowing recognition elements to be proposed for tRNA(Asp) and tRNA(Asn). It is proposed that a long loop (Arg77-Lys90) in this H. pylori domain influences its relaxed tRNA specificity, such that it is classified as nondiscriminating. A structural comparison between D-AspRS from E. coli and ND-AspRS from P. aeruginosa suggests that turns E and F (78GAGL81 and 83NPKL86) in H. pylori ND-AspRS play a crucial role in anticodon recognition. Accordingly, the conserved Pro84 in turn F facilitates the recognition of the anticodons of tRNA(Asp) ((34)GUC(36)) and tRNA(Asn) ((34)GUU(36)). The absence of the amide H atom allows both C and U bases to be accommodated in the tRNA-recognition site.

  4. Kinetic Analysis of tRNA Methylfransferases

    PubMed Central

    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

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

    PubMed

    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.

  6. Magnesium dependence of the measured equilibrium constants of aminoacyl-tRNA synthetases.

    PubMed

    Airas, R Kalervo

    2007-12-01

    The apparent equilibrium constants (K') for six reactions catalyzed by aminoacyl-tRNA synthetases from Escherichia coli were measured, the equations for the magnesium dependence of the equilibrium constants were derived, and best-fit analyses between the measured and calculated values were used. The K' values at 1 mM Mg(2+) ranged from 0.49 to 1.13. The apparent equilibrium constants increased with increasing Mg(2+) concentrations. The values were 2-3 times higher at 20 mM Mg(2+) than at 1 mM Mg(2+), and the dependence was similar in the class I and class II synthetases. The main reason for the Mg(2+) dependence is the existence of PP(i) as two magnesium complexes, but only one of them is the real product. AMP exists either as free AMP or as MgAMP, and therefore also has some effect on the measured equilibrium constant. However, these dependences alone cannot explain the measured results. The measured dependence of the K' on the Mg(2+) concentration is weaker than that caused by PP(i) and AMP. Different bindings of the Mg(2+) ions to the substrate tRNA and product aminoacyl-tRNA can explain this observation. The best-fit analysis suggests that tRNA reacts as a magnesium complex in the forward aminoacylation direction but this given Mg(2+) ion is not bound to aminoacyl-tRNA at the start of the reverse reaction. Thus Mg(2+) ions seem to have an active catalytic role, not only in the activation of the amino acid, but in the posttransfer steps of the aminoacyl-tRNA synthetase reaction, too.

  7. Nucleotide sequence of Neurospora crassa cytoplasmic initiator tRNA.

    PubMed Central

    Gillum, A M; Hecker, L I; Silberklang, M; Schwartzbach, S D; RajBhandary, U L; Barnett, W E

    1977-01-01

    Initiator methionine tRNA from the cytoplasm of Neurospora crassa has been purified and sequenced. The sequence is: pAGCUGCAUm1GGCGCAGCGGAAGCGCM22GCY*GGGCUCAUt6AACCCGGAGm7GU (or D) - CACUCGAUCGm1AAACGAG*UUGCAGCUACCAOH. Similar to initiator tRNAs from the cytoplasm of other eukaryotes, this tRNA also contains the sequence -AUCG- instead of the usual -TphiCG (or A)- found in loop IV of other tRNAs. The sequence of the N. crassa cytoplasmic initiator tRNA is quite different from that of the corresponding mitochondrial initiator tRNA. Comparison of the sequence of N. crassa cytoplasmic initiator tRNA to those of yeast, wheat germ and vertebrate cytoplasmic initiator tRNA indicates that the sequences of the two fungal tRNAs are no more similar to each other than they are to those of other initiator tRNAs. Images PMID:146192

  8. Stochastic context-free grammars for tRNA modeling.

    PubMed Central

    Sakakibara, Y; Brown, M; Hughey, R; Mian, I S; Sjölander, K; Underwood, R C; Haussler, D

    1994-01-01

    Stochastic context-free grammars (SCFGs) are applied to the problems of folding, aligning and modeling families of tRNA sequences. SCFGs capture the sequences' common primary and secondary structure and generalize the hidden Markov models (HMMs) used in related work on protein and DNA. Results show that after having been trained on as few as 20 tRNA sequences from only two tRNA subfamilies (mitochondrial and cytoplasmic), the model can discern general tRNA from similar-length RNA sequences of other kinds, can find secondary structure of new tRNA sequences, and can produce multiple alignments of large sets of tRNA sequences. Our results suggest potential improvements in the alignments of the D- and T-domains in some mitochondrial tRNAs that cannot be fit into the canonical secondary structure. PMID:7800507

  9. Telomerase reverse transcriptase promotes cancer cell proliferation by augmenting tRNA expression

    PubMed Central

    Khattar, Ekta; Kumar, Pavanish; Liu, Chia Yi; Akıncılar, Semih Can; Raju, Anandhkumar; Lakshmanan, Manikandan; Maury, Julien Jean Pierre; Qiang, Yu; Li, Shang; Tan, Ern Yu; Hui, Kam M.; Loh, Yuin Han

    2016-01-01

    Transcriptional reactivation of telomerase reverse transcriptase (TERT) reconstitutes telomerase activity in the majority of human cancers. Here, we found that ectopic TERT expression increases cell proliferation, while acute reductions in TERT levels lead to a dramatic loss of proliferation without any change in telomere length, suggesting that the effects of TERT could be telomere independent. We observed that TERT determines the growth rate of cancer cells by directly regulating global protein synthesis independently of its catalytic activity. Genome-wide TERT binding across 5 cancer cell lines and 2 embryonic stem cell lines revealed that endogenous TERT, driven by mutant promoters or oncogenes, directly associates with the RNA polymerase III (pol III) subunit RPC32 and enhances its recruitment to chromatin, resulting in increased RNA pol III occupancy and tRNA expression in cancers. TERT-deficient mice displayed marked delays in polyomavirus middle T oncogene–induced (PyMT-induced) mammary tumorigenesis, increased survival, and reductions in tRNA levels. Ectopic expression of either RPC32 or TERT restored tRNA levels and proliferation defects in TERT-depleted cells. Finally, we determined that levels of TERT and tRNA correlated in breast and liver cancer samples. Together, these data suggest the existence of a unifying mechanism by which TERT enhances translation in cells to regulate cancer cell proliferation. PMID:27643433

  10. Limitations to the development of recombinant human embryonic kidney 293E cells using glutamine synthetase-mediated gene amplification: Methionine sulfoximine resistance.

    PubMed

    Yu, Da Young; Noh, Soo Min; Lee, Gyun Min

    2016-08-10

    To investigate the feasibility of glutamine synthetase (GS)-mediated gene amplification in HEK293 cells for the high-level stable production of therapeutic proteins, HEK293E cells were transfected by the GS expression vector containing antibody genes and were selected at various methionine sulfoximine (MSX) concentrations in 96-well plates. For a comparison, CHOK1 cells were transfected by the same GS expression vector and selected at various MSX concentrations. Unlike CHOK1 cells, HEK293E cells producing high levels of antibodies were not selected at all. For HEK293E cells, the number of wells with the cell pool did not decrease with an increase in the concentration of MSX up to 500μM MSX. A q-RT-PCR analysis confirmed that the antibody genes in the HEK293E cells, unlike the CHOK1 cells, were not amplified after increasing the MSX concentration. It was found that the GS activity in HEK293E cells was much higher than that in CHOK1 cells (P<0.05). In a glutamine-free medium, the GS activity of HEK293E cells was approximately 4.8 times higher than that in CHOK1 cells. Accordingly, it is inferred that high GS activity of HEK293E cells results in elevated resistance to MSX and therefore hampers GS-mediated gene amplification by MSX. Thus, in order to apply the GS-mediated gene amplification system to HEK293 cells, the endogenous GS expression level in HEK293 cells needs to be minimized by knock-out or down-regulation methods.

  11. Hepatocytes explanted in the spleen preferentially express carbamoylphosphate synthetase rather than glutamine synthetase.

    PubMed

    Lamers, W H; Been, W; Charles, R; Moorman, A F

    1990-10-01

    Urea cycle enzymes and glutamine synthetase are essential for NH3 detoxification and systemic pH homeostasis in mammals. Carbamoylphosphate synthetase, the first and flux-determining enzyme of the cycle, is found only in a large periportal compartment, and glutamine synthetase is found only in a small, complementary pericentral compartment. Because it is not possible to manipulate experimentally the intrahepatic distribution of carbamoylphosphate synthetase and glutamine synthetase, we looked for conditions in which explanted hepatocytes would exhibit either the carbamoylphosphate synthetase phenotype or glutamine synthetase phenotype. In the spleen hepatocytes either settle as individual cells or in small agglomerates. The dispersed cells only express the carbamoylphosphate synthetase phenotype. Within the agglomerates, sinusoids that drain on venules develop. Hepatocytes surrounding the venules stain only weakly for carbamoylphosphate synthetase but are strongly positive for glutamine synthetase. These observations were made for explanted embryonic hepatocytes (no prior expression of either carbamoylphosphate synthetase or glutamine synthetase), neonatal hepatocytes (compartments of gene expression not yet established) and adult periportal and pericentral hepatocytes.

  12. The Bacillus subtilis and Bacillus halodurans Aspartyl-tRNA Synthetases Retain Recognition of tRNA(Asn).

    PubMed

    Nair, Nilendra; Raff, Hannah; Islam, Mohammed Tarek; Feen, Melanie; Garofalo, Denise M; Sheppard, Kelly

    2016-02-13

    Synthesis of asparaginyl-tRNA (Asn-tRNA(Asn)) in bacteria can be formed either by directly ligating Asn to tRNA(Asn) using an asparaginyl-tRNA synthetase (AsnRS) or by synthesizing Asn on the tRNA. In the latter two-step indirect pathway, a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) attaches Asp to tRNA(Asn) and the amidotransferase GatCAB transamidates the Asp to Asn on the tRNA. GatCAB can be similarly used for Gln-tRNA(Gln) formation. Most bacteria are predicted to use only one route for Asn-tRNA(Asn) formation. Given that Bacillus halodurans and Bacillus subtilis encode AsnRS for Asn-tRNA(Asn) formation and Asn synthetases to synthesize Asn and GatCAB for Gln-tRNA(Gln) synthesis, their AspRS enzymes were thought to be specific for tRNA(Asp). However, we demonstrate that the AspRSs are non-discriminating and can be used with GatCAB to synthesize Asn. The results explain why B. subtilis with its Asn synthetase genes knocked out is still an Asn prototroph. Our phylogenetic analysis suggests that this may be common among Firmicutes and 30% of all bacteria. In addition, the phylogeny revealed that discrimination toward tRNA(Asp) by AspRS has evolved independently multiple times. The retention of the indirect pathway in B. subtilis and B. halodurans likely reflects the ancient link between Asn biosynthesis and its use in translation that enabled Asn to be added to the genetic code.

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

    PubMed

    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.

  14. CCA addition to tRNA: implications for tRNA quality control.

    PubMed

    Hou, Ya-Ming

    2010-04-01

    The CCA sequence is conserved at the 3' end of all mature tRNA molecules to function as the site of amino acid attachment. This sequence is acquired and maintained by stepwise nucleotide addition by the ubiquitous CCA enzyme, which is an unusual RNA polymerase that does not use a nucleic acid template for nucleotide addition. Crystal structural work has divided CCA enzymes into two structurally distinct classes, which differ in the mechanism of template-independent nucleotide selection. Recent kinetic work of the class II E. coli CCA enzyme has demonstrated a rapid and uniform rate constant for the chemistry of nucleotide addition at each step of CCA synthesis, although the enzyme uses different determinants to control the rate of each step. Importantly, the kinetic work reveals that, at each step of CCA synthesis, E. coli CCA enzyme has an innate ability to discriminate against tRNA backbone damage. This discrimination suggests the possibility of a previously unrecognized quality control mechanism that would prevent damaged tRNA from CCA maturation and from entering the ribosome machinery of protein synthesis. This quality control is relevant to cellular stress conditions that damage tRNA backbone and predicts a role of CCA addition in stress response.

  15. The CCA-end of P-tRNA Contacts Both the Human RPL36AL and the A-site Bound Translation Termination Factor eRF1 at the Peptidyl Transferase Center of the Human 80S Ribosome.

    PubMed

    Hountondji, Codjo; Bulygin, Konstantin; Créchet, Jean-Bernard; Woisard, Anne; Tuffery, Pierre; Nakayama, Jun-Ichi; Frolova, Ludmila; Nierhaus, Knud H; Karpova, Galina; Baouz, Soria

    2014-01-01

    We have demonstrated previously that the E-site specific protein RPL36AL present in human ribosomes can be crosslinked with the CCA-end of a P-tRNA in situ. Here we report the following: (i) We modeled RPL36AL into the structure of the archaeal ortholog RPL44E extracted from the known X-ray structure of the 50S subunit of Haloarcula marismortui. Superimposing the obtained RPL36AL structure with that of P/E tRNA observed in eukaryotic 80S ribosomes suggested that RPL36AL might in addition to its CCA neighbourhood interact with the inner site of the tRNA elbow similar to an interaction pattern known from tRNA•synthetase pairs. (ii) Accordingly, we detected that the isolated recombinant protein RPL36AL can form a tight binary complex with deacylated tRNA, and even tRNA fragments truncated at their CCA end showed a high affinity in the nanomolar range supporting a strong interaction outside the CCA end. (iii) We constructed programmed 80S complexes containing the termination factor eRF1 (stop codon UAA at the A-site) and a 2',3'-dialdehyde tRNA (tRNAox) analog at the P-site. Surprisingly, we observed a crosslinked ternary complex containing the tRNA, eRF1 and RPL36AL crosslinked both to the aldehyde groups of tRNAox at the 2'- and 3'-positions of the ultimate A. We also demonstrated that, upon binding to the ribosomal A-site, eRF1 induces an alternative conformation of the ribosome and/or the tRNA, leading to a novel crosslink of tRNAox to another large-subunit ribosomal protein (namely L37) rather than to RPL36AL, both ribosomal proteins being labeled in a mutually exclusive fashion. Since the human 80S ribosome in complex with P-site bound tRNAox and A-site bound eRF1 corresponds to the post-termination state of the ribosome, the results represent the first biochemical evidence for the positioning of the CCA-arm of the P-tRNA in close proximity to both RPL36AL and eRF1 at the end of the translation process.

  16. Regulation of active site coupling in glutamine-dependent NAD[superscript +] synthetase

    SciTech Connect

    LaRonde-LeBlanc, Nicole; Resto, Melissa; Gerratana, Barbara

    2009-05-21

    NAD{sup +} is an essential metabolite both as a cofactor in energy metabolism and redox homeostasis and as a regulator of cellular processes. In contrast to humans, Mycobacterium tuberculosis NAD{sup +} biosynthesis is absolutely dependent on the activity of a multifunctional glutamine-dependent NAD{sup +} synthetase, which catalyzes the ATP-dependent formation of NAD{sup +} at the synthetase domain using ammonia derived from L-glutamine in the glutaminase domain. Here we report the kinetics and structural characterization of M. tuberculosis NAD{sup +} synthetase. The kinetics data strongly suggest tightly coupled regulation of the catalytic activities. The structure, the first of a glutamine-dependent NAD{sup +} synthetase, reveals a homooctameric subunit organization suggesting a tight dependence of catalysis on the quaternary structure, a 40-{angstrom} intersubunit ammonia tunnel and structural elements that may be involved in the transfer of information between catalytic sites.

  17. tRNA Shifts the G-quadruplex-Hairpin Conformational Equilibrium in RNA towards the Hairpin Conformer.

    PubMed

    Rode, Ambadas B; Endoh, Tamaki; Sugimoto, Naoki

    2016-11-07

    Non-coding RNAs play important roles in cellular homeostasis and are involved in many human diseases including cancer. Intermolecular RNA-RNA interactions are the basis for the diverse functions of many non-coding RNAs. Herein, we show how the presence of tRNA influences the equilibrium between hairpin and G-quadruplex conformations in the 5' untranslated regions of oncogenes and model sequences. Kinetic and equilibrium analyses of the hairpin to G-quadruplex conformational transition of purified RNA as well as during co-transcriptional folding indicate that tRNA significantly shifts the equilibrium toward the hairpin conformer. The enhancement of relative translation efficiency in a reporter gene assay is shown to be due to the tRNA-mediated shift in hairpin-G-quadruplex equilibrium of oncogenic mRNAs. Our findings suggest that tRNA is a possible therapeutic target in diseases in which RNA conformational equilibria is dysregulated.

  18. Argonaute 2 Binds Directly to tRNA Genes and Promotes Gene Repression in cis

    PubMed Central

    Woolnough, Jessica L.; Atwood, Blake L.

    2015-01-01

    To further our understanding of the RNAi machinery within the human nucleus, we analyzed the chromatin and RNA binding of Argonaute 2 (AGO2) within human cancer cell lines. Our data indicated that AGO2 binds directly to nascent tRNA and 5S rRNA, and to the genomic loci from which these RNAs are transcribed, in a small RNA- and DICER-independent manner. AGO2 chromatin binding was not observed at non-TFIIIC-dependent RNA polymerase III (Pol III) genes or at extra-TFIIIC (ETC) sites, indicating that the interaction is specific for TFIIIC-dependent Pol III genes. A genome-wide analysis indicated that loss of AGO2 caused a global increase in mRNA expression level among genes that flank AGO2-bound tRNA genes. This effect was shown to be distinct from that of the disruption of DICER, DROSHA, or CTCF. We propose that AGO2 binding to tRNA genes has a novel and important regulatory role in human cells. PMID:25918241

  19. Substrate and Enzyme Functional Groups Contribute to Translational Quality Control by Bacterial Prolyl-tRNA Synthetase

    PubMed Central

    Kumar, Sandeep; Das, Mom; Hadad, Christopher M.; Musier-Forsyth, Karin

    2012-01-01

    Aminoacyl-tRNA synthetases activate specific amino acid substrates and attach them via an ester linkage to cognate tRNA molecules. In addition to cognate proline, prolyl-tRNA synthetase (ProRS) can activate cysteine and alanine and misacylate tRNAPro. Editing of the misacylated aminoacyl-tRNA is required for error-free protein synthesis. An editing domain (INS) appended to bacterial ProRS selectively hydrolyzes Ala-tRNAPro, whereas Cys-tRNAPro is cleared by a freestanding editing domain, YbaK, through a unique mechanism involving substrate sulfhydryl chemistry. The detailed mechanism of catalysis by INS is currently unknown. To understand the alanine specificity and mechanism of catalysis by INS, we have explored several possible mechanisms of Ala-tRNAPro deacylation via hybrid QM/MM calculations. Experimental studies were also performed to test the role of several residues in the INS active site, as well as various substrate functional groups in catalysis. Our results support a critical role for the tRNA 2′-OH group in substrate binding and catalytic water activation. A role is also proposed for the protein’s conserved GXXXP loop in transition state stabilization and for the main chain atoms of Gly261 in a proton relay that contributes substantially to catalysis. PMID:22458656

  20. Handling tRNA introns, archaeal way and eukaryotic way

    PubMed Central

    Yoshihisa, Tohru

    2014-01-01

    Introns are found in various tRNA genes in all the three kingdoms of life. Especially, archaeal and eukaryotic genomes are good sources of tRNA introns that are removed by proteinaceous splicing machinery. Most intron-containing tRNA genes both in archaea and eukaryotes possess an intron at a so-called canonical position, one nucleotide 3′ to their anticodon, while recent bioinformatics have revealed unusual types of tRNA introns and their derivatives especially in archaeal genomes. Gain and loss of tRNA introns during various stages of evolution are obvious both in archaea and eukaryotes from analyses of comparative genomics. The splicing of tRNA molecules has been studied extensively from biochemical and cell biological points of view, and such analyses of eukaryotic systems provided interesting findings in the past years. Here, I summarize recent progresses in the analyses of tRNA introns and the splicing process, and try to clarify new and old questions to be solved in the next stages. PMID:25071838

  1. Small-angle X-ray Solution Scattering Study of the Multi-aminoacyl-tRNA Synthetase Complex Reveals an Elongated and Multi-armed particle*

    PubMed Central

    Dias, José; Renault, Louis; Pérez, Javier; Mirande, Marc

    2013-01-01

    In animal cells, nine aminoacyl-tRNA synthetases are associated with the three auxiliary proteins p18, p38, and p43 to form a stable and conserved large multi-aminoacyl-tRNA synthetase complex (MARS), whose molecular mass has been proposed to be between 1.0 and 1.5 MDa. The complex acts as a molecular hub for coordinating protein synthesis and diverse regulatory signal pathways. Electron microscopy studies defined its low resolution molecular envelope as an overall rather compact, asymmetric triangular shape. Here, we have analyzed the composition and homogeneity of the native mammalian MARS isolated from rabbit liver and characterized its overall internal structure, size, and shape at low resolution by hydrodynamic methods and small-angle x-ray scattering in solution. Our data reveal that the MARS exhibits a much more elongated and multi-armed shape than expected from previous reports. The hydrodynamic and structural features of the MARS are large compared with other supramolecular assemblies involved in translation, including ribosome. The large dimensions and non-compact structural organization of MARS favor a large protein surface accessibility for all its components. This may be essential to allow structural rearrangements between the catalytic and cis-acting tRNA binding domains of the synthetases required for binding the bulky tRNA substrates. This non-compact architecture may also contribute to the spatiotemporal controlled release of some of its components, which participate in non-canonical functions after dissociation from the complex. PMID:23836901

  2. Gene encoding plant asparagine synthetase

    DOEpatents

    Coruzzi, Gloria M.; Tsai, Fong-Ying

    1993-10-26

    The identification and cloning of the gene(s) for plant asparagine synthetase (AS), an important enzyme involved in the formation of asparagine, a major nitrogen transport compound of higher plants is described. Expression vectors constructed with the AS coding sequence may be utilized to produce plant AS; to engineer herbicide resistant plants, salt/drought tolerant plants or pathogen resistant plants; as a dominant selectable marker; or to select for novel herbicides or compounds useful as agents that synchronize plant cells in culture. The promoter for plant AS, which directs high levels of gene expression and is induced in an organ specific manner and by darkness, is also described. The AS promoter may be used to direct the expression of heterologous coding sequences in appropriate hosts.

  3. Structural modeling of tissue-specific mitochondrial alanyl-tRNA synthetase (AARS2) defects predicts differential effects on aminoacylation.

    PubMed

    Euro, Liliya; Konovalova, Svetlana; Asin-Cayuela, Jorge; Tulinius, Már; Griffin, Helen; Horvath, Rita; Taylor, Robert W; Chinnery, Patrick F; Schara, Ulrike; Thorburn, David R; Suomalainen, Anu; Chihade, Joseph; Tyynismaa, Henna

    2015-01-01

    The accuracy of mitochondrial protein synthesis is dependent on the coordinated action of nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mtARSs) and the mitochondrial DNA-encoded tRNAs. The recent advances in whole-exome sequencing have revealed the importance of the mtARS proteins for mitochondrial pathophysiology since nearly every nuclear gene for mtARS (out of 19) is now recognized as a disease gene for mitochondrial disease. Typically, defects in each mtARS have been identified in one tissue-specific disease, most commonly affecting the brain, or in one syndrome. However, mutations in the AARS2 gene for mitochondrial alanyl-tRNA synthetase (mtAlaRS) have been reported both in patients with infantile-onset cardiomyopathy and in patients with childhood to adulthood-onset leukoencephalopathy. We present here an investigation of the effects of the described mutations on the structure of the synthetase, in an effort to understand the tissue-specific outcomes of the different mutations. The mtAlaRS differs from the other mtARSs because in addition to the aminoacylation domain, it has a conserved editing domain for deacylating tRNAs that have been mischarged with incorrect amino acids. We show that the cardiomyopathy phenotype results from a single allele, causing an amino acid change R592W in the editing domain of AARS2, whereas the leukodystrophy mutations are located in other domains of the synthetase. Nevertheless, our structural analysis predicts that all mutations reduce the aminoacylation activity of the synthetase, because all mtAlaRS domains contribute to tRNA binding for aminoacylation. According to our model, the cardiomyopathy mutations severely compromise aminoacylation whereas partial activity is retained by the mutation combinations found in the leukodystrophy patients. These predictions provide a hypothesis for the molecular basis of the distinct tissue-specific phenotypic outcomes.

  4. An ENU-induced mutation in mouse glycyl-tRNA synthetase (GARS) causes peripheral sensory and motor phenotypes creating a model of Charcot-Marie-Tooth type 2D peripheral neuropathy

    PubMed Central

    Achilli, Francesca; Bros-Facer, Virginie; Williams, Hazel P.; Banks, Gareth T.; AlQatari, Mona; Chia, Ruth; Tucci, Valter; Groves, Michael; Nickols, Carole D.; Seburn, Kevin L.; Kendall, Rachel; Cader, Muhammed Z.; Talbot, Kevin; van Minnen, Jan; Burgess, Robert W.; Brandner, Sebastian; Martin, Joanne E.; Koltzenburg, Martin; Greensmith, Linda; Nolan, Patrick M.; Fisher, Elizabeth M. C.

    2009-01-01

    SUMMARY Mutations in the enzyme glycyl-tRNA synthetase (GARS) cause motor and sensory axon loss in the peripheral nervous system in humans, described clinically as Charcot-Marie-Tooth type 2D or distal spinal muscular atrophy type V. Here, we characterise a new mouse mutant, GarsC201R, with a point mutation that leads to a non-conservative substitution within GARS. Heterozygous mice with a C3H genetic background have loss of grip strength, decreased motor flexibility and disruption of fine motor control; this relatively mild phenotype is more severe on a C57BL/6 background. Homozygous mutants have a highly deleterious set of features, including movement difficulties and death before weaning. Heterozygous animals have a reduction in axon diameter in peripheral nerves, slowing of nerve conduction and an alteration in the recovery cycle of myelinated axons, as well as innervation defects. An assessment of GARS levels showed increased protein in 15-day-old mice compared with controls; however, this increase was not observed in 3-month-old animals, indicating that GARS function may be more crucial in younger animals. We found that enzyme activity was not reduced detectably in heterozygotes at any age, but was diminished greatly in homozygous mice compared with controls; thus, homozygous animals may suffer from a partial loss of function. The GarsC201R mutation described here is a contribution to our understanding of the mechanism by which mutations in tRNA synthetases, which are fundamentally important, ubiquitously expressed enzymes, cause axonopathy in specific sets of neurons. PMID:19470612

  5. Genetics Home Reference: holocarboxylase synthetase deficiency

    MedlinePlus

    ... use of biotin, a B vitamin found in foods such as liver, egg yolks, and milk. Holocarboxylase synthetase attaches biotin to certain enzymes that are essential for the normal production and breakdown of proteins, fats, and carbohydrates in ...

  6. tRNA nucleotide 47: an evolutionary enigma.

    PubMed

    Cermakian, N; McClain, W H; Cedergren, R

    1998-08-01

    A previous analysis of tRNA sequences suggested a correlation between the absence of a nucleotide at position 47 (nt 47) in the extra loop and the presence of a U13:G22 base pair in the D-stem. We have evaluated the significance of this correlation by determining the in vivo activity of tRNAs containing either a C13:G22 or a U13:G22 pair in tRNA molecules with or without nt 47. Although this correlation might reflect some malfunction of tRNAs lacking nt 47, but containing the C13:G22, assays of the in vivo suppressor activity showed that this tRNA is actually more active than the tRNA with the features found in the database, i.e., a U13:G22 base pair and no nt 47. Moreover, analogous constructs with a GGC anticodon permitted the growth of an Escherichia coli strain deleted for tRNA(Ala)GGC genes equally well. On the other hand, long-term growth experiments with competing E. coli strains harboring the tRNA lacking nt 47, either with the C13:G22 or the U13:G22 base pair demonstrated that the U13:G22 tRNA overtook the C13:G22 strain even when the starting proportion of strains favored the C13:G22 strain. Thus, the preference for the U13:G22 tRNA lacking nt 47 in the sequence database is most likely due to factors that come into play during extended growth or latency rather than to the ability of the tRNA to engage in protein synthesis.

  7. Unique pathway of expression of an opal suppressor phosphoserine tRNA

    SciTech Connect

    Lee, B.J.; De La Pena, P.; Tobian, J.A.; Zasloff, M.; Hatfield, D.

    1987-09-01

    An opal suppressor phosphoserine tRNA gene is present in single copy in the genomes of higher vertebrates. The authors have shown that the product of this gene functions as a suppressor in an in vitro assay, and they have proposed that it may donate a modified amino acid directly to protein in response to specific UGA codons. In this report, they show through in vitro and in vivo studies that the human and Xenopus opal suppressor phosphoserine tRNAs are synthesized by a pathway that is, to the best of our knowledge, unlike that of nay know eukaryotic tRNA. The primary transcript of this gene does not contain a 5'-leader sequence; and, therefore, transcription of this suppressor is initiated at the first nucleotide within the coding sequence. The 5'-terminal triphosphate, present on the primary transcript, remains intact through 3'-terminal maturation and through subsequent transport of the tRNA to the cytoplasm. The unique biosynthetic pathway of this opal suppressor may underlie its distinctive role in eukaryotic cells.

  8. Structural basis for tRNA modification by Elp3 from Dehalococcoides mccartyi

    PubMed Central

    Glatt, Sebastian; Onuma, Osita F.; Baudin, Florence; Graziadei, Andrea; Taverniti, Valerio; Lin, Ting-Yu; Baymann, Frauke; Seraphin, Bertrand; Breunig, Karin D.; Müller, Christoph W.

    2016-01-01

    During translation elongation decoding is based on the recognition of codons by corresponding tRNA anticodon triplets. Molecular mechanisms that regulate global protein synthesis via specific base modifications in tRNA anticodons have recently received increasing attention. The conserved eukaryotic Elongator complex specifically modifies uridines located in the wobble base position of tRNAs. Here, we present the crystal structure of Dehalococcoides mccartyi Elp3 (DmcElp3) at 2.15 Å resolution. Our results reveal the unexpected arrangement of Elp3 lysine acetyl transferase (KAT) and radical S-adenosyl-methionine (SAM) domains that share a large interface to form a composite active site and tRNA binding pocket with an iron sulfur cluster located in the dimerization interface of two DmcElp3 molecules. Structure-guided mutagenesis studies of yeast Elp3 confirm the relevance of our findings for eukaryotic Elp3s and for understanding Elongator’s role in the onset of various neurodegenerative diseases and cancer in humans. PMID:27455459

  9. Vanderwaltozyma polyspora possesses two glycyl-tRNA synthetase genes: one constitutive and one inducible.

    PubMed

    Chien, Chin-I; Chen, Yueh-Lin; Chen, Shun-Jia; Chou, Chi-Mao; Chen, Chin-Yu; Wang, Chien-Chia

    2015-03-01

    Aminoacyl-tRNA synthetases are housekeeping enzymes essential for protein synthesis. We herein present evidence that the yeast Vanderwaltozyma polyspora possesses two paralogous glycyl-tRNA synthetase (GlyRS) genes-GRS1 and GRS2. Paradoxically, GRS1 provided functions in both the cytoplasm and mitochondria, while GRS2 was essentially silent under normal growth conditions. Expression of GRS2 could be activated by stresses such as high pH or ethanol and most effectively by high temperature. The expressed GlyRS2 protein was exclusively found in the cytoplasm and more stable under heat-shock conditions (37°C) than under normal growth conditions (30°C) in vivo. In addition, GRS2 effectively rescued the cytoplasmic defect of a Saccharomyces cerevisiae GRS1 knockout strain when expressed from a constitutive promoter. Moreover, the purified GlyRS2 enzyme was fairly active at both 30°C and 37°C in glycylation of yeast tRNA in vitro. However, unexpectedly, the purified GlyRS2 enzyme was practically inactive at temperature above 40°C in vitro. Our study suggests that GRS2 is an inducible gene that acts under stress conditions where GlyRS1 may be insufficient, unavailable, or rendered inactive.

  10. Molecular basis of the inhibition of human aromatase (estrogen synthetase) by flavone and isoflavone phytoestrogens: A site-directed mutagenesis study.

    PubMed

    Kao, Y C; Zhou, C; Sherman, M; Laughton, C A; Chen, S

    1998-02-01

    Flavone and isoflavone phytoestrogens are plant chemicals and are known to be competitive inhibitors of cytochrome P450 aromatase with respect to the androgen substrate. Aromatase is the enzyme that converts androgen to estrogen; therefore, these plant chemicals are thought to be capable of modifying the estrogen level in women. In this study, the inhibition profiles of four flavones [chrysin (5, 7-dihydroxyflavone), 7,8-dihydroxyflavone, baicalein (5,6,7-trihydroxyflavone), and galangin (3,5,7-trihydroxyflavone)], two isoflavones [genistein (4,5,7-trihydroxyisoflavone) and biochanin A (5,7-dihydroxy-4-methoxyisoflavone)], one flavanone [naringenin (4, 5,7-trihydroxyflavanone)], and one naphthoflavone (alpha-naphthoflavone) on the wild-type and six human aromatase mutants (I133Y, P308F, D309A, T310S, I395F, and I474Y) were determined. In combination with computer modeling, the binding characteristics and the structure requirement for flavone and isoflavone phytoestrogens to inhibit human aromatase were obtained. These compounds were found to bind to the active site of aromatase in an orientation in which rings A and C mimic rings D and C of the androgen substrate, respectively. This study also provides a molecular basis as to why isoflavones are significantly poorer inhibitors of aromatase than flavones.

  11. Molecular basis of the inhibition of human aromatase (estrogen synthetase) by flavone and isoflavone phytoestrogens: A site-directed mutagenesis study.

    PubMed Central

    Kao, Y C; Zhou, C; Sherman, M; Laughton, C A; Chen, S

    1998-01-01

    Flavone and isoflavone phytoestrogens are plant chemicals and are known to be competitive inhibitors of cytochrome P450 aromatase with respect to the androgen substrate. Aromatase is the enzyme that converts androgen to estrogen; therefore, these plant chemicals are thought to be capable of modifying the estrogen level in women. In this study, the inhibition profiles of four flavones [chrysin (5, 7-dihydroxyflavone), 7,8-dihydroxyflavone, baicalein (5,6,7-trihydroxyflavone), and galangin (3,5,7-trihydroxyflavone)], two isoflavones [genistein (4,5,7-trihydroxyisoflavone) and biochanin A (5,7-dihydroxy-4-methoxyisoflavone)], one flavanone [naringenin (4, 5,7-trihydroxyflavanone)], and one naphthoflavone (alpha-naphthoflavone) on the wild-type and six human aromatase mutants (I133Y, P308F, D309A, T310S, I395F, and I474Y) were determined. In combination with computer modeling, the binding characteristics and the structure requirement for flavone and isoflavone phytoestrogens to inhibit human aromatase were obtained. These compounds were found to bind to the active site of aromatase in an orientation in which rings A and C mimic rings D and C of the androgen substrate, respectively. This study also provides a molecular basis as to why isoflavones are significantly poorer inhibitors of aromatase than flavones. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:9435150

  12. tRNA is entrapped in similar, but distinct, nuclear and cytoplasmic ribonucleoprotein complexes, both of which contain vigilin and elongation factor 1 alpha.

    PubMed Central

    Kruse, C; Grünweller, A; Willkomm, D K; Pfeiffer, T; Hartmann, R K; Müller, P K

    1998-01-01

    Vigilin, which is found predominantly in cells and tissues with high levels of protein biosynthesis, was isolated in its native form from human HEp-2 cells (A.T.C.C. CCL23) by immunoaffinity chromatography. Here we demonstrate that vigilin is part of a novel large tRNA-binding ribonucleoprotein complex (tRNP), found not only in the cytoplasm, but also in the nuclei of human cells. Compositional differences in the protein pattern were detected between the nuclear and cytoplasmic tRNPs, although some properties of the purified nuclear tRNP, such as tRNA protection against nuclease attack, were identical with those of the cytoplasmic tRNP. By using either a pool of total human nuclear RNA or radioactively labelled yeast tRNAAsp in rebinding experiments, we could show that tRNA is specifically recaptured by the RNA-depleted, vigilin-containing nuclear complex. We could also show that vigilin is capable of binding tRNA in vitro. Another tRNA-binding protein is elongation factor 1 alpha, which appears to be enriched in the cytoplasmic and nuclear tRNP complexes. This suggests that the cytoplasmic tRNP may be involved in the channelled tRNA cycle in the cytoplasm of eukaryotic cells. Our results also suggest that the nuclear vigilin-containing tRNP may be related to the nuclear export of tRNA. PMID:9445390

  13. A miRNA-tRNA mix-up: tRNA origin of proposed miRNA.

    PubMed

    Schopman, Nick C T; Heynen, Stephan; Haasnoot, Joost; Berkhout, Ben

    2010-01-01

    The rapid release of new data from DNA genome sequencing projects has led to a variety of misannotations in public databases. Our results suggest that next generation sequencing approaches are particularly prone to such misannotations. Two related miRNA candidates did recently enter the miRBase database, miR-1274b and miR-1274a, but they share identical 18-nucleotide stretches with tRNA (Lys3) and tRNA (Lys5) , respectively. The possibility that the small RNA fragments that led to the description of these two miRNAs originated from the two tRNAs was examined. The ratio of the miR-1274b:miR-1274a fragments does closely resemble the known tRNA lys3:lys5 ratio in the cell. Furthermore, the proposed miRNA hairpins have a very low prediction score and the proposed miRNA genes are in fact endogenous retroviral elements. We searched for other miRNA-mimics in the human genome and found more examples of tRNA-miRNA mimicry. We propose that the corresponding miRNAs should be validated in more detail, as the small RNA fragments that led to their description are likely derived from tRNA processing.

  14. On origin of genetic code and tRNA before translation

    PubMed Central

    2011-01-01

    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-long cloverleaf

  15. Modelling of vitamin A binding to tRNA.

    PubMed

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

    2014-10-01

    The binding sites of retinol and retinoic acid with tRNA are located in aqueous solution at physiological conditions using constant tRNA concentration and various retinoid contents. FTIR, CD, fluorescence spectroscopic methods and molecular modelling were used to determine retinoid binding sites, the binding constant and the effects of retinol and retinoic acid complexation on tRNA conformation and aggregation. Structural analysis showed that retinol and retinoic acid bind tRNA via G-C and A-U base pairs with overall binding constants of Kret-tRNA=2.0 (±0.40)×10(4)M(-1) and Kretac-tRNA=6.0 (±1)×10(4)M(-1). The number of binding sites occupied by retinoids on tRNA were 1.4 for retinol-tRNA and 1.7 for retinoic acid-tRNA complexes. Hydrophobic interactions were also observed at high retinol and retinoic acid contents. Molecular modelling showed the participation of several nucleobases in retinoid-tRNA complexation with free binding energy of -4.36 for retinol-tRNA and -4.53kcal/mol for retinoic acid-tRNA adducts.

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

    SciTech Connect

    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.

    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.

  17. Cloning and characterization of the C. elegans histidyl-tRNA synthetase gene.

    PubMed Central

    Amaar, Y G; Baillie, D L

    1993-01-01

    In this paper, we report the cloning and sequencing of the C. elegans histidyl-tRNA synthetase gene. The complete genomic sequence, and most of the cDNA sequence, of this gene is now determined. The gene size including flanking and coding regions is 2230 nucleotides long. Three small introns (45-50 bp long) are found to interrupt the open reading frame. The open reading frame translates to 523 amino acids. This putative protein sequence shows extensive homology with the human and yeast histidyl-tRNA the histidyl-tRNA synthetase gene is a single copy gene. Hence, it is very likely that it encodes both the cytoplasmic and the mitochondrial histidyl-tRNA synthetases. It is likely to be trans-spliced since it contains a trans-splice site in its 5' untranslated region. PMID:8414990

  18. Fatty Acid Synthetase of Saccharomyces cerevisiae

    PubMed Central

    Klein, Harold P.; Volkmann, Carol M.; Chao, Fu-Chuan

    1967-01-01

    A light particle fraction of Saccharomyces cerevisiae, obtained from the crude ribosomal material, and containing the fatty acid synthetase, consisted primarily of 27S and 47S components. This fraction has a protein-ribonucleic acid ratio of about 13. Electron micrographs showed particles ranging in diameter between 100 and 300 A in this material. By use of density gradient analysis, the fatty acid synthetase was found in the 47S component. This component contained particles which were predominantly 300 A in diameter and which were considerably flatter than ribosomes, and it consisted almost entirely of protein. Images PMID:6025308

  19. Semisynthetic tRNA complement mediates in vitro protein synthesis.

    PubMed

    Cui, Zhenling; Stein, Viktor; Tnimov, Zakir; Mureev, Sergey; Alexandrov, Kirill

    2015-04-08

    Genetic code expansion is a key objective of synthetic biology and protein engineering. Most efforts in this direction are focused on reassigning termination or decoding quadruplet codons. While the redundancy of genetic code provides a large number of potentially reassignable codons, their utility is diminished by the inevitable interaction with cognate aminoacyl-tRNAs. To address this problem, we sought to establish an in vitro protein synthesis system with a simplified synthetic tRNA complement, thereby orthogonalizing some of the sense codons. This quantitative in vitro peptide synthesis assay allowed us to analyze the ability of synthetic tRNAs to decode all of 61 sense codons. We observed that, with the exception of isoacceptors for Asn, Glu, and Ile, the majority of 48 synthetic Escherichia coli tRNAs could support protein translation in the cell-free system. We purified to homogeneity functional Asn, Glu, and Ile tRNAs from the native E. coli tRNA mixture, and by combining them with synthetic tRNAs, we formulated a semisynthetic tRNA complement for all 20 amino acids. We further demonstrated that this tRNA complement could restore the protein translation activity of tRNA-depleted E. coli lysate to a level comparable to that of total native tRNA. To confirm that the developed system could efficiently synthesize long polypeptides, we expressed three different sequences coding for superfolder GFP. This novel semisynthetic translation system is a powerful tool for tRNA engineering and potentially enables the reassignment of at least 9 sense codons coding for Ser, Arg, Leu, Pro, Thr, and Gly.

  20. A conserved and essential basic region mediates tRNA binding to the Elp1 subunit of the Saccharomyces cerevisiae Elongator complex.

    PubMed

    Di Santo, Rachael; Bandau, Susanne; Stark, Michael J R

    2014-06-01

    Elongator is a conserved, multi-protein complex discovered in Saccharomyces cerevisiae, loss of which confers a range of pleiotropic phenotypes. Elongator in higher eukaryotes is required for normal growth and development and a mutation in the largest subunit of human Elongator (Elp1) causes familial dysautonomia, a severe recessive neuropathy. Elongator promotes addition of mcm(5) and ncm(5) modifications to uridine in the tRNA anticodon 'wobble' position in both yeast and higher eukaryotes. Since these modifications are required for the tRNAs to function efficiently, a translation defect caused by hypomodified tRNAs may therefore underlie the variety of phenotypes associated with Elongator dysfunction. The Elp1 carboxy-terminal domain contains a highly conserved arginine/lysine-rich region that resembles a nuclear localization sequence (NLS). Using alanine substitution mutagenesis, we show that this region is essential for Elongator's function in tRNA wobble uridine modification. However, rather than acting to determine the nucleo-cytoplasmic distribution of Elongator, we find that the basic region plays a critical role in a novel interaction between tRNA and the Elp1 carboxy-terminal domain. Thus the conserved basic region in Elp1 may be essential for tRNA wobble uridine modification by acting as tRNA binding motif.

  1. A recurrent loss-of-function alanyl-tRNA synthetase (AARS) mutation in patients with Charcot-Marie-Tooth disease type 2N (CMT2N).

    PubMed

    McLaughlin, Heather M; Sakaguchi, Reiko; Giblin, William; Wilson, Thomas E; Biesecker, Leslie; Lupski, James R; Talbot, Kevin; Vance, Jeffery M; Züchner, Stephan; Lee, Yi-Chung; Kennerson, Marina; Hou, Ya-Ming; Nicholson, Garth; Antonellis, Anthony

    2012-01-01

    Charcot-Marie-Tooth (CMT) disease comprises a heterogeneous group of peripheral neuropathies characterized by muscle weakness and wasting, and impaired sensation in the extremities. Four genes encoding an aminoacyl-tRNA synthetase (ARS) have been implicated in CMT disease. ARSs are ubiquitously expressed, essential enzymes that ligate amino acids to cognate tRNA molecules. Recently, a p.Arg329His variant in the alanyl-tRNA synthetase (AARS) gene was found to segregate with dominant axonal CMT type 2N (CMT2N) in two French families; however, the functional consequence of this mutation has not been determined. To investigate the role of AARS in CMT, we performed a mutation screen of the AARS gene in patients with peripheral neuropathy. Our results showed that p.Arg329His AARS also segregated with CMT disease in a large Australian family. Aminoacylation and yeast viability assays showed that p.Arg329His AARS severely reduces enzyme activity. Genotyping analysis indicated that this mutation arose on three distinct haplotypes, and the results of bisulfite sequencing suggested that methylation-mediated deamination of a CpG dinucleotide gives rise to the recurrent p.Arg329His AARS mutation. Together, our data suggest that impaired tRNA charging plays a role in the molecular pathology of CMT2N, and that patients with CMT should be directly tested for the p.Arg329His AARS mutation.

  2. Probing tRNA interaction with biogenic polyamines.

    PubMed

    Ouameur, Amin Ahmed; Bourassa, Philippe; Tajmir-Riahi, Heidar-Ali

    2010-10-01

    Biogenic polyamines are found to modulate protein synthesis at different levels. This effect may be explained by the ability of polyamines to bind and influence the secondary structure of tRNA, mRNA, and rRNA. We report the interaction between tRNA and the three biogenic polyamines putrescine, spermidine, spermine, and cobalt(III)hexamine at physiological conditions, using FTIR spectroscopy, capillary electrophoresis, and molecular modeling. The results indicated that tRNA was stabilized at low biogenic polyamine concentration, as a consequence of polyamine interaction with the backbone phosphate group. The main tRNA reactive sites for biogenic polyamine at low concentration were guanine-N7/O6, uracil-O2/O4, adenine-N3, and 2'OH of the ribose. At high polyamine concentration, the interaction involves guanine-N7/O6, adenine-N7, uracil-O2 reactive sites, and the backbone phosphate group. The participation of the polycation primary amino group, in the interaction and the presence of the hydrophobic contact, are also shown. The binding affinity of biogenic polyamine to tRNA molecule was in the order of spermine > spermidine > putrescine with K(Spm) = 8.7 × 10(5) M(-1), K(Spd) = 6.1 × 10(5) M(-1), and K(Put) = 1.0 × 10(5) M(-1), which correlates with their positively charged amino group content. Hill analysis showed positive cooperativity for the biogenic polyamines and negative cooperativity for cobalt-hexamine. Cobalt(III)hexamine contains high- and low-affinity sites in tRNA with K(1) = 3.2 × 10(5) M(-1) and K(2) = 1.7 × 10(5) M(-1), that have been attributed to the interactions with guanine-N7 sites and the backbone PO(2) group, respectively. This mechanism of tRNA binding could explain the condensation phenomenon observed at high Co(III) content, as previously shown in the Co(III)-DNA complexes.

  3. Discovery of ATP-Competitive Inhibitors of tRNAIle Lysidine Synthetase (TilS) by High-Throughput Screening.

    PubMed

    Shapiro, Adam B; Plant, Helen; Walsh, Jarrod; Sylvester, Mark; Hu, Jun; Gao, Ning; Livchak, Stephania; Tentarelli, Sharon; Thresher, Jason

    2014-09-01

    A novel, ultrahigh-throughput, fluorescence anisotropy-based assay was developed and used to screen a 1.4-million-sample library for compounds that compete with adenosine triphosphate (ATP) for binding to Escherichia coli tRNA(Ile) lysidine synthetase (TilS), an essential, conserved, ATP-dependent, tRNA-modifying enzyme of bacterial pathogens. TilS modifies a cytidine base in the anticodon loop of Ile2 tRNA by attaching lysine, thereby altering codon recognition of the CAU anticodon from AUG (methionine) to AUA (isoleucine). A scintillation proximity assay for the incorporation of lysine into Ile2 tRNA was used to eliminate false positives in the initial screen resulting from detection artifacts as well as compounds competitive with the fluorescent label instead of ATP, and to measure inhibitor potencies against E. coli and Pseudomonas aeruginosa TilS isozymes. The tRNA(Ile) substrate for P. aeruginosa TilS was identified for the first time to enable these measurements. ATP-competitive binding of inhibitors was confirmed by one-dimensional ligand-observe nuclear magnetic resonance. A preliminary structure-activity relationship is shown for two inhibitor series.

  4. Crystal structures of three protozoan homologs of tryptophanyl-tRNA synthetase.

    PubMed

    Merritt, Ethan A; Arakaki, Tracy L; Gillespie, Robert; Napuli, Alberto J; Kim, Jessica E; Buckner, Frederick S; Van Voorhis, Wesley C; Verlinde, Christophe L M J; Fan, Erkang; Zucker, Frank; Hol, Wim G J

    2011-05-01

    Tryptophanyl-tRNA synthetase (TrpRS) is an essential enzyme that is recognizably conserved across all forms of life. It is responsible for activating and attaching tryptophan to a cognate tRNA(Trp) molecule for use in protein synthesis. In some eukaryotes this original core function has been supplemented or modified through the addition of extra domains or the expression of variant TrpRS isoforms. The three TrpRS structures from pathogenic protozoa described here represent three illustrations of this malleability in eukaryotes. The Cryptosporidium parvum genome contains a single TrpRS gene, which codes for an N-terminal domain of uncertain function in addition to the conserved core TrpRS domains. Sequence analysis indicates that this extra domain, conserved among several apicomplexans, is related to the editing domain of some AlaRS and ThrRS. The C. parvum enzyme remains fully active in charging tRNA(Trp) after truncation of this extra domain. The crystal structure of the active, truncated enzyme is presented here at 2.4Å resolution. The Trypanosoma brucei genome contains separate cytosolic and mitochondrial isoforms of TrpRS that have diverged in their respective tRNA recognition domains. The crystal structure of the T. brucei cytosolic isoform is presented here at 2.8Å resolution. The Entamoeba histolytica genome contains three sequences that appear to be TrpRS homologs. However one of these, whose structure is presented here at 3.0Å resolution, has lost the active site motifs characteristic of the Class I aminoacyl-tRNA synthetase catalytic domain while retaining the conserved features of a fully formed tRNA(Trp) recognition domain. The biological function of this variant E. histolytica TrpRS remains unknown, but, on the basis of a completely conserved tRNA recognition region and evidence for ATP but not tryptophan binding, it is tempting to speculate that it may perform an editing function. Together with a previously reported structure of an unusual Trp

  5. Inhibition of selenocysteine tRNA[Ser]Sec aminoacylation provides evidence that aminoacylation is required for regulatory methylation of this tRNA

    PubMed Central

    Kim, Jin Young; Carlson, Bradley A.; Xu, Xue-Ming; Zeng, Yu; Chen, Shawn; Gladyshev, Vadim N.; Lee, Byeong Jae; Hatfield, Dolph L.

    2011-01-01

    There are two isoforms of selenocysteine (Sec) tRNA[Ser]Sec that differ by a single methyl group, Um34. The non-Um34 isoform supports the synthesis of a subclass of selenoproteins, designated housekeeping, while the Um34 isoform supports the expression of another subclass, designated stress-related selenoproteins. Herein, we investigated the relationship between tRNA[Ser]Sec aminoacylation and Um34 synthesis which is the last step in the maturation of this tRNA. Mutation of the discriminator base at position 73 in tRNA[Ser]Sec dramatically reduced aminoacylation with serine, as did an inhibitor of seryl-tRNA synthetase, SB-217452. Although both the mutation and the inhibitor prevented Um34 synthesis, neither precluded the synthesis of any other of the known base modifications on tRNA[Ser]Sec following microinjection and incubation of the mutant tRNA[Ser]Sec transcript, or the wild type transcript along with inhibitor, in Xenopus oocytes. The data demonstrate that Sec tRNA[Ser]Sec must be aminoacylated for Um34 addition. The fact that selenium is required for Um34 methylation suggests that Sec must be attached to its tRNA for Um34 methylation. This would explain why selenium is essential for the function of Um34 methylase and provides further insights into the hierarchy of selenoprotein expression. PMID:21624347

  6. Ancient origin of the divergent forms of leucyl-tRNA synthetases in the Halobacteriales

    PubMed Central

    2012-01-01

    Background Horizontal gene transfer (HGT) has greatly impacted the genealogical history of many lineages, particularly for prokaryotes, with genes frequently moving in and out of a line of descent. Many genes that were acquired by a lineage in the past likely originated from ancestral relatives that have since gone extinct. During the course of evolution, HGT has played an essential role in the origin and dissemination of genetic and metabolic novelty. Results Three divergent forms of leucyl-tRNA synthetase (LeuRS) exist in the archaeal order Halobacteriales, commonly known as haloarchaea. Few haloarchaeal genomes have the typical archaeal form of this enzyme and phylogenetic analysis indicates it clusters within the Euryarchaeota as expected. The majority of sequenced halobacterial genomes possess a bacterial form of LeuRS. Phylogenetic reconstruction puts this larger group of haloarchaea at the base of the bacterial domain. The most parsimonious explanation is that an ancient transfer of LeuRS took place from an organism related to the ancestor of the bacterial domain to the haloarchaea. The bacterial form of LeuRS further underwent gene duplications and/or gene transfers within the haloarchaea, with some genomes possessing two distinct types of bacterial LeuRS. The cognate tRNALeu also reveals two distinct clusters for the haloarchaea; however, these tRNALeu clusters do not coincide with the groupings found in the LeuRS tree, revealing that LeuRS evolved independently of its cognate tRNA. Conclusions The study of leucyl-tRNA synthetase in haloarchaea illustrates the importance of gene transfer originating in lineages that went extinct since the transfer occurred. The haloarchaeal LeuRS and tRNALeu did not co-evolve. PMID:22694720

  7. Leucyl-tRNA synthetase editing domain functions as a molecular rheostat to control codon ambiguity in Mycoplasma pathogens.

    PubMed

    Li, Li; Palencia, Andrés; Lukk, Tiit; Li, Zhi; Luthey-Schulten, Zaida A; Cusack, Stephen; Martinis, Susan A; Boniecki, Michal T

    2013-03-05

    Mycoplasma leucyl-tRNA synthetases (LeuRSs) have been identified in which the connective polypeptide 1 (CP1) amino acid editing domain that clears mischarged tRNAs are missing (Mycoplasma mobile) or highly degenerate (Mycoplasma synoviae). Thus, these enzymes rely on a clearance pathway called pretransfer editing, which hydrolyzes misactivated aminoacyl-adenylate intermediate via a nebulous mechanism that has been controversial for decades. Even as the sole fidelity pathway for clearing amino acid selection errors in the pathogenic M. mobile, pretransfer editing is not robust enough to completely block mischarging of tRNA(Leu), resulting in codon ambiguity and statistical proteins. A high-resolution X-ray crystal structure shows that M. mobile LeuRS structurally overlaps with other LeuRS cores. However, when CP1 domains from different aminoacyl-tRNA synthetases and origins were fused to this common LeuRS core, surprisingly, pretransfer editing was enhanced. It is hypothesized that the CP1 domain evolved as a molecular rheostat to balance multiple functions. These include distal control of specificity and enzyme activity in the ancient canonical core, as well as providing a separate hydrolytic active site for clearing mischarged tRNA.

  8. Stimulation of Ribonucleic Acid Synthesis by Chloramphenicol in a rel+ Aminoacyl-Transfer Ribonucleic Acid Synthetase Mutant of Escherichia coli

    PubMed Central

    Yegian, Charles D.; Vanderslice, Rebecca W.

    1971-01-01

    Escherichia coli strain 9D3 possesses a highly temperature-sensitive valyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.9). Since 9D3 is a rel+ strain, it cannot carry out net RNA synthesis at high temperature. A 100-μg amount of chloramphenicol (CAP) per ml added in the absence of valine cannot stimulate RNA synthesis. Either 300 μg of CAP or 100 μg of CAP plus 50 μg of valine per ml, however, promotes nearly maximal RNA synthesis. These results can be understood as follows. (i) Valyl-tRNA is required for net RNA synthesis, (ii) the synthetase lesion is incomplete, (iii) the rate of mutant acylation of tRNAval at high temperature is valine-dependent, and (iv) the CAP concentration determines the rate of residual protein synthesis. Data are also presented which demonstrate that the rate of net RNA synthesis can greatly increase long after the addition of CAP, if the amount of valyl-tRNA increases. PMID:4942766

  9. Pathogenic mechanism of a human mitochondrial tRNAPhe mutation associated with myoclonic epilepsy with ragged red fibers syndrome.

    PubMed

    Ling, Jiqiang; Roy, Hervé; Qin, Daoming; Rubio, Mary Anne T; Alfonzo, Juan D; Fredrick, Kurt; Ibba, Michael

    2007-09-25

    Human mitochondrial tRNA (hmt-tRNA) mutations are associated with a variety of diseases including mitochondrial myopathies, diabetes, encephalopathies, and deafness. Because the current understanding of the precise molecular mechanisms of these mutations is limited, there is no efficient method to treat their associated mitochondrial diseases. Here, we use a variety of known mutations in hmt-tRNA(Phe) to investigate the mechanisms that lead to malfunctions. We tested the impact of hmt-tRNA(Phe) mutations on aminoacylation, structure, and translation elongation-factor binding. The majority of the mutants were pleiotropic, exhibiting defects in aminoacylation, global structure, and elongation-factor binding. One notable exception was the G34A anticodon mutation of hmt-tRNA(Phe) (mitochondrial DNA mutation G611A), which is associated with MERRF (myoclonic epilepsy with ragged red fibers). In vitro, the G34A mutation decreases aminoacylation activity by 100-fold, but does not affect global folding or recognition by elongation factor. Furthermore, G34A hmt-tRNA(Phe) does not undergo adenosine-to-inosine (A-to-I) editing, ruling out miscoding as a possible mechanism for mitochondrial malfunction. To improve the aminoacylation state of the mutant tRNA, we modified the tRNA binding domain of the nucleus-encoded human mitochondrial phenylalanyl-tRNA synthetase, which aminoacylates hmt-tRNA(Phe) with cognate phenylalanine. This variant enzyme displayed significantly improved aminoacylation efficiency for the G34A mutant, suggesting a general strategy to treat certain classes of mitochondrial diseases by modification of the corresponding nuclear gene.

  10. Protozoan ALKBH8 oxygenases display both DNA repair and tRNA modification activities.

    PubMed

    Zdżalik, Daria; Vågbø, Cathrine B; Kirpekar, Finn; Davydova, Erna; Puścian, Alicja; Maciejewska, Agnieszka M; Krokan, Hans E; Klungland, Arne; Tudek, Barbara; van den Born, Erwin; Falnes, Pål Ø

    2014-01-01

    The ALKBH family of Fe(II) and 2-oxoglutarate dependent oxygenases comprises enzymes that display sequence homology to AlkB from E. coli, a DNA repair enzyme that uses an oxidative mechanism to dealkylate methyl and etheno adducts on the nucleobases. Humans have nine different ALKBH proteins, ALKBH1-8 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 previously demonstrated for one such protein. To further explore this apparent functional duality of the ALKBH8 proteins, we have here enzymatically characterized a panel of such proteins, originating from bacteria, protozoa and mimivirus. All the enzymes showed DNA repair activity in vitro, but, 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 different from that of their eukaryotic counterparts. The present study provides new insights on the function and evolution of the ALKBH8 family of proteins.

  11. AlkB homolog 3-mediated tRNA demethylation promotes protein synthesis in cancer cells

    PubMed Central

    Ueda, Yuko; Ooshio, Ikumi; Fusamae, Yasuyuki; Kitae, Kaori; Kawaguchi, Megumi; Jingushi, Kentaro; Hase, Hiroaki; Harada, Kazuo; Hirata, Kazumasa; Tsujikawa, Kazutake

    2017-01-01

    The mammalian AlkB homolog (ALKBH) family of proteins possess a 2-oxoglutarate- and Fe(II)-dependent oxygenase domain. A similar domain in the Escherichia coli AlkB protein catalyzes the oxidative demethylation of 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) in both DNA and RNA. AlkB homolog 3 (ALKBH3) was also shown to demethylate 1-meA and 3-meC (induced in single-stranded DNA and RNA by a methylating agent) to reverse the methylation damage and retain the integrity of the DNA/RNA. We previously reported the high expression of ALKBH3 in clinical tumor specimens and its involvement in tumor progression. In this study, we found that ALKBH3 effectively demethylated 1-meA and 3-meC within endogenously methylated RNA. Moreover, using highly purified recombinant ALKBH3, we identified N6-methyladenine (N6-meA) in mammalian transfer RNA (tRNA) as a novel ALKBH3 substrate. An in vitro translation assay showed that ALKBH3-demethylated tRNA significantly enhanced protein translation efficiency. In addition, ALKBH3 knockdown in human cancer cells impaired cellular proliferation and suppressed the nascent protein synthesis that is usually accompanied by accumulation of the methylated RNAs. Thus, our data highlight a novel role for ALKBH3 in tumor progression via RNA demethylation and subsequent protein synthesis promotion. PMID:28205560

  12. The Selenocysteine tRNA STAF-Binding Region is Essential for Adequate Selenocysteine tRNA Status, Selenoprotein Expression and Early Age Survival of Mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    STAF is a transcription activating factor for a number of RNA Pol III-and RNA Pol II-dependent genes including the selenocysteine (Sec) tRNA gene. Here, the role of STAF in regulating expression of Sec tRNA and selenoproteins was examined in an invivo model. Heterozygous inactivation of the Staf gen...

  13. Dexamethasone regulates glutamine synthetase expression in rat skeletal muscles

    NASA Technical Reports Server (NTRS)

    Max, Stephen R.; Konagaya, Masaaki; Konagaya, Yoko; Thomas, John W.; Banner, Carl; Vitkovic, Ljubisa

    1986-01-01

    The regulation of glutamine synthetase by glucocorticoids in rat skeletal muscles was studied. Administration of dexamethasone strikingly enhanced glutamine synthetase activity in plantaris and soleus muscles. The dexamethasone-mediated induction of glutamine synthetase activity was blocked to a significant extent by orally administered RU38486, a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves dramatically increased levels of glutamine synthetase mRNA. The induction of glutamine synthetase was selective in that glutaminase activity of soleus and plantaris muscles was not increased by dexamethasone. Furthermore, dexamethasone treatment resulted in only a small increase in glutamine synthetase activity in the heart. Accordingly, there was only a slight change in glutamine synthetase mRNA level in this tissue. Thus, glucocorticoids regulate glutamine synthetase gene expression in rat muscles at the transcriptional level via interaction with intracellular glutamine production by muscle and to mechanisms underlying glucocorticoid-induced muscle atrophy.

  14. Specificity determinants for the two tRNA substrates of the cyclodipeptide synthase AlbC from Streptomyces noursei

    PubMed Central

    Moutiez, Mireille; Seguin, Jérôme; Fonvielle, Matthieu; Belin, Pascal; Jacques, Isabelle Béatrice; Favry, Emmanuel; Arthur, Michel; Gondry, Muriel

    2014-01-01

    Cyclodipeptide synthases (CDPSs) use two aminoacyl-tRNA substrates in a sequential ping-pong mechanism to form a cyclodipeptide. The crystal structures of three CDPSs have been determined and all show a Rossmann-fold domain similar to the catalytic domain of class-I aminoacyl-tRNA synthetases (aaRSs). Structural features and mutational analyses however suggest that CDPSs and aaRSs interact differently with their tRNA substrates. We used AlbC from Streptomyces noursei that mainly produces cyclo(l-Phe-l-Leu) to investigate the interaction of a CDPS with its substrates. We demonstrate that Phe-tRNAPhe is the first substrate accommodated by AlbC. Its binding to AlbC is dependent on basic residues located in the helix α4 that form a basic patch at the surface of the protein. AlbC does not use all of the Leu-tRNALeu isoacceptors as a second substrate. We show that the G1-C72 pair of the acceptor stem is essential for the recognition of the second substrate. Substitution of D163 located in the loop α6–α7 or D205 located in the loop β6–α8 affected Leu-tRNALeu isoacceptors specificity, suggesting the involvement of these residues in the binding of the second substrate. This is the first demonstration that the two substrates of CDPSs are accommodated in different binding sites. PMID:24782519

  15. The effect of chemical modification of 3-(3-amino-3-carboxypropyl)uridine on tRNA function.

    PubMed

    Friedman, S

    1979-08-10

    The minor base 3-(3-amino-3-carboxypropyl)uridine (acp3U) in Escherichia coli tRNAPhe was acylated with the N-hydroxysuccinimide esters of acetic, phenoxy-acetic, and naphthoxyacetic acid, as well as the ester of 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-glycine. The derivatives of tRNAPhe formed were all capable of accepting phenylalanine. There were only minor effects on the kinetic parameters of these derivatives for E. coli phenylalanyl-tRNA synthetase. There was no effect on the ability of tRNAPhe to participate in poly(U)- or poly(ACU)-directed polypeptide synthesis or in the poly(U)-stimulated binding to E. coli ribosomes. The rate of photodynamic cross-linking of 4-Srd 8 to Cyd 13 was decreased in tRNAs containing the acetyl and dansyl-glycyl derivatives of acp3U, indicating that acylation of this base may perturb the tertiary structure of the tRNA. This base in tRNAPhe does not appear to play any role in the known biological functions of tRNAPhe.

  16. In vitro effects of metal pollution on Mediterranean sponges: species-specific inhibition of 2',5'-oligoadenylate synthetase.

    PubMed

    Saby, Emilie; Justesen, Just; Kelve, Merike; Uriz, Maria J

    2009-09-14

    Heavy metals are among the main pollutants of the Mediterranean coastal waters where they can harm sublittoral biota. Filter-feeder, long-living invertebrates that remain fixed to the rocky bottom, such as sponges, are good targets to metal contamination studies since they may be exposed to potential low levels of contamination for years. Several molecular and biochemical mechanisms are developed by sponges to counteract the effects of noxious metals. As a result, some of the normal cell functions can be altered. Here we show that the main heavy metals that can be found in marine sublittoral waters (i.e. copper, iron, zinc and manganese) may alter the immune system of sponges by inhibiting the activity of the sponge 2',5'-oligoadenylate synthetase (2-5A synthetase), which is an enzyme involved in the immune system of vertebrates. We selected the widespread Mediterranean sponges Geodia cydonium, Crella elegans and Chondrosia reniformis for the study. They exerted a high 2-5A synthetase activity and gave a unique profile of 2',5'-oligoadenylate product production. Several metals alter the 2-5A synthetase activity differently, in a species-specific manner. 2-5A synthetases from G. cydonium and C. elegans were inhibited by all the metal ions assayed. However, in C. reniformis, 2-5A synthetase was either activated or inhibited by the same ions depending on their final concentrations. Like in humans, metal contamination may have an effect on the OAS activity and thus it might alter the sponge immune system. However, since the effects are species-specific, 2-5A synthetase cannot be used as general biomarker of metal pollutions.

  17. Elevated levels of interferon-induced 2'-5' oligoadenylate synthetase in generalized persistent lymphadenopathy and the acquired immunodeficiency syndrome.

    PubMed

    Read, S E; Williams, B R; Coates, R A; Evans, W K; Fanning, M M; Garvey, M B; Shepherd, F A

    1985-09-01

    The levels of the 2'-5' oligoadenylate enzyme synthetase in extracts of peripheral blood mononuclear cells from individuals with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC) were measured and compared with synthetase levels in peripheral blood mononuclear cells (PBMs) from healthy heterosexual and homosexual controls. The mean basal synthetase level in heterosexual and homosexual controls was 14 +/- 13 and 12 +/- 9 pmol per hr/10(5) PBMs, respectively. Thirteen individuals with AIDS had a mean basal level of 129 +/- 75 pmol. Serial levels were persistently elevated in six of these individuals over a one- to 10-month period. Twelve of the 13 individuals had antibodies to human T cell lymphotrophic virus-III/lymphadenopathy-associated virus (HTLV-III/LAV). Thirty-three individuals with ARC had a mean basal synthetase level of 68 +/- 84 pmol. Thirty-two of the 33 had antibodies to HTLV-III/LAV. Eleven (33%) have had consistently normal synthetase levels (less than 2 SD above the mean for the homosexual controls, i.e., 30 pmol) over a three- to nine-month follow-up period. Fourteen (42%) had persistently elevated levels over the same period; four (29%) of these developed AIDS during the follow-up period. Eight have had fluctuating levels but have remained clinically well. These studies suggest that persistently elevated synthetase levels in individuals with ARC and antibodies to HTLV-III/LAV indicate progressive virus-induced disease activity. Elevated synthetase levels may be an important prognostic indicator of increased risk of progression to AIDS.

  18. Interaction of tRNA with antitumor polyamine analogues.

    PubMed

    N'soukpoé-Kossi, C N; Ahmed Ouameur, A; Thomas, T; Thomas, T J; Tajmir-Riahi, H A

    2009-08-01

    We studied the interaction between tRNA and three polyamine analogues (1,11-diamino-4,8-diazaundecane.4HCl (333), 3,7,11,15-tetrazaheptadecane.4HCl (BE-333), and 3,7,11,15,19-pentazahenicosane.5HCl (BE-3333)) using FTIR, UV-visible, and CD spectroscopic methods. Spectroscopic evidence showed that polyamine analogues bound tRNA via guanine N7, adenine, uracil O2, and the backbone phosphate (PO2-) groups, while the most reactive sites for biogenic polyamines were guanine N7/O6, adenine N7, uracil O2, and sugar 2'-OH groups as well as the backbone phosphate group. The binding constants of polyamine analogue-tRNA recognition were lower than those of the biogenic polyamine-tRNA complexes, with K333 = 2.8 (+/-0.5) x 10(4), K(BE-333) = 3.7 (+/-0.7) x 10(4), K(BE-3333) = 4.0 (+/-0.9) x 10(4), K(spm) = 8.7 (+/-0.9) x 10(5), K(spd) = 6.1 (+/-0.7) x 10(5), and K(put) = 1.0 (+/-0.3) x 10(5) mol/L. tRNA remained in the A-family conformation; however, it aggregated at high polyamine analogue concentrations.

  19. tRNA hopping: enhancement by an expanded anticodon.

    PubMed Central

    O'Connor, M; Gesteland, R F; Atkins, J F

    1989-01-01

    At a low level wild-type tRNA(1Val) inserts a single amino acid (valine) for the five nucleotide sequence GUGUA which has overlapping valine codons. Mutants of tRNA(1Val) with an insertion of A or U between positions 34 and 35 of their anticodons have enhanced reading of the quintuplet sequences. We propose that this decoding occurs by a hopping mechanism rather than by quintuplet pairing. Such hopping involves disengagement of the paired codon and anticodon with the mRNA slipping two (or more) bases along the ribosomal--peptidyl tRNA complex and subsequently re-pairing at a second codon--the landing site. The mutant with the anticodon sequence 3'CAAU5' 'hops' over the stop codon in the mRNA sequence GUG UAA GUU with the insertion of a single amino acid (valine). In contrast, in reading the same sequence, the mutant with the anticodon 3'CAUU5' hops onto the stop with the insertion of two valine residues. It is likely that in some instances of hopping alternate anticodon bases are used for the initial pairing and at the landing site. PMID:2686986

  20. Structural analysis of the active site geometry of N5-carboxyaminoimidazole ribonucleotide synthetase from Escherichia coli.

    PubMed

    Thoden, James B; Holden, Hazel M; Firestine, Steven M

    2008-12-16

    N(5)-Carboxyaminoimidazole ribonucleotide synthetase (N(5)-CAIR synthetase) converts 5-aminoimidazole ribonucleotide (AIR), MgATP, and bicarbonate into N(5)-CAIR, MgADP, and P(i). The enzyme is required for de novo purine biosynthesis in microbes yet is not found in humans suggesting that it represents an ideal and unexplored target for antimicrobial drug design. Here we report the X-ray structures of N(5)-CAIR synthetase from Escherichia coli with either MgATP or MgADP/P(i) bound in the active site cleft. These structures, determined to 1.6-A resolution, provide detailed information regarding the active site geometry before and after ATP hydrolysis. In both structures, two magnesium ions are observed. Each of these is octahedrally coordinated, and the carboxylate side chain of Glu238 bridges them. For the structure of the MgADP/P(i) complex, crystals were grown in the presence of AIR and MgATP. No electron density was observed for AIR, and the electron density corresponding to the nucleotide clearly revealed the presence of ADP and P(i) rather than ATP. The bound P(i) shifts by approximately 3 A relative to the gamma-phosphoryl group of ATP and forms electrostatic interactions with the side chains of Arg242 and His244. Since the reaction mechanism of N(5)-CAIR synthetase is believed to proceed via a carboxyphosphate intermediate, we propose that the location of the inorganic phosphate represents the binding site for stabilization of this reactive species. Using the information derived from the two structures reported here, coupled with molecular modeling, we propose a catalytic mechanism for N(5)-CAIR synthetase.

  1. Site Directed Mutagenesis of Schizosaccharomyces pombe Glutathione Synthetase Produces an Enzyme with Homoglutathione Synthetase Activity

    PubMed Central

    Dworeck, Tamara; Zimmermann, Martin

    2012-01-01

    Three different His-tagged, mutant forms of the fission yeast glutathione synthetase (GSH2) were derived by site-directed mutagenesis. The mutant and wild-type enzymes were expressed in E. coli DH5α and affinity purified in a two-step procedure. Analysis of enzyme activity showed that it was possible to shift the substrate specificity of GSH2 from Gly (km 0,19; wild-type) to β-Ala or Ser. One mutation (substitution of Ile471, Cy472 to Met and Val and Ala 485 and Thr486 to Leu and Pro) increased the affinity of GSH2 for β-Ala (km 0,07) and lowered the affinity for Gly (km 0,83), which is a characteristic of the enzyme homoglutathione synthetase found in plants. Substitution of Ala485 and Thr486 to Leu and Pro only, increased instead the affinity of GSH2 for Ser (km 0,23) as a substrate, while affinity to Gly was preserved (km 0,12). This provides a new biosynthetic pathway for hydroxymethyl glutathione, which is known to be synthesized from glutathione and Ser in a reaction catalysed by carboxypeptidase Y. The reported findings provide further insight into how specific amino acids positioned in the GSH2 active site facilitate the recognition of different amino acid substrates, furthermore they support the evolutionary theory that homoglutathione synthetase evolved from glutathione synthetase by a single gene duplication event. PMID:23091597

  2. Retinal Vasculitis in Anti-Synthetase Syndrome.

    PubMed

    Donovan, Christopher P; Pecen, Paula E; Baynes, Kimberly; Ehlers, Justis P; Srivastava, Sunil K

    2016-09-01

    A 31-year-old woman with a history of anti-synthetase syndrome-related myositis and interstitial lung disease presented with acute-onset blurry vision and rash on her hands and feet. Visual acuity was hand motion in her right eye and 20/40 in her left eye. Dilated fundus exam showed extensive retinal vasculitis, diffuse intraretinal hemorrhages, and subretinal fluid. Optical coherence tomography revealed significant macular thickening, and fluorescein angiography revealed vascular leakage with peripheral nonperfusion. Aggressive systemic immunosuppression was initiated, with gradual resolution of her disease during 8 months of follow-up. [Ophthalmic Surg Lasers Imaging Retina. 2016;47:874-879.].

  3. Characterization of Cereulide Synthetase, a Toxin-Producing Macromolecular Machine

    PubMed Central

    Alonzo, Diego A.; Magarvey, Nathan A.; Schmeing, T. Martin

    2015-01-01

    Cereulide synthetase is a two-protein nonribosomal peptide synthetase system that produces a potent emetic toxin in virulent strains of Bacillus cereus. The toxin cereulide is a depsipeptide, as it consists of alternating aminoacyl and hydroxyacyl residues. The hydroxyacyl residues are derived from keto acid substrates, which cereulide synthetase selects and stereospecifically reduces with imbedded ketoreductase domains before incorporating them into the growing depsipeptide chain. We present an in vitro biochemical characterization of cereulide synthetase. We investigate the kinetics and side chain specificity of α-keto acid selection, evaluate the requirement of an MbtH-like protein for adenylation domain activity, assay the effectiveness of vinylsulfonamide inhibitors on ester-adding modules, perform NADPH turnover experiments and evaluate in vitro depsipeptide biosynthesis. This work also provides biochemical insight into depsipeptide-synthesizing nonribosomal peptide synthetases responsible for other bioactive molecules such as valinomycin, antimycin and kutzneride. PMID:26042597

  4. The microsomal dicarboxylyl-CoA synthetase.

    PubMed Central

    Vamecq, J; de Hoffmann, E; Van Hoof, F

    1985-01-01

    Dicarboxylic acids are products of the omega-oxidation of monocarboxylic acids. We demonstrate that in rat liver dicarboxylic acids (C5-C16) can be converted into their CoA esters by a dicarboxylyl-CoA synthetase. During this activation ATP, which cannot be replaced by GTP, is converted into AMP and PPi, both acting as feedback inhibitors of the reaction. Thermolabile at 37 degrees C, and optimally active at pH 6.5, dicarboxylyl-CoA synthetase displays the highest activity on dodecanedioic acid (2 micromol/min per g of liver). Cell-fractionation studies indicate that this enzyme belongs to the hepatic microsomal fraction. Investigations about the fate of dicarboxylyl-CoA esters disclosed the existence of an oxidase, which could be measured by monitoring the production of H2O2. In our assay conditions this H2O2 production is dependent on and closely follows the CoA consumption. It appears that the chain-length specificity of the handling of dicarboxylic acids by this catabolic pathway (activation to acyl-CoA and oxidation with H2O2 production) parallels the pattern of the degradation of exogenous dicarboxylic acids in vivo. PMID:4062873

  5. tRNA concentration fine tunes protein solubility.

    PubMed

    Fedyunin, Ivan; Lehnhardt, Lothar; Böhmer, Nadine; Kaufmann, Paul; Zhang, Gong; Ignatova, Zoya

    2012-09-21

    Clusters of codons pairing to low-abundance tRNAs synchronize the translation with co-translational folding of single domains in multidomain proteins. Although proven with some examples, the impact of the ribosomal speed on the folding and solubility on a global, cell-wide level remains elusive. Here we show that upregulation of three low-abundance tRNAs in Escherichia coli increased the aggregation propensity of several cellular proteins as a result of an accelerated elongation rate. Intriguingly, alterations in the concentration of the natural tRNA pool compromised the solubility of various chaperones consequently rendering the solubility of some chaperone-dependent proteins.

  6. An alanine tRNA gene cluster from Nephila clavipes.

    PubMed

    Luciano, E; Candelas, G C

    1996-06-01

    We report the sequence of a 2.3-kb genomic DNA fragment from the orb-web spider, Nephila clavipes (Nc). The fragment contains four regions of high homology to tRNA(Ala). The members of this irregularly spaced cluster of genes are oriented in the same direction and have the same anticodon (GCA), but their sequence differs at several positions. Initiation and termination signals, as well as consensus intragenic promoter sequences characteristic of tRNA genes, have been identified in all genes. tRNA(Ala) are involved in the regulation of the fibroin synthesis in the large ampullate Nc glands.

  7. Aminoacyl-tRNA synthetase dependent angiogenesis revealed by a bioengineered macrolide inhibitor.

    PubMed

    Mirando, Adam C; Fang, Pengfei; Williams, Tamara F; Baldor, Linda C; Howe, Alan K; Ebert, Alicia M; Wilkinson, Barrie; Lounsbury, Karen M; Guo, Min; Francklyn, Christopher S

    2015-08-14

    Aminoacyl-tRNA synthetases (AARSs) catalyze an early step in protein synthesis, but also regulate diverse physiological processes in animal cells. These include angiogenesis, and human threonyl-tRNA synthetase (TARS) represents a potent pro-angiogenic AARS. Angiogenesis stimulation can be blocked by the macrolide antibiotic borrelidin (BN), which exhibits a broad spectrum toxicity that has discouraged deeper investigation. Recently, a less toxic variant (BC194) was identified that potently inhibits angiogenesis. Employing biochemical, cell biological, and biophysical approaches, we demonstrate that the toxicity of BN and its derivatives is linked to its competition with the threonine substrate at the molecular level, which stimulates amino acid starvation and apoptosis. By separating toxicity from the inhibition of angiogenesis, a direct role for TARS in vascular development in the zebrafish could be demonstrated. Bioengineered natural products are thus useful tools in unmasking the cryptic functions of conventional enzymes in the regulation of complex processes in higher metazoans.

  8. Aminoacyl-tRNA synthetase dependent angiogenesis revealed by a bioengineered macrolide inhibitor

    PubMed Central

    Mirando, Adam C.; Fang, Pengfei; Williams, Tamara F.; Baldor, Linda C.; Howe, Alan K.; Ebert, Alicia M.; Wilkinson, Barrie; Lounsbury, Karen M.; Guo, Min; Francklyn, Christopher S.

    2015-01-01

    Aminoacyl-tRNA synthetases (AARSs) catalyze an early step in protein synthesis, but also regulate diverse physiological processes in animal cells. These include angiogenesis, and human threonyl-tRNA synthetase (TARS) represents a potent pro-angiogenic AARS. Angiogenesis stimulation can be blocked by the macrolide antibiotic borrelidin (BN), which exhibits a broad spectrum toxicity that has discouraged deeper investigation. Recently, a less toxic variant (BC194) was identified that potently inhibits angiogenesis. Employing biochemical, cell biological, and biophysical approaches, we demonstrate that the toxicity of BN and its derivatives is linked to its competition with the threonine substrate at the molecular level, which stimulates amino acid starvation and apoptosis. By separating toxicity from the inhibition of angiogenesis, a direct role for TARS in vascular development in the zebrafish could be demonstrated. Bioengineered natural products are thus useful tools in unmasking the cryptic functions of conventional enzymes in the regulation of complex processes in higher metazoans. PMID:26271225

  9. Towards a comprehensive picture of alloacceptor tRNA remolding in metazoan mitochondrial genomes

    PubMed Central

    Sahyoun, Abdullah H.; Hölzer, Martin; Jühling, Frank; Höner zu Siederdissen, Christian; Al-Arab, Marwa; Tout, Kifah; Marz, Manja; Middendorf, Martin; Stadler, Peter F.; Bernt, Matthias

    2015-01-01

    Remolding of tRNAs is a well-documented process in mitochondrial genomes that changes the identity of a tRNA. It involves a duplication of a tRNA gene, a mutation that changes the anticodon and the loss of the ancestral tRNA gene. The net effect is a functional tRNA that is more closely related to tRNAs of a different alloacceptor family than to tRNAs with the same anticodon in related species. Beyond being of interest for understanding mitochondrial tRNA function and evolution, tRNA remolding events can lead to artifacts in the annotation of mitogenomes and thus in studies of mitogenomic evolution. Therefore, it is important to identify and catalog these events. Here we describe novel methods to detect tRNA remolding in large-scale data sets and apply them to survey tRNA remolding throughout animal evolution. We identify several novel remolding events in addition to the ones previously mentioned in the literature. A detailed analysis of these remoldings showed that many of them are derived from ancestral events. PMID:26227972

  10. Dynamics of Hydrated tRNA on Nanodiamond Surface Studied by Quasi-elastic Neutron Scattering

    NASA Astrophysics Data System (ADS)

    Dhindsa, Gurpreet; Mochalin, Vadym N.; O'Neill, Hugh; Gogotsi, Yury; Chu, Xiang Qiang

    2014-03-01

    Diamond is an outstanding material in many aspects, and nanodiamond (ND) inherits most of the superior properties of bulk diamond and delivers them at the nanoscale. ND has excellent properties that can be applied in biomedical field such as a good platform for drug delivery. In this study, we show that hydrated tRNA can be adsorbed on the surfaces of nanodiamonds and further demonstrate specific properties in its dynamics. We investigate the dynamics of the system by Quasielastic neutron scattering (QENS) technique. The dynamics of hydrated tRNA on ND surfaces exhibits a logarithmic-like decay within the time range of 10 ps to 1 ns, which has also been observed in the freestanding proteins and other biopolymers. We further compare the dynamics of tRNA hydrated with D2O on ND surface with that of freestanding hydrated tRNA molecules. Our results show that the relaxational dynamics of tRNA on ND surface is much faster than that of the freestanding tRNA molecules. This gives the hint that the folded states of tRNA is modified by ND surfaces to engage faster dynamics. The difference in the dynamics of the hydration water modified by ND is another possible reason which causes the faster dynamics in tRNA on ND surface.

  11. Evolutionary constraints on the plastid tRNA set decoding methionine and isoleucine

    PubMed Central

    Alkatib, Sibah; Fleischmann, Tobias T.; Scharff, Lars B.; Bock, Ralph

    2012-01-01

    The plastid (chloroplast) genomes of seed plants typically encode 30 tRNAs. Employing wobble and superwobble mechanisms, most codon boxes are read by only one or two tRNA species. The reduced set of plastid tRNAs follows the evolutionary trend of organellar genomes to shrink in size and coding capacity. A notable exception is the AUN codon box specifying methionine and isoleucine, which is decoded by four tRNA species in nearly all seed plants. However, three of these four tRNA genes were lost from the genomes of some parasitic plastid-containing lineages, possibly suggesting that less than four tRNA species could be sufficient to decode the triplets in the AUN box. To test this hypothesis, we have performed knockout experiments for the four AUN-decoding tRNAs in tobacco (Nicotiana tabacum) plastids. We find that all four tRNA genes are essential under both autotrophic and heterotrophic growth conditions, possibly suggesting tRNA import into plastids of parasitic plastid-bearing species. Phylogenetic analysis of the four plastid tRNA genes reveals striking conservation of all those bacterial features that are involved in discrimination between the different tRNA species containing CAU anticodons. PMID:22553362

  12. Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids.

    PubMed

    Melton, Elaina M; Cerny, Ronald L; DiRusso, Concetta C; Black, Paul N

    2013-11-01

    In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of

  13. Overexpression of Human Fatty Acid Transport Protein 2/Very Long Chain Acyl-CoA Synthetase 1 (FATP2/Acsvl1) Reveals Distinct Patterns of Trafficking of Exogenous Fatty Acids

    PubMed Central

    Melton, Elaina M.; Cerny, Ronald L.; DiRusso, Concetta C.; Black, Paul N.

    2014-01-01

    In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4hr. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of

  14. Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids

    SciTech Connect

    Melton, Elaina M.; Cerny, Ronald L.; DiRusso, Concetta C.; Black, Paul N.

    2013-11-01

    Highlights: •Roles of FATP2 in fatty acid transport/activation contribute to lipid homeostasis. •Use of 13C- and D-labeled fatty acids provide novel insights into FATP2 function. •FATP2-dependent trafficking of FA into phospholipids results in distinctive profiles. •FATP2 functions in the transport and activation pathways for exogenous fatty acids. -- Abstract: In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4 h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The

  15. Quality Control Pathways for Nucleus-Encoded Eukaryotic tRNA Biosynthesis and Subcellular Trafficking

    PubMed Central

    Huang, Hsiao-Yun

    2015-01-01

    tRNAs perform an essential role in translating the genetic code. They are long-lived RNAs that are generated via numerous posttranscriptional steps. Eukaryotic cells have evolved numerous layers of quality control mechanisms to ensure that the tRNAs are appropriately structured, processed, and modified. We describe the known tRNA quality control processes that check tRNAs and correct or destroy aberrant tRNAs. These mechanisms employ two types of exonucleases, CCA end addition, tRNA nuclear aminoacylation, and tRNA subcellular traffic. We arrange these processes in order of the steps that occur from generation of precursor tRNAs by RNA polymerase (Pol) III transcription to end maturation and modification in the nucleus to splicing and additional modifications in the cytoplasm. Finally, we discuss the tRNA retrograde pathway, which allows tRNA reimport into the nucleus for degradation or repair. PMID:25848089

  16. Antitumor/Antifungal Celecoxib Derivative AR-12 is a Non-Nucleoside Inhibitor of the ANL-Family Adenylating Enzyme Acetyl CoA Synthetase

    PubMed Central

    2016-01-01

    AR-12/OSU-03012 is an antitumor celecoxib-derivative that has progressed to Phase I clinical trial as an anticancer agent and has activity against a number of infectious agents including fungi, bacteria and viruses. However, the mechanism of these activities has remained unclear. Based on a chemical-genetic profiling approach in yeast, we have found that AR-12 is an ATP-competitive, time-dependent inhibitor of yeast acetyl coenzyme A synthetase. AR-12-treated fungal cells show phenotypes consistent with the genetic reduction of acetyl CoA synthetase activity, including induction of autophagy, decreased histone acetylation, and loss of cellular integrity. In addition, AR-12 is a weak inhibitor of human acetyl CoA synthetase ACCS2. Acetyl CoA synthetase activity is essential in many fungi and parasites. In contrast, acetyl CoA is primarily synthesized by an alternate enzyme, ATP-citrate lyase, in mammalian cells. Taken together, our results indicate that AR-12 is a non-nucleoside acetyl CoA synthetase inhibitor and that acetyl CoA synthetase may be a feasible antifungal drug target. PMID:27088128

  17. Genome-wide screen uncovers novel pathways for tRNA processing and nuclear–cytoplasmic dynamics

    PubMed Central

    Wu, Jingyan; Bao, Alicia; Chatterjee, Kunal; Wan, Yao; Hopper, Anita K.

    2015-01-01

    Transfer ribonucleic acids (tRNAs) are essential for protein synthesis. However, key gene products involved in tRNA biogenesis and subcellular movement remain to be discovered. We conducted the first comprehensive unbiased analysis of the role of nearly an entire proteome in tRNA biology and describe 162 novel and 12 previously known Saccharomyces cerevisiae gene products that function in tRNA processing, turnover, and subcellular movement. tRNA nuclear export is of particular interest because it is essential, but the known tRNA exporters (Los1 [exportin-t] and Msn5 [exportin-5]) are unessential. We report that mutations of CRM1 (Exportin-1), MEX67/MTR2 (TAP/p15), and five nucleoporins cause accumulation of unspliced tRNA, a hallmark of defective tRNA nuclear export. CRM1 mutation genetically interacts with los1Δ and causes altered tRNA nuclear–cytoplasmic distribution. The data implicate roles for the protein and mRNA nuclear export machineries in tRNA nuclear export. Mutations of genes encoding actin cytoskeleton components and mitochondrial outer membrane proteins also cause accumulation of unspliced tRNA, likely due to defective splicing on mitochondria. Additional gene products, such as chromatin modification enzymes, have unanticipated effects on pre-tRNA end processing. Thus, this genome-wide screen uncovered putative novel pathways for tRNA nuclear export and extensive links between tRNA biology and other aspects of cell physiology. PMID:26680305

  18. Mechanism of 3'-Matured tRNA Discrimination from 3'-Immature tRNA by Class-II CCA-Adding Enzyme.

    PubMed

    Yamashita, Seisuke; Tomita, Kozo

    2016-06-07

    CCA-adding enzyme adds the 3'-CCA of tRNA, using CTP and ATP as substrates, and terminates RNA synthesis after completion of CCA addition, without using a nucleic acid template. The complex structure of class-II Thermotoga maritima CCA-adding enzyme and mature tRNA with 3'-CCA revealed the mechanisms by which the enzyme terminates RNA synthesis after completion of 3'-CCA addition, and discriminates 3'-mature tRNA from 3'-immature tRNA. After completion of 3'-CCA addition at the catalytic site, the 3'-CCA refolds and relocates to the release site, which is discrete from the catalytic site. The 3'-CCA forms a continuously stacked, stable conformation together with the enzyme. Consequently, the 3'-mature tRNA rotates relative to the surface of the enzyme, and only the 3'-mature tRNA is ready for release. The 3'-regions of immature tRNAs cannot form the stable stacking conformation in the release site; thus, the 3' end is relocated in the catalytic site, and the 3'-CCA is reconstructed.

  19. tRNA evolution from the proto-tRNA minihelix world

    PubMed Central

    Root-Bernstein, Robert; Kim, Yunsoo; Sanjay, Adithya; Burton, Zachary F.

    2016-01-01

    ABSTRACT Multiple models have been advanced for the evolution of cloverleaf tRNA. Here, the conserved archaeal tRNA core (75-nt) is posited to have evolved from ligation of three proto-tRNA minihelices (31-nt) and two-symmetrical 9-nt deletions within joined acceptor stems (93 – 18 = 75-nt). The primary evidence for this conclusion is that the 5-nt stem 7-nt anticodon loop and the 5-nt stem 7-nt T loop are structurally homologous and related by coding sequence. We posit that the D loop was generated from a third minihelix (31-nt) in which the stem and loop became rearranged after 9-nt acceptor stem deletions and cloverleaf folding. The most 3´-5-nt segment of the D loop and the 5-nt V loop are apparent remnants of the joined acceptor stems (14 – 9 = 5-nt). Before refolding in the tRNA cloverleaf, we posit that the 3′-5-nt segment of the D loop and the 5-nt V loop were paired, and, in the tRNA cloverleaf, frequent pairing of positions 29 (D loop) and 47 (V loop) remains (numbered on a 75-nt tRNA cloverleaf core). Amazingly, after >3.5 billion years of evolutionary pressure on the tRNA cloverleaf structure, a model can be constructed that convincingly describes the genesis of 75/75-nt conserved archaeal tRNA core positions. Judging from the tRNA structure, cloverleaf tRNA appears to represent at least a second-generation scheme (and possibly a third-generation scheme) that replaced a robust 31-nt minihelix protein-coding system, evidence for which is preserved in the cloverleaf structure. Understanding tRNA evolution provides insights into ribosome and rRNA evolution. PMID:27636862

  20. Mechanistic issues in asparagine synthetase catalysis.

    PubMed

    Richards, N G; Schuster, S M

    1998-01-01

    The enzymatic synthesis of asparagine is an ATP-dependent process that utilizes the nitrogen atom derived from either glutamine or ammonia. Despite a long history of kinetic and mechanistic investigation, there is no universally accepted catalytic mechanism for this seemingly straightforward carboxyl group activating enzyme, especially as regards those steps immediately preceding amide bond formation. This chapter considers four issues dealing with the mechanism: (a) the structural organization of the active site(s) partaking in glutamine utilization and aspartate activation; (b) the relationship of asparagine synthetase to other amidotransferases; (c) the way in which ATP is used to activate the beta-carboxyl group; and (d) the detailed mechanism by which nitrogen is transferred.

  1. Glutamine Synthetase: Role in Neurological Disorders.

    PubMed

    Jayakumar, Arumugam R; Norenberg, Michael D

    2016-01-01

    Glutamine synthetase (GS) is an ATP-dependent enzyme found in most species that synthesizes glutamine from glutamate and ammonia. In brain, GS is exclusively located in astrocytes where it serves to maintain the glutamate-glutamine cycle, as well as nitrogen metabolism. Changes in the activity of GS, as well as its gene expression, along with excitotoxicity, have been identified in a number of neurological conditions. The literature describing alterations in the activation and gene expression of GS, as well as its involvement in different neurological disorders, however, is incomplete. This review summarizes changes in GS gene expression/activity and its potential contribution to the pathogenesis of several neurological disorders, including hepatic encephalopathy, ischemia, epilepsy, Alzheimer's disease, amyotrophic lateral sclerosis, traumatic brain injury, Parkinson's disease, and astroglial neoplasms. This review also explores the possibility of targeting GS in the therapy of these conditions.

  2. The enterococcal cytolysin synthetase has an unanticipated lipid kinase fold.

    PubMed

    Dong, Shi-Hui; Tang, Weixin; Lukk, Tiit; Yu, Yi; Nair, Satish K; van der Donk, Wilfred A

    2015-07-30

    The enterococcal cytolysin is a virulence factor consisting of two post-translationally modified peptides that synergistically kill human immune cells. Both peptides are made by CylM, a member of the LanM lanthipeptide synthetases. CylM catalyzes seven dehydrations of Ser and Thr residues and three cyclization reactions during the biosynthesis of the cytolysin large subunit. We present here the 2.2 Å resolution structure of CylM, the first structural information on a LanM. Unexpectedly, the structure reveals that the dehydratase domain of CylM resembles the catalytic core of eukaryotic lipid kinases, despite the absence of clear sequence homology. The kinase and phosphate elimination active sites that affect net dehydration are immediately adjacent to each other. Characterization of mutants provided insights into the mechanism of the dehydration process. The structure is also of interest because of the interactions of human homologs of lanthipeptide cyclases with kinases such as mammalian target of rapamycin.

  3. Parallel loss of nuclear-encoded mitochondrial aminoacyl-tRNA synthetases and mtDNA-encoded tRNAs in Cnidaria.

    PubMed

    Haen, Karri M; Pett, Walker; Lavrov, Dennis V

    2010-10-01

    Unlike most animal mitochondrial (mt) genomes, which encode a set of 22 transfer RNAs (tRNAs) sufficient for mt protein synthesis, those of cnidarians have only retained one or two tRNA genes. Whether the missing cnidarian mt-tRNA genes relocated outside the main mt chromosome or were lost remains unclear. It is also unknown what impact the loss of tRNA genes had on other components of the mt translational machinery. Here, we explored the nuclear genome of the cnidarian Nematostella vectensis for the presence of mt-tRNA genes and their corresponding mt aminoacyl-tRNA synthetases (mt-aaRS). We detected no candidates for mt-tRNA genes and only two mt-aaRS orthologs. At the same time, we found that all but one cytosolic aaRS appear to be targeted to mitochondria. These results indicate that the loss of mt-tRNAs in Cnidaria is genuine and occurred in parallel with the loss of nuclear-encoded mt-aaRS. Our phylogenetic analyses of individual aaRS revealed that although the nearly total loss of mt-aaRS is rare, aaRS gene deletion and replacement have occurred throughout the evolution of Metazoa.

  4. Regulation of 2', 5'-oligoadenylate synthetase gene expression by interferons and platelet-derived growth factor

    SciTech Connect

    Garcia-Blanco, M.A. ); Lengyel, P. . Dept. of Molecular Biophysics and Biochemistry); Morrison, E.; BrownLee, C.; Stiles, C.D. ); Williams, B.R.G. )

    1989-03-01

    In murine BALB/c 3T3 cell cultures, either beta interferon or platelet-derived growth factor (PDGF) enhanced expression of the 2', 5-oligoadenylate synthetase mRNA and protein. The time course of induction in response to beta inteferon was similar to that in response to PDGF. Of several growth factors known to be present in clotted blood serum (i.e., epidermal growth factor, transforming growth factor beta, and PDGF), only PDGF enhanced expression of 2', 5-oligoadenylate synthetase. The linkage of an interferon response element-containing segment from the 5'-flanking region of a human or murine 2'-5'-oligoadenylate synthetase gene made a heterologous gene responsive to interferon. The expression of such a gene construct in transfected cells was also induced by PDGF. Induction by PDGF was inhibited by mono- or polyclonal antibodies to murine interferon, which suggested that induction by PDGF requires interferon. Both PDGF and interferon induced nuclear factors that bound to this interferon response element-containing segment in vitro.

  5. Dual binding sites for translocation catalysis by Escherichia coli glutathionylspermidine synthetase.

    PubMed

    Pai, Chien-Hua; Chiang, Bing-Yu; Ko, Tzu-Ping; Chou, Chia-Cheng; Chong, Cheong-Meng; Yen, Fang-Jiun; Chen, Shoujun; Coward, James K; Wang, Andrew H-J; Lin, Chun-Hung

    2006-12-13

    Most organisms use glutathione to regulate intracellular thiol redox balance and protect against oxidative stress; protozoa, however, utilize trypanothione for this purpose. Trypanothione biosynthesis requires ATP-dependent conjugation of glutathione (GSH) to the two terminal amino groups of spermidine by glutathionylspermidine synthetase (GspS) and trypanothione synthetase (TryS), which are considered as drug targets. GspS catalyzes the penultimate step of the biosynthesis-amide bond formation between spermidine and the glycine carboxylate of GSH. We report herein five crystal structures of Escherichia coli GspS in complex with substrate, product or inhibitor. The C-terminal of GspS belongs to the ATP-grasp superfamily with a similar fold to the human glutathione synthetase. GSH is likely phosphorylated at one of two GSH-binding sites to form an acylphosphate intermediate that then translocates to the other site for subsequent nucleophilic addition of spermidine. We also identify essential amino acids involved in the catalysis. Our results constitute the first structural information on the biochemical features of parasite homologs (including TryS) that underlie their broad specificity for polyamines.

  6. TRYPTOPHANASE-TRYPTOPHAN SYNTHETASE SYSTEMS IN ESCHERICHIA COLI III.

    PubMed Central

    Freundlich, Martin; Lichstein, Herman C.

    1962-01-01

    Freundlich, Martin (University of Minnesota, Minneapolis) and Herman C. Lichstein. Tryptophanase-tryptophan synthetase systems in Escherichia coli. III. Requirements for enzyme synthesis. J. Bacteriol. 84:996–1006. 1962.—The requirements for the formation of tryptophanase and tryptophan synthetase in Escherichia coli during repression release were studied. The kinetics of the formation of tryptophan synthetase differed in the two strains examined; this was attributed to differences in the endogenous level of tryptophan in the bacterial cells. The formation of both enzymes was inhibited by chloramphenicol, and by the absence of arginine in an arginine-requiring mutant. These results are indicative of a requirement for protein synthesis for enzyme formation. Requirements for nucleic acid synthesis were examined by use of a uracil- and thymine-requiring mutant, and with purine and pyrimidine analogues. The results obtained suggest that some type of ribonucleic acid synthesis was necessary for the formation of tryptophanase and tryptophan synthetase. PMID:13959620

  7. A novel, enigmatic histone modification: biotinylation of histones by holocarboxylase synthetase.

    PubMed

    Hassan, Yousef I; Zempleni, Janos

    2008-12-01

    Holocarboxylase synthetase catalyzes the covalent binding of biotin to histones in humans and other eukaryotes. Eleven biotinylation sites have been identified in histones H2A, H3, and H4. K12-biotinylated histone H4 is enriched in heterochromatin, repeat regions, and plays a role in gene repression. About 30% of the histone H4 molecules are biotinylated at K12 in histone H4 in human fibroblast telomeres. The abundance of biotinylated histones at distinct genomic loci depends on biotin availability. Decreased histone biotinylation decreases life span and stress resistance in Drosophila. Low enrichment of biotinylated histones at transposable elements impairs repression of these elements.

  8. tRNA conjugation with chitosan nanoparticles: An AFM imaging study.

    PubMed

    Agudelo, D; Kreplak, L; Tajmir-Riahi, H A

    2016-04-01

    The conjugation of tRNA with chitosan nanoparticles of different sizes 15,100 and 200 kDa was investigated in aqueous solution using multiple spectroscopic methods and atomic force microscopy (AFM). Structural analysis showed that chitosan binds tRNA via G-C and A-U base pairs as well as backbone PO2 group, through electrostatic, hydrophilic and H-bonding contacts with overall binding constants of KCh-15-tRNA=4.1 (±0.60)×10(3)M(-1), KCh-100-tRNA=5.7 (±0.8)×10(3)M(-1) and KCh-200-tRNA=1.2 (±0.3)×10(4)M(-1). As chitosan size increases more stable polymer-tRNA conjugate is formed. AFM images showed major tRNA aggregation and particle formation occurred as chitosan concentration increased. Even though chitosan induced major biopolymer structural changes, tRNA remains in A-family structure.

  9. In vivo single-RNA tracking shows that most tRNA diffuses freely in live bacteria

    PubMed Central

    Plochowietz, Anne; Farrell, Ian; Smilansky, Zeev; Cooperman, Barry S.; Kapanidis, Achillefs N.

    2017-01-01

    Transfer RNA (tRNA) links messenger RNA nucleotide sequence with amino acid sequence during protein synthesis. Despite the importance of tRNA for translation, its subcellular distribution and diffusion properties in live cells are poorly understood. Here, we provide the first direct report on tRNA diffusion localization in live bacteria. We internalized tRNA labeled with organic fluorophores into live bacteria, applied single-molecule fluorescence imaging with single-particle tracking and localized and tracked single tRNA molecules over seconds. We observed two diffusive species: fast (with a diffusion coefficient of ∼8 μm2/s, consistent with free tRNA) and slow (consistent with tRNA bound to larger complexes). Our data indicate that a large fraction of internalized fluorescent tRNA (>70%) appears to diffuse freely in the bacterial cell. We also obtained the subcellular distribution of fast and slow diffusing tRNA molecules in multiple cells by normalizing for cell morphology. While fast diffusing tRNA is not excluded from the bacterial nucleoid, slow diffusing tRNA is localized to the cell periphery (showing a 30% enrichment versus a uniform distribution), similar to non-uniform localizations previously observed for mRNA and ribosomes. PMID:27625389

  10. TRYPTOPHANASE-TRYPTOPHAN SYNTHETASE SYSTEMS IN ESCHERICHIA COLI I.

    PubMed Central

    Freundlich, Martin; Lichstein, Herman C.

    1962-01-01

    Freundlich, Martin (University of Minnesota, Minneapolis) and Herman C. Lichstein. Tryptophanase-tryptophan synthetase systems in Escherichia coli. I. Effect of tryptophan and related compounds. J. Bacteriol. 84:979–987. 1962.—The effect of tryptophan and related compounds on tryptophanase and tryptophan synthetase formation in Escherichia coli was determined. Several of these compounds stimulated the formation of tryptophanase while concomitantly decreasing the production of synthetase. A number of tryptophan analogues were found to inhibit growth. The possible mode of action of these substances was examined further. 5-Hydroxytryptophan greatly inhibited the formation of synthetase and also reduced growth. Its inhibitory action on growth was attributed, at least partially, to the false feedback inhibition of anthranilic acid formation. Tryptamine was found to be a potent inhibitor of the activity of synthetase, as well as of the enzyme(s) involved in the synthesis of anthranilic acid from shikimic acid. However, growth reduction was only partially reversed by tryptophan. Indole-3-acetic acid and indole-3-propionic acid decreased growth and increased the formation of synthetase six- to eightfold. The action of these compounds was ascribed to their ability to block the endogenous formation of tryptophan. PMID:13959621

  11. Activation of 2',5'-oligoadenylate synthetase activity on induction of HL-60 leukemia cell differentiation.

    PubMed Central

    Schwartz, E L; Nilson, L A

    1989-01-01

    A 27-fold increase in 2',5'-oligoadenylate synthetase activity, an enzyme associated with the antiproliferative actions of interferon (IFN), was observed after treatment of HL-60 human leukemia cells with dimethyl sulfoxide (DMSO), an inducer of granulocytic differentiation of the cells. Enzyme activity was elevated after 24 h of exposure to DMSO, was maximal at 48 hours, and declined thereafter. A comparable increase was observed after treatment with 1 U of alpha interferon (IFN-alpha) per ml or 8 U of beta interferon (IFN-beta) per ml. Elevated levels of expression of other IFN-inducible genes, including type I histocompatibility antigen (HLA-B) mRNA and 2',5'-oligoadenylate phosphodiesterase activity, were also observed with DMSO treatment. DMSO-treated HL-60 cells had an increased amount of a 1.8-kilobase mRNA for oligoadenylate [oligo(A)] synthetase when compared with that of control cells; both DMSO- and IFN-treated HL-60 cells also expressed 1.6-, 3.4-, and 4.3-kilobase mRNA. The increase in both oligo(A) synthetase activity and mRNA levels was inhibited by polyclonal antiserum to human IFN-alpha; however, no IFN-alpha mRNA could be detected in the cells. Antiserum to IFN-beta or gamma interferon (IFN-gamma) had no effect on oligo(A) synthetase expression or activity nor was there any detectable IFN-beta 1 or IFN-beta 2 mRNA in the cells. The anti-IFN-alpha serum did not block the elevation of HLA-B mRNA in DMSO-treated cells. These observations suggest that the increased expression of oligo(A) synthetase in DMSO-treated cells may be mediated by the release of an IFN-alpha-like factor; however, the levels of any IFN-alpha mRNA produced in the cells were extremely low. Images PMID:2476665

  12. Evolution of aminoacyl-tRNA synthetase quaternary structure and activity: Saccharomyces cerevisiae mitochondrial phenylalanyl-tRNA synthetase.

    PubMed Central

    Sanni, A; Walter, P; Boulanger, Y; Ebel, J P; Fasiolo, F

    1991-01-01

    Phenylalanyl-tRNA synthetases [L-phenylalanine:tRNAPhe ligase (AMP-forming), EC 6.1.1.20] from Escherichia coli, yeast cytoplasm, and mammalian cytoplasm have an unusual conserved alpha 2 beta 2 quaternary structure that is shared by only one other aminoacyl-tRNA synthetase. Both subunits are required for activity. We show here that a single mitochondrial polypeptide from Saccharomyces cerevisiae is an active phenylalanyl-tRNA synthetase. This protein (the MSF1 gene product) is active as a monomer. It has all three characteristic sequence motifs of the class II aminoacyl-tRNA synthetases, and its activity may result from the recruitment of additional sequences into an alpha-subunit-like structure. Images PMID:1924298

  13. Enhancement of lysyl-tRNA synthetase activity in the Enterobacteriaceae

    SciTech Connect

    Hickey, E.W.; Hirshfield, I.

    1987-05-01

    Lysyl-tRNA synthetase (LRS) in E. coli is coded by two genes, one constitutive, and the other inducible; the latter is a cell stress protein. To determine if this system is wide spread in prokaryotes, the inducibility of LRS was first tested in eight members of the Enterobacteriaceae using cultural conditions known to induce the enzyme in E. coli K-12. Uninduced control cultures were grown to an O.D. of 0.2 at 580 nm in a supplemented minimal medium (SMM), pH 7.0 at 37/sup 0/C. Induction stimuli include: growth in SMM with 3mM Gly-L-Leu; growth in SMM as above, but with the initial pH adjusted to 5.0; or growth in Difco AC Broth to early stationary phase with a concomitant drop in the pH of the medium below 5.5. LRS activity was assayed in whole-cell sonic extracts by the aminoacylation of crude E. coli tRNA by /sup 14/C-lysine at pH 7.8 for three minutes. When E. aerogenes, K. pneumoniae, C. freundii, and S. typhimurium were grown in AC Broth, LRS activity was enhanced 2 to 4 fold. The enzyme is induced 2 to 4 fold in C. freundii and S. typhimurium upon growth at pH 5.0, whereas E. coli, K.; pneumoniae, and E. aerogenes show only a 1.5 fold induction. The peptide Gly-L-Leu enhanced LRS activity only in E. coli. LRS was not found to be inducible in S. marcescens, M. morganii, P. mirabilis, or P. vulgaris by any of the stimuli.

  14. Dual targeting of aminoacyl-tRNA synthetases to the mitochondrion and complex plastid in chlorarachniophytes.

    PubMed

    Hirakawa, Yoshihisa; Burki, Fabien; Keeling, Patrick J

    2012-12-15

    In plants, many nucleus-encoded proteins are targeted to both mitochondria and plastids, and this process is generally mediated by ambiguous N-terminal targeting sequences that are recognized by receptors on both organelles. In many algae, however, plastids were acquired by secondarily engulfing green or red algae, which were retained within the endomembrane system. Protein targeting to these secondary plastids is more complex, and because they do not reside directly in the cytoplasm, dual targeting cannot function as it does in plant cells. Here we investigate dual targeting of aminoacyl-tRNA synthetases (aaRSs) in chlorarachniophytes, which are complex algae that possess secondary plastids and a relict nucleus derived from a green algal endosymbiont. Chlorarachniophytes have four genome-containing compartments, but almost all the aaRSs are nucleus-encoded and present in fewer than four copies (some as few as two), suggesting multiple targeting. We characterized the subcellular localization of two classes, HisRS (three copies) and GlyRS (two copies), using GFP fusion proteins. In both cases, one copy was dually targeted to mitochondria and plastids, but unlike plants this was mediated by translation initiation variants. We also found that the periplastidal compartment (the relict green algal cytoplasm) lacks both GlyRS and a cognate tRNA, suggesting that pre-charged host tRNAs are imported into this compartment. Leader analysis of other aaRSs suggests that alternative translation is a common strategy for dual targeting in these complex cells. Overall, dual targeting to mitochondria and plastids is a shared feature of plastid-bearing organisms, but the increased complexity of trafficking into secondary plastids requires a different strategy.

  15. Chitin synthetase in encysting Giardia lamblia and Entamoeba invadens

    SciTech Connect

    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.

  16. The prokaryotic FAD synthetase family: a potential drug target.

    PubMed

    Serrano, Ana; Ferreira, Patricia; Martínez-Júlvez, Marta; Medina, Milagros

    2013-01-01

    Disruption of cellular production of the flavin cofactors, flavin adenine mononucleotide (FMN) and flavin adenine dinucleotide(FAD) will prevent the assembly of a large number of flavoproteins and flavoenzymes involved in key metabolic processes in all types of organisms. The enzymes responsible for FMN and FAD production in prokaryotes and eukaryotes exhibit various structural characteristics to catalyze the same chemistry, a fact that converts the prokaryotic FAD synthetase (FADS) in a potential drug target for the development of inhibitors endowed with anti-pathogenic activity. The first step before searching for selective inhibitors of FADS is to understand the structural and functional mechanisms for the riboflavin kinase and FMN adenylyltransferase activities of the prokaryotic enzyme, and particularly to identify their differential functional characteristics with regard to the enzymes performing similar functions in other organisms, particularly humans. In this paper, an overview of the current knowledge of the structure-function relationships in prokaryotic FADS has been presented, as well as of the state of the art in the use of these enzymes as drug targets.

  17. The aminoacyl-tRNA synthetases of Drosophila melanogaster

    PubMed Central

    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

  18. Secondary NAD+ deficiency in the inherited defect of glutamine synthetase.

    PubMed

    Hu, Liyan; Ibrahim, Khalid; Stucki, Martin; Frapolli, Michele; Shahbeck, Noora; Chaudhry, Farrukh A; Görg, Boris; Häussinger, Dieter; Penberthy, W Todd; Ben-Omran, Tawfeg; Häberle, Johannes

    2015-11-01

    Glutamine synthetase (GS) deficiency is an ultra-rare inborn error of amino acid metabolism that has been described in only three patients so far. The disease is characterized by neonatal onset of severe encephalopathy, low levels of glutamine in blood and cerebrospinal fluid, chronic moderate hyperammonemia, and an overall poor prognosis in the absence of an effective treatment. Recently, enteral glutamine supplementation was shown to be a safe and effective therapy for this disease but there are no data available on the long-term effects of this intervention. The amino acid glutamine, severely lacking in this disorder, is central to many metabolic pathways in the human organism and is involved in the synthesis of nicotinamide adenine dinucleotide (NAD(+)) starting from tryptophan or niacin as nicotinate, but not nicotinamide. Using fibroblasts, leukocytes, and immortalized peripheral blood stem cells (PBSC) from a patient carrying a GLUL gene point mutation associated with impaired GS activity, we tested whether glutamine deficiency in this patient results in NAD(+) depletion and whether it can be rescued by supplementation with glutamine, nicotinamide or nicotinate. The present study shows that congenital GS deficiency is associated with NAD(+) depletion in fibroblasts, leukocytes and PBSC, which may contribute to the severe clinical phenotype of the disease. Furthermore, it shows that NAD(+) depletion can be rescued by nicotinamide supplementation in fibroblasts and leukocytes, which may open up potential therapeutic options for the treatment of this disorder.

  19. Improved systematic tRNA gene annotation allows new insights into the evolution of mitochondrial tRNA structures and into the mechanisms of mitochondrial genome rearrangements

    PubMed Central

    Jühling, Frank; Pütz, Joern; Bernt, Matthias; Donath, Alexander; Middendorf, Martin; Florentz, Catherine; Stadler, Peter F.

    2012-01-01

    Transfer RNAs (tRNAs) are present in all types of cells as well as in organelles. tRNAs of animal mitochondria show a low level of primary sequence conservation and exhibit ‘bizarre’ secondary structures, lacking complete domains of the common cloverleaf. Such sequences are hard to detect and hence frequently missed in computational analyses and mitochondrial genome annotation. Here, we introduce an automatic annotation procedure for mitochondrial tRNA genes in Metazoa based on sequence and structural information in manually curated covariance models. The method, applied to re-annotate 1876 available metazoan mitochondrial RefSeq genomes, allows to distinguish between remaining functional genes and degrading ‘pseudogenes’, even at early stages of divergence. The subsequent analysis of a comprehensive set of mitochondrial tRNA genes gives new insights into the evolution of structures of mitochondrial tRNA sequences as well as into the mechanisms of genome rearrangements. We find frequent losses of tRNA genes concentrated in basal Metazoa, frequent independent losses of individual parts of tRNA genes, particularly in Arthropoda, and wide-spread conserved overlaps of tRNAs in opposite reading direction. Direct evidence for several recent Tandem Duplication-Random Loss events is gained, demonstrating that this mechanism has an impact on the appearance of new mitochondrial gene orders. PMID:22139921

  20. Glucocorticoid receptor-mediated induction of glutamine synthetase in skeletal muscle cells in vitro

    NASA Technical Reports Server (NTRS)

    Max, Stephen R.; Thomas, John W.; Banner, Carl; Vitkovic, Ljubisa; Konagaya, Masaaki

    1987-01-01

    The regulation by glucocorticoids of glutamine synthetase in L6 muscle cells in culture is studied. Glutamine synthetase activity was strikingly enhanced by dexamethasone. The dexamethasone-mediated induction of glutamine synthetase activity was blocked by RU38486, a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction process. RU38486 alone was without effect. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves increased levels of glutamine synthetase mRNA. Glucocorticoids regulate the expression of glutamine synthetase mRNA in cultured muscle cells via interaction with intracellular receptors. Such regulation may be relevant to control of glutamine production by muscle.

  1. Mutations activating the yeast eIF-2 alpha kinase GCN2: isolation of alleles altering the domain related to histidyl-tRNA synthetases.

    PubMed Central

    Ramirez, M; Wek, R C; Vazquez de Aldana, C R; Jackson, B M; Freeman, B; Hinnebusch, A G

    1992-01-01

    The protein kinase GCN2 stimulates expression of the yeast transcriptional activator GCN4 at the translational level by phosphorylating the alpha subunit of translation initiation factor 2 (eIF-2 alpha) in amino acid-starved cells. Phosphorylation of eIF-2 alpha reduces its activity, allowing ribosomes to bypass short open reading frames present in the GCN4 mRNA leader and initiate translation at the GCN4 start codon. We describe here 17 dominant GCN2 mutations that lead to derepression of GCN4 expression in the absence of amino acid starvation. Seven of these GCN2c alleles map in the protein kinase moiety, and two in this group alter the presumed ATP-binding domain, suggesting that ATP binding is a regulated aspect of GCN2 function. Six GCN2c alleles map in a region related to histidyl-tRNA synthetases, and two in this group alter a sequence motif conserved among class II aminoacyl-tRNA synthetases that directly interacts with the acceptor stem of tRNA. These results support the idea that GCN2 kinase function is activated under starvation conditions by binding uncharged tRNA to the domain related to histidyl-tRNA synthetase. The remaining GCN2c alleles map at the extreme C terminus, a domain required for ribosome association of the protein. Representative mutations in each domain were shown to depend on the phosphorylation site in eIF-2 alpha for their effects on GCN4 expression and to increase the level of eIF-2 alpha phosphorylation in the absence of amino acid starvation. Synthetic GCN2c double mutations show greater derepression of GCN4 expression than the parental single mutations, and they have a slow-growth phenotype that we attribute to inhibition of general translation initiation. The phenotypes of the GCN2c alleles are dependent on GCN1 and GCN3, indicating that these two positive regulators of GCN4 expression mediate the inhibitory effects on translation initiation associated with activation of the yeast eIF-2 alpha kinase GCN2. Images PMID:1448107

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

    SciTech Connect

    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

    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.

  3. Extensive and evolutionarily persistent mitochondrial tRNA editing in Velvet Worms (phylum Onychophora).

    PubMed

    Segovia, Romulo; Pett, Walker; Trewick, Steve; Lavrov, Dennis V

    2011-10-01

    Mitochondrial genomes of onychophorans (velvet worms) present an interesting problem: Some previous studies reported them lacking several transfer RNA (tRNA) genes, whereas others found that all their tRNA genes were present but severely reduced. To resolve this discrepancy, we determined complete mitochondrial DNA (mtDNA) sequences of the onychophorans Oroperipatus sp. and Peripatoides sympatrica as well as cDNA sequences from 14 and 10 of their tRNAs, respectively. We show that tRNA genes in these genomes are indeed highly reduced and encode truncated molecules, which are restored to more conventional structures by extensive tRNA editing. During this editing process, up to 34 nucleotides are added to the tRNA sequences encoded in Oroperipatus sp. mtDNA, rebuilding the aminoacyl acceptor stem, the TΨC arm, and in some extreme cases, the variable arm and even a part of the anticodon stem. The editing is less extreme in P. sympatrica in which at least a part of the TΨC arm is always encoded in mtDNA. When the entire TΨC arm is added de novo in Oroperipatus sp., the sequence of this arm is either identical or similar among different tRNA species, yet the sequences show substantial variation for each tRNA. These observations suggest that the arm is rebuilt, at least in part, by a template-independent mechanism and argue against the alternative possibility that tRNA genes or their parts are imported from the nucleus. By contrast, the 3' end of the aminoacyl acceptor stem is likely restored by a template-dependent mechanism. The extreme tRNA editing reported here has been preserved for >140 My as it was found in both extant families of onychophorans. Furthermore, a similar type of tRNA editing may be present in several other groups of arthropods, which show a high degree of tRNA gene reduction in their mtDNA.

  4. Autosomal-Recessive Mutations in the tRNA Splicing Endonuclease Subunit TSEN15 Cause Pontocerebellar Hypoplasia and Progressive Microcephaly.

    PubMed

    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

    2016-07-07

    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.

  5. Three-Dimensional Algebraic Models of the tRNA Code and 12 Graphs for Representing the Amino Acids

    PubMed Central

    José, Marco V.; Morgado, Eberto R.; Guimarães, Romeu Cardoso; Zamudio, Gabriel S.; de Farías, Sávio Torres; Bobadilla, Juan R.; Sosa, Daniela

    2014-01-01

    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. PMID:25370377

  6. Dihydrofolate synthetase and folylpolyglutamate synthetase: direct evidence for intervention of acyl phosphate intermediates

    SciTech Connect

    Banerjee, R.V.; Shane, B.; McGuire, J.J.; Coward, J.K.

    1988-12-13

    The transfer of /sup 17/O and/or /sup 18/O from (COOH-/sup 17/O or -/sup 18/O) enriched substrates to inorganic phosphate (P/sub i/) has been demonstrated for two enzyme-catalyzed reactions involved in folate biosynthesis and glutamylation. COOH-/sup 18/O-labeled folate, methotrexate, and dihydropteroate, in addition to (/sup 17/O)-glutamate, were synthesized and used as substrates for folylpolyglutamate synthetase (FPGS) isolated from Escherichia coli, hog liver, and rat liver and for dihydrofolate synthetase (DHFS) isolated from E. coli. P/sub i/ was purified from the reaction mixtures and converted to trimethyl phosphate (TMP), which was then analyzed for /sup 17/O and /sup 18/O enrichment by nuclear magnetic resonance (NMR) spectroscopy and/or mass spectroscopy. In the reactions catalyzed by the E. coli enzymes, both NMR and quantitative mass spectral analyses established that transfer of the oxygen isotope from the substrate /sup 18/O-enriched carboxyl group to P/sub i/ occurred, thereby providing strong evidence for an acyl phosphate intermediate in both the FPGS- and DHFS-catalyzed reactions. Similar oxygen-transfer experiments were carried out by use of two mammalian enzymes. The small amounts of P/sub i/ obtained from reactions catalyzed by these less abundant FPGS proteins precluded the use of NMR techniques. However, mass spectral analysis of the TMP derived from the mammalian FPGS-catalyzed reactions showed clearly that /sup 18/O transfer had occurred.

  7. Effect of PEG and mPEG-anthracene on tRNA aggregation and particle formation.

    PubMed

    Froehlich, E; Mandeville, J S; Arnold, D; Kreplak, L; Tajmir-Riahi, H A

    2012-01-09

    Poly(ethylene glycol) (PEG) and its derivatives are synthetic polymers with major applications in gene and drug delivery systems. Synthetic polymers are also used to transport miRNA and siRNA in vitro. We studied the interaction of tRNA with several PEGs of different compositions, such as PEG 3350, PEG 6000, and mPEG-anthracene under physiological conditions. FTIR, UV-visible, CD, and fluorescence spectroscopic methods as well as atomic force microscopy (AFM) were used to analyze the PEG binding mode, the binding constant, and the effects of polymer complexation on tRNA stability, aggregation, and particle formation. Structural analysis showed that PEG-tRNA interaction occurs via RNA bases and the backbone phosphate group with both hydrophilic and hydrophobic contacts. The overall binding constants of K(PEG 3350-tRNA)= 1.9 (±0.5) × 10(4) M(-1), K(PEG 6000-tRNA) = 8.9 (±1) × 10(4) M(-1), and K(mPEG-anthracene)= 1.2 (±0.40) × 10(3) M(-1) show stronger polymer-RNA complexation by PEG 6000 and by PEG 3350 than the mPEG-anthracene. AFM imaging showed that PEG complexes contain on average one tRNA with PEG 3350, five tRNA with PEG 6000, and ten tRNA molecules with mPEG-anthracene. tRNA aggregation and particle formation occurred at high polymer concentrations, whereas it remains in A-family structure.

  8. Flipping of the ribosomal A-site adenines provides a basis for tRNA selection

    PubMed Central

    Zeng, Xiancheng; Chugh, Jeetender; Casiano-Negroni, Anette; Al-Hashimi, Hashim M.; Brooks, Charles L.

    2014-01-01

    Ribosomes control the missense error rate of ~10−4 during translation though quantitative contributions of individual mechanistic steps of the conformational changes yet to be fully determined. Biochemical and biophysical studies led to a qualitative tRNA selection model in which ribosomal A-site residues A1492 and A1493 (A1492/3) flip out in response to cognate tRNA binding, promoting the subsequent reactions, but not in the case of near cognate or non-cognate tRNA. However, this model was recently questioned by X-ray structures revealing conformations of extrahelical A1492/3 and domain closure of the decoding center in both cognate and near-cognate tRNA bound ribosome complexes, suggesting that the non-specific flipping of A1492/3 has no active role in tRNA selection. We explore this question by carrying out molecular dynamics (MD) simulations, aided with fluorescence and NMR experiments, to probe the free energy cost of extrahelical flipping of 1492/3 and the strain energy associated with domain conformational change. Our rigorous calculations demonstrate that the A1492/3 flipping is indeed a specific response to the binding of cognate tRNA, contributing 3 kcal/mol to the specificity of tRNA selection. Furthermore, the different A-minor interactions in cognate and near-cognate complexes propagate into the conformational strain and contribute another 4 kcal/mol in domain closure. The recent structure of ribosome with features of extrahelical A1492/3 and closed domain in near-cognate complex is reconciled by possible tautomerization of the wobble base pair in mRNA-tRNA. These results quantitatively rationalize other independent experimental observations and explain the ribosomal discrimination mechanism of selecting cognate versus near-cognate tRNA. PMID:24813122

  9. P-body components, Dhh1 and Pat1, are involved in tRNA nuclear-cytoplasmic dynamics

    PubMed Central

    Hurto, Rebecca L.; Hopper, Anita K.

    2011-01-01

    The nuclear-cytoplasmic distribution of tRNA depends on the balance between tRNA nuclear export/re-export and retrograde tRNA nuclear import in Saccharomyces cerevisiae. The distribution of tRNA is sensitive to nutrient availability as cells deprived of various nutrients exhibit tRNA nuclear accumulation. Starvation induces numerous events that result in translational repression and P-body formation. This study investigated the possible coordination of these responses with tRNA nuclear-cytoplasmic distribution. Dhh1 and Pat1 function in parallel to promote translation repression and P-body formation in response to starvation. Loss of both, Dhh1 and Pat1, results in a failure to repress translation and to induce P-body formation in response to glucose starvation. This study reports that nutrient deprived dhh1 pat1 cells also fail to accumulate tRNA within nuclei. Conversely, inhibition of translation initiation and induction of P-body formation by overproduction of Dhh1 or Pat1 cause tRNA nuclear accumulation in nutrient-replete conditions. Also, loss of the mRNA decapping activator, Lsm1, causes tRNA nuclear accumulation. However, the coordination between P-body formation, translation repression, and tRNA distribution is limited to the early part of the P-body formation/translation repression pathway as loss of mRNA decapping or 5′ to 3′ degradation does not influence tRNA nuclear-cytoplasmic dynamics. The data provide the first link between P-body formation/translation initiation and tRNA nuclear-cytoplasmic dynamics. The current model is that Dhh1 and Pat1 function in parallel to promote starvation-induced tRNA nuclear accumulation. PMID:21398402

  10. Structural Analysis of the Active Site Geometry of N[superscript 5]-Carboxyaminoimidazole Ribonucleotide Synthetase from Escherichia coli

    SciTech Connect

    Thoden, James B.; Holden, Hazel M.; Firestine, Steven M.

    2009-09-11

    N{sub 5}-Carboxyaminoimidazole ribonucleotide synthetase (N{sub 5}-CAIR synthetase) converts 5-aminoimidazole ribonucleotide (AIR), MgATP, and bicarbonate into N{sub 5}-CAIR, MgADP, and P{sub i}. The enzyme is required for de novo purine biosynthesis in microbes yet is not found in humans suggesting that it represents an ideal and unexplored target for antimicrobial drug design. Here we report the X-ray structures of N{sub 5}-CAIR synthetase from Escherichia coli with either MgATP or MgADP/P{sub i} bound in the active site cleft. These structures, determined to 1.6-{angstrom} resolution, provide detailed information regarding the active site geometry before and after ATP hydrolysis. In both structures, two magnesium ions are observed. Each of these is octahedrally coordinated, and the carboxylate side chain of Glu238 bridges them. For the structure of the MgADP/P{sub i} complex, crystals were grown in the presence of AIR and MgATP. No electron density was observed for AIR, and the electron density corresponding to the nucleotide clearly revealed the presence of ADP and P{sub i} rather than ATP. The bound P{sub i} shifts by approximately 3 {angstrom} relative to the {gamma}-phosphoryl group of ATP and forms electrostatic interactions with the side chains of Arg242 and His244. Since the reaction mechanism of N{sub 5}-CAIR synthetase is believed to proceed via a carboxyphosphate intermediate, we propose that the location of the inorganic phosphate represents the binding site for stabilization of this reactive species. Using the information derived from the two structures reported here, coupled with molecular modeling, we propose a catalytic mechanism for N{sub 5}-CAIR synthetase.

  11. Crystal structure of aspartyl-tRNA synthetase from Pyrococcus kodakaraensis KOD: archaeon specificity and catalytic mechanism of adenylate formation.

    PubMed Central

    Schmitt, E; Moulinier, L; Fujiwara, S; Imanaka, T; Thierry, J C; Moras, D

    1998-01-01

    The crystal structure of aspartyl-tRNA synthetase (AspRS) from Pyrococcus kodakaraensis was solved at 1.9 A resolution. The sequence and three-dimensional structure of the catalytic domain are highly homologous to those of eukaryotic AspRSs. In contrast, the N-terminal domain, whose function is to bind the tRNA anticodon, is more similar to that of eubacterial enzymes. Its structure explains the unique property of archaeal AspRSs of accommodating both tRNAAsp and tRNAAsn. Soaking the apo-enzyme crystals with ATP and aspartic acid both separately and together allows the adenylate formation to be followed. Due to the asymmetry of the dimeric enzyme in the crystalline state, different steps of the reaction could be visualized within the same crystal. Four different states of the aspartic acid activation reaction could thus be characterized, revealing the functional correlation of the observed conformational changes. The binding of the amino acid substrate induces movement of two invariant loops which secure the position of the peptidyl moiety for adenylate formation. An unambiguous spatial and functional assignment of three magnesium ion cofactors can be made. This study shows the important role of residues present in both archaeal and eukaryotic AspRSs, but absent from the eubacterial enzymes. PMID:9724658

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

    PubMed Central

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

    2015-01-01

    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. PMID:26220470

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

    NASA Astrophysics Data System (ADS)

    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.

  14. Cleavage of tRNA within the mature tRNA sequence by the catalytic RNA of RNase P: implication for the formation of the primer tRNA fragment for reverse transcription in copia retrovirus-like particles.

    PubMed Central

    Kikuchi, Y; Sasaki, N; Ando-Yamagami, Y

    1990-01-01

    The retrovirus-like particles of Drosophila are intermediates of retrotransposition of the transposable element copia. In these particles, a 39-nucleotide-long fragment from the 5' region of Drosophila initiator methionine tRNA (tRNA(iMet) is used as the primer for copia minus-strand reverse transcription. To function as primer for this reverse transcription, the Drosophila tRNA(iMet) must be cleaved in vivo at the site between nucleotides 39 and 40. When a synthetic Drosophila tRNA(iMet) precursor was incubated with M1RNA, the catalytic RNA of Escherichia coli RNase P, other cleavages within the mature tRNA sequence were detected in addition to the efficient removal of the 5' leader sequence of this tRNA precursor. One of these cleavage sites is between nucleotides 39 and 40 of Drosophila tRNA(iMet). Based on this result, we propose a model for formation of the primer tRNA fragment for reverse transcription in copia retrovirus-like particles. Images PMID:1700426

  15. Inhibitors of Methionyl-tRNA Synthetase Have Potent Activity against Giardia intestinalis Trophozoites

    PubMed Central

    Ranade, Ranae M.; Zhang, Zhongsheng; Gillespie, J. Robert; Shibata, Sayaka; Verlinde, Christophe L. M. J.; Hol, Wim G. J.; Fan, Erkang

    2015-01-01

    The methionyl-tRNA synthetase (MetRS) is a novel drug target for the protozoan pathogen Giardia intestinalis. This protist contains a single MetRS that is distinct from the human cytoplasmic MetRS. A panel of MetRS inhibitors was tested against recombinant Giardia MetRS, Giardia trophozoites, and mammalian cell lines. The best compounds inhibited trophozoite growth at 500 nM (metronidazole did so at ∼5,000 nM) and had low cytotoxicity against mammalian cells, indicating excellent potential for further development as anti-Giardia drugs. PMID:26324270

  16. Peculiarities of interaction of porphyrins with tRNA at low ionic strength.

    PubMed

    Dalyan, Y; Vardanyan, I; Chavushyan, A; Balayan, G

    2010-08-01

    The interaction of meso-tetra-(4N-oxyethylpyridyl)porphyrin (TOEPyP4) and its Zn(II)-, Cu(II)-, Mn(III)-derivatives with tRNA from E.Coli at low ionic strength (micro=0.02M) was studied using UV/Vis spectrophotometry and Circular Dichroism (CD) methods. An unusual Induced Circular Dichroism (ICD) spectra profile of the ZnTOEPyP4-tRNA complex is found. It is demonstrated that ZnTOEPyP4 is ordered in a stack, not only on helical sites, but also on loops of a hairpin form of tRNA. TOEPyP4 and CuTOEPyP4 are able to intercalate in the helical sites of this form of tRNA. MnTOEPyP4 interacts with tRNA via external non-ordered mechanism. It is established that all porphyrins are bound with tRNA more strongly than with DNA.

  17. tRNA fluorescent labeling at 3' end inducing an aminoacyl-tRNA-like behavior.

    PubMed

    Servillo, L; Balestrieri, C; Quagliuolo, L; Iorio, E L; Giovane, A

    1993-04-01

    A fluorescent tRNA derivative labeled at 3'-O position of the ultimate adenosine residue by reaction, under mild conditions, of tRNA with isatoic anhydride [3,1-benzoxazine-2,4(1H)-dione] was obtained. The labeling selectivity was determined by several criteria: digestion with RNase, followed by HPLC of the digest, produces only one labeled nucleoside, identified as 3'-O-anthraniloyladenosine; the ratio of the absorbance at 260 nm to 332 nm also suggests a 1:1 molar ratio between the nucleic acid and the fluorophore; finally, the incapacity of the labeled tRNA to be charged by the specific aminoacyltransferase further demonstrates the engagement of the 3'-O position. Although the 3'-O-anthraniloyl-labeled tRNA does not seem to be functionally active, as far as the aminoacyl charging activity is concerned, surprisingly we found that it is able to form the ternary complex with elongation factor Tu (EF-Tu) and GTP with an affinity consistently higher than uncharged tRNA. From fluorescence anisotropy measurements the ternary complex dissociation constant was estimated as 73 nM for Escherichia coli and 140 nM for yeast anthraniloyl-tRNA(Phe). These results may be interpreted in terms of the particular structure of the anthraniloyl group that makes the labeled tRNA similar to an aminoacyl-tRNA.

  18. tRNA thiolation links translation to stress responses in Saccharomyces cerevisiae.

    PubMed

    Damon, Jadyn R; Pincus, David; Ploegh, Hidde L

    2015-01-15

    Although tRNA modifications have been well catalogued, the precise functions of many modifications and their roles in mediating gene expression are still being elucidated. Whereas tRNA modifications were long assumed to be constitutive, it is now apparent that the modification status of tRNAs changes in response to different environmental conditions. The URM1 pathway is required for thiolation of the cytoplasmic tRNAs tGlu(UUC), tGln(UUG), and tLys(UUU) in Saccharomyces cerevisiae. We demonstrate that URM1 pathway mutants have impaired translation, which results in increased basal activation of the Hsf1-mediated heat shock response; we also find that tRNA thiolation levels in wild-type cells decrease when cells are grown at elevated temperature. We show that defects in tRNA thiolation can be conditionally advantageous, conferring resistance to endoplasmic reticulum stress. URM1 pathway proteins are unstable and hence are more sensitive to changes in the translational capacity of cells, which is decreased in cells experiencing stresses. We propose a model in which a stress-induced decrease in translation results in decreased levels of URM1 pathway components, which results in decreased tRNA thiolation levels, which further serves to decrease translation. This mechanism ensures that tRNA thiolation and translation are tightly coupled and coregulated according to need.

  19. Nuclear pore proteins are involved in the biogenesis of functional tRNA.

    PubMed Central

    Simos, G; Tekotte, H; Grosjean, H; Segref, A; Sharma, K; Tollervey, D; Hurt, E C

    1996-01-01

    Los1p and Pus1p, which are involved in tRNA biogenesis, were found in a genetic screen for components interacting with the nuclear pore protein Nsp1p. LOS1, PUS1 and NSP1 interact functionally, since the combination of mutations in the three genes causes synthetic lethality. Pus1p is an intranuclear protein which exhibits a nucleotide-specific and intron-dependent tRNA pseudouridine synthase activity. Los1p was shown previously to be required for efficient pre-tRNA splicing; we report here that Los1p localizes to the nuclear pores and is linked functionally to several components of the tRNA biogenesis machinery including Pus1p and Tfc4p. When the formation of functional tRNA was analyzed by an in vivo assay, the los1(-) pus1(-) double mutant, as well as several thermosensitive nucleoporin mutants including nsp1, nup116, nup133 and nup85, exhibited loss of suppressor tRNA activity even at permissive temperatures. These data suggest that nuclear pore proteins are required for the biogenesis of functional tRNA. Images PMID:8641292

  20. Exportin-5-mediated nuclear export of eukaryotic elongation factor 1A and tRNA.

    PubMed

    Calado, Angelo; Treichel, Nathalie; Müller, Eva-Christina; Otto, Albrecht; Kutay, Ulrike

    2002-11-15

    Transport of proteins and RNA into and out of the cell nucleus is mediated largely by a family of RanGTP-binding transport receptors. Export receptors (exportins) need to bind RanGTP for efficient loading of their export cargo. We have identified eukaryotic elongation factor 1A (eEF1A) and tRNA as RanGTP-dependent binding partners of exportin-5 (Exp5). Exp5 stimulates nuclear export of eEF1A when microinjected into the nucleus of Xenopus laevis oocytes. Surprisingly, the interaction between eEF1A and Exp5 is dependent on tRNA that can interact directly with Exp5 and, if aminoacylated, recruits eEF1A into the export complex. These data suggested to us that Exp5 might support tRNA export. Indeed, not only the canonical tRNA export receptor, exportin-t, but also Exp5 can drive nuclear export of tRNA. Taken together, we show that there exists an alternative tRNA export pathway which can be exploited to keep eEF1A out of the cell nucleus.

  1. Functional linkage between the glutaminase and synthetase domains of carbamoyl-phosphate synthetase. Role of serine 44 in carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (cad).

    PubMed

    Hewagama, A; Guy, H I; Vickrey, J F; Evans, D R

    1999-10-01

    Mammalian carbamoyl-phosphate synthetase is part of carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (CAD), a multifunctional protein that also catalyzes the second and third steps of pyrimidine biosynthesis. Carbamoyl phosphate synthesis requires the concerted action of the glutaminase (GLN) and carbamoyl-phosphate synthetase domains of CAD. There is a functional linkage between these domains such that glutamine hydrolysis on the GLN domain does not occur at a significant rate unless ATP and HCO(3)(-), the other substrates needed for carbamoyl phosphate synthesis, bind to the synthetase domain. The GLN domain consists of catalytic and attenuation subdomains. In the separately cloned GLN domain, the catalytic subdomain is down-regulated by interactions with the attenuation domain, a process thought to be part of the functional linkage. Replacement of Ser(44) in the GLN attenuation domain with alanine increases the k(cat)/K(m) for glutamine hydrolysis 680-fold. The formation of a functional hybrid between the mammalian Ser(44) GLN domain and the Escherichia coli carbamoyl-phosphate synthetase large subunit had little effect on glutamine hydrolysis. In contrast, ATP and HCO(3)(-) did not stimulate the glutaminase activity, indicating that the interdomain linkage had been disrupted. In accord with this interpretation, the rate of glutamine hydrolysis and carbamoyl phosphate synthesis were no longer coordinated. Approximately 3 times more glutamine was hydrolyzed by the Ser(44) --> Ala mutant than that needed for carbamoyl phosphate synthesis. Ser(44), the only attenuation subdomain residue that extends into the GLN active site, appears to be an integral component of the regulatory circuit that phases glutamine hydrolysis and carbamoyl phosphate synthesis.

  2. Near-UV stress in salmonella typhimurium: 4-thiouridine in tRNA, ppGpp, and ApppGpp as components of an adaptive response

    SciTech Connect

    Kramer, G.F.; Baker, J.C.; Ames, B.N.

    1988-05-01

    We have examined the role of 4-thiouridine in the responses of Salmonella typhimurium to near-UV irradiation. Mutants lacking 4-thiouridine (nuv) and mutants defective in the synthesis of ppGpp (guanosine 5'-diphosphate-3'-diphosphate) (relA) were found to be sensitive to killing by near-UV. Near-UV induced the synthesis of a set of proteins that were not induced in the nuv mutant. Some of these proteins were identified as oxidative defense proteins, and others were identified as ppGpp-inducible proteins. Over 100-fold increases in ApppGpp (adenoisine 5', 5'''-triphosphoguanosine-3'''-diphosphate, the adenylylated form of ppGpp) were observed in wild-type cells after near-UV irradiation but not in the 4-thiouridine-deficient mutant. These data support a model in which ppGpp and ApppGpp, a dinucleotide proposed to be synthesized by tRNA-aminoacyl synthetases as a response to the cross-linking of 4-thiouridine in tRNA by near-UV, induce the synthesis of proteins necessary for resistance to near-UV irradiation.

  3. The glutamine synthetase gene family in Populus

    PubMed Central

    2011-01-01

    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. PMID:21867507

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

    NASA Astrophysics Data System (ADS)

    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

  5. Heterogeneity of Glutamine Synthetase Polypeptides in Phaseolus vulgaris L. 1

    PubMed Central

    Lara, Miguel; Porta, Helena; Padilla, Jaime; Folch, Jorge; Sánchez, Federico

    1984-01-01

    Glutamine synthetases from roots, nodules, and leaves of Phaseolus vulgaris L. have been purified to homogeneity and their polypeptide composition determined. The leaf enzyme is composed of six polypeptides. The cytosolic fraction contains two 43,000 dalton polypeptides and the chloroplastic enzyme is formed by four 45,000 dalton polypeptides. Root glutamine synthetase consists only of the same two polypeptides of 43,000 dalton that are present in the leaf enzyme. The nodule enzyme is formed by two polypeptides of 43,000 dalton, one is common to the leaf and root enzyme but the other is specific for N2-fixing nodule tissue. The two glutamine synthetase forms of the nodule contain a different proportion of the 43,000 dalton polypeptides. Images Fig. 1 Fig. 2 Fig. 4 PMID:16663942

  6. Enzymology of tRNA modification in the bacterial MnmEG pathway.

    PubMed

    Armengod, M-Eugenia; Moukadiri, Ismaïl; Prado, Silvia; Ruiz-Partida, Rafael; Benítez-Páez, Alfonso; Villarroya, Magda; Lomas, Rodrigo; Garzón, María J; Martínez-Zamora, Ana; Meseguer, Salvador; Navarro-González, Carmen

    2012-07-01

    Among all RNAs, tRNA exhibits the largest number and the widest variety of post-transcriptional modifications. Modifications within the anticodon stem loop, mainly at the wobble position and purine-37, collectively contribute to stabilize the codon-anticodon pairing, maintain the translational reading frame, facilitate the engagement of the ribosomal decoding site and enable translocation of tRNA from the A-site to the P-site of the ribosome. Modifications at the wobble uridine (U34) of tRNAs reading two degenerate codons ending in purine are complex and result from the activity of two multi-enzyme pathways, the IscS-MnmA and MnmEG pathways, which independently work on positions 2 and 5 of the U34 pyrimidine ring, respectively, and from a third pathway, controlled by TrmL (YibK), that modifies the 2'-hydroxyl group of the ribose. MnmEG is the only common pathway to all the mentioned tRNAs, and involves the GTP- and FAD-dependent activity of the MnmEG complex and, in some cases, the activity of the bifunctional enzyme MnmC. The Escherichia coli MnmEG complex catalyzes the incorporation of an aminomethyl group into the C5 atom of U34 using methylene-tetrahydrofolate and glycine or ammonium as donors. The reaction requires GTP hydrolysis, probably to assemble the active site of the enzyme or to carry out substrate recognition. Inactivation of the evolutionarily conserved MnmEG pathway produces a pleiotropic phenotype in bacteria and mitochondrial dysfunction in human cell lines. While the IscS-MnmA pathway and the MnmA-mediated thiouridylation reaction are relatively well understood, we have limited information on the reactions mediated by the MnmEG, MnmC and TrmL enzymes and on the precise role of proteins MnmE and MnmG in the MnmEG complex activity. This review summarizes the present state of knowledge on these pathways and what we still need to know, with special emphasis on the MnmEG pathway.

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

    PubMed Central

    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

    2014-01-01

    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

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

    PubMed

    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.

  9. A newly discovered tRNA(1Asp) gene (aspV) of Escherichia coli K12.

    PubMed

    Horiuchi, T; Nagasawa, T; Takano, K; Sekiguchi, M

    1987-02-01

    We report a new tRNA(1Asp) gene near the dnaQ gene, which is located at 5 min on the Escherichia coli linkage map. We named it aspV. The sequence corresponding to the mature tRNA is identical with that of the two previously identified tRNA(1Asp) genes (aspT and aspU), but there is no homology in the sequences of their 3'- and 5'-flanking regions.

  10. The role of mitochondrial tRNA mutations in lung cancer

    PubMed Central

    Wang, Lie; Chen, Zhi-Jun; Zhang, Yong-Kui; Le, Han-Bo

    2015-01-01

    Alternations in mitochondrial genome resulting in mitochondrial dysfunction have long been hypothesized to be involved in tumorigenesis. Mitochondrial tRNA (mt-tRNA) is known for its high frequencies of polymorphisms and mutations, however, the roles of these mutations and polymorphisms in lung cancer are among heated debates. To evaluate the possible roles of reported mt-tRNA mutations in lung cancer, we examine recent published paper concerning three mt-tRNA mutations with lung cancer: A7460G in tRNASer (UCN) gene, G5563A in tRNATrp gene and A12172G in tRNAHis gene. We perform the phylogenetic approach to investigate the deleterious roles of these mutations in lung cancer, moreover, we use bioinformatics tool to predict the secondary structure of mt-tRNAs with and without these mutations. In addition, through the application of pathogenicity scoring system, we find that only the A12172G mutation is regarded as a pathogenic mutation, whereas other mutations may act as neutral polymorphisms in human population. Thus, our study provides the novel insight into the molecular pathogenesis of mt-tRNA mutations in lung cancer. PMID:26550263

  11. The role of mitochondrial tRNA mutations in lung cancer.

    PubMed

    Wang, Lie; Chen, Zhi-Jun; Zhang, Yong-Kui; Le, Han-Bo

    2015-01-01

    Alternations in mitochondrial genome resulting in mitochondrial dysfunction have long been hypothesized to be involved in tumorigenesis. Mitochondrial tRNA (mt-tRNA) is known for its high frequencies of polymorphisms and mutations, however, the roles of these mutations and polymorphisms in lung cancer are among heated debates. To evaluate the possible roles of reported mt-tRNA mutations in lung cancer, we examine recent published paper concerning three mt-tRNA mutations with lung cancer: A7460G in tRNA(Ser (UCN)) gene, G5563A in tRNA(Trp) gene and A12172G in tRNA(His) gene. We perform the phylogenetic approach to investigate the deleterious roles of these mutations in lung cancer, moreover, we use bioinformatics tool to predict the secondary structure of mt-tRNAs with and without these mutations. In addition, through the application of pathogenicity scoring system, we find that only the A12172G mutation is regarded as a pathogenic mutation, whereas other mutations may act as neutral polymorphisms in human population. Thus, our study provides the novel insight into the molecular pathogenesis of mt-tRNA mutations in lung cancer.

  12. Mode of action of RNase BN/RNase Z on tRNA precursors: RNase BN does not remove the CCA sequence from tRNA.

    PubMed

    Dutta, Tanmay; Deutscher, Murray P

    2010-07-23

    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 purified RNase BN can process both CCA-less and CCA-containing tRNA precursors. On CCA-less precursors, RNase BN cleaved endonucleolytically after the discriminator nucleotide to allow subsequent CCA addition. On CCA-containing precursors, RNase BN acted as either an exoribonuclease or endoribonuclease depending on the nature of the added divalent cation. Addition of Co(2+) resulted in higher activity and predominantly exoribonucleolytic activity, whereas in the presence of Mg(2+), RNase BN was primarily an endoribonuclease. In no case was any evidence obtained for removal of the CCA sequence. Certain tRNA precursors were extremely poor substrates under any conditions tested. These findings provide important information on the ability of RNase BN to process tRNA precursors and help explain the known physiological properties of this enzyme. In addition, they call into question the removal of CCA sequences by RNase BN.

  13. The heteromeric Nanoarchaeum equitans splicing endonuclease cleaves noncanonical bulge–helix–bulge motifs of joined tRNA halves

    PubMed Central

    Randau, Lennart; Calvin, Kate; Hall, Michelle; Yuan, Jing; Podar, Mircea; Li, Hong; Söll, Dieter

    2005-01-01

    Among the tRNA population of the archaeal parasite Nanoarchaeum equitans are five species assembled from separate 5′ and 3′ tRNA halves and four species derived from tRNA precursors containing introns. In both groups an intervening sequence element must be removed during tRNA maturation. A bulge–helix–bulge (BHB) motif is the hallmark structure required by the archaeal splicing endonuclease for recognition and excision of all introns. BHB motifs are recognizable at the joining sites of all five noncontinuous tRNA species, although deviations from the canonical BHB motif are clearly present in at least two of them. Here, we show that the N. equitans splicing endonuclease cleaves tRNA precursors containing normal introns, as well as all five noncontinuous precursor tRNAs, at the predicted splice sites, indicating the enzyme's dual role in the removal of tRNA introns and processing of tRNA halves to be joined in trans. The cleavage activity on a set of synthetic canonical and noncanonical BHB constructs showed that the N. equitans splicing endonuclease accepts a broader range of substrates than the homodimeric Archaeoglobus fulgidus enzyme. In contrast to the A. fulgidus endonuclease, the N. equitans splicing enzyme possesses two different subunits. This heteromeric endonuclease type, found in N. equitans, in all Crenarchaeota, and in Methanopyrus kandleri, is able to act on the noncanonical tRNA introns present only in these organisms, which suggests coevolution of enzyme and substrate. PMID:16330750

  14. Minireview on Glutamine Synthetase Deficiency, an Ultra-Rare Inborn Error of Amino Acid Biosynthesis

    PubMed Central

    Spodenkiewicz, Marta; Diez-Fernandez, Carmen; Rüfenacht, Véronique; Gemperle-Britschgi, Corinne; Häberle, Johannes

    2016-01-01

    Glutamine synthetase (GS) is a cytosolic enzyme that produces glutamine, the most abundant free amino acid in the human body. Glutamine is a major substrate for various metabolic pathways, and is thus an important factor for the functioning of many organs; therefore, deficiency of glutamine due to a defect in GS is incompatible with normal life. Mutations in the human GLUL gene (encoding for GS) can cause an ultra-rare recessive inborn error of metabolism—congenital glutamine synthetase deficiency. This disease was reported until now in only three unrelated patients, all of whom suffered from neonatal onset severe epileptic encephalopathy. The hallmark of GS deficiency in these patients was decreased levels of glutamine in body fluids, associated with chronic hyperammonemia. This review aims at recapitulating the clinical history of the three known patients with congenital GS deficiency and summarizes the findings from studies done along with the work-up of these patients. It is the aim of this paper to convince the reader that (i) this disorder is possibly underdiagnosed, since decreased concentrations of metabolites do not receive the attention they deserve; and (ii) early detection of GS deficiency may help to improve the outcome of patients who could be treated early with metabolites that are lacking in this condition. PMID:27775558

  15. Minireview on Glutamine Synthetase Deficiency, an Ultra-Rare Inborn Error of Amino Acid Biosynthesis.

    PubMed

    Spodenkiewicz, Marta; Diez-Fernandez, Carmen; Rüfenacht, Véronique; Gemperle-Britschgi, Corinne; Häberle, Johannes

    2016-10-19

    Glutamine synthetase (GS) is a cytosolic enzyme that produces glutamine, the most abundant free amino acid in the human body. Glutamine is a major substrate for various metabolic pathways, and is thus an important factor for the functioning of many organs; therefore, deficiency of glutamine due to a defect in GS is incompatible with normal life. Mutations in the human GLUL gene (encoding for GS) can cause an ultra-rare recessive inborn error of metabolism-congenital glutamine synthetase deficiency. This disease was reported until now in only three unrelated patients, all of whom suffered from neonatal onset severe epileptic encephalopathy. The hallmark of GS deficiency in these patients was decreased levels of glutamine in body fluids, associated with chronic hyperammonemia. This review aims at recapitulating the clinical history of the three known patients with congenital GS deficiency and summarizes the findings from studies done along with the work-up of these patients. It is the aim of this paper to convince the reader that (i) this disorder is possibly underdiagnosed, since decreased concentrations of metabolites do not receive the attention they deserve; and (ii) early detection of GS deficiency may help to improve the outcome of patients who could be treated early with metabolites that are lacking in this condition.

  16. Elongator function in tRNA wobble uridine modification is conserved between yeast and plants

    PubMed Central

    Mehlgarten, Constance; Jablonowski, Daniel; Wrackmeyer, Uta; Tschitschmann, Susan; Sondermann, David; Jäger, Gunilla; Gong, Zhizhong; Byström, Anders S; Schaffrath, Raffael; Breunig, Karin D

    2010-01-01

    Based on studies in yeast and mammalian cells the Elongator complex has been implicated in functions as diverse as histone acetylation, polarized protein trafficking and tRNA modification. Here we show that Arabidopsis mutants lacking the Elongator subunit AtELP3/ELO3 have a defect in tRNA wobble uridine modification. Moreover, we demonstrate that yeast elp3 and elp1 mutants expressing the respective Arabidopsis Elongator homologues AtELP3/ELO3 and AtELP1/ELO2 assemble integer Elongator complexes indicating a high degree of structural conservation. Surprisingly, in vivo complementation studies based on Elongator-dependent tRNA nonsense suppression and zymocin tRNase toxin assays indicated that while AtELP1 rescued defects of a yeast elp1 mutant, the most conserved Elongator gene AtELP3, failed to complement an elp3 mutant. This lack of complementation is due to incompatibility with yeast ELP1 as coexpression of both plant genes in an elp1 elp3 yeast mutant restored Elongator's tRNA modification function in vivo. Similarly, AtELP1, not ScELP1 also supported partial complementation by yeast–plant Elp3 hybrids suggesting that AtElp1 has less stringent sequence requirements for Elp3 than ScElp1. We conclude that yeast and plant Elongator share tRNA modification roles and propose that this function might be conserved in Elongator from all eukaryotic kingdoms of life. PMID:20398216

  17. Determinants of tRNA Recognition by the Radical SAM Enzyme RlmN

    PubMed Central

    Fitzsimmons, Christina M.; Fujimori, Danica Galonić

    2016-01-01

    RlmN, a bacterial radical SAM methylating enzyme, has the unusual ability to modify two distinct types of RNA: 23S rRNA and tRNA. In rRNA, RlmN installs a methyl group at the C2 position of A2503 of 23S rRNA, while in tRNA the modification occurs at nucleotide A37, immediately adjacent to the anticodon triplet. Intriguingly, only a subset of tRNAs that contain an adenosine at position 37 are substrates for RlmN, suggesting that the enzyme carefully probes the highly conserved tRNA fold and sequence features to identify its targets. Over the past several years, multiple studies have addressed rRNA modification by RlmN, while relatively few investigations have focused on the ability of this enzyme to modify tRNAs. In this study, we utilized in vitro transcribed tRNAs as model substrates to interrogate RNA recognition by RlmN. Using chimeras and point mutations, we probed how the structure and sequence of RNA influences methylation, identifying position 38 of tRNAs as a critical determinant of substrate recognition. We further demonstrate that, analogous to previous mechanistic studies with fragments of 23S rRNA, tRNA methylation requirements are consistent with radical SAM reactivity. Together, our findings provide detailed insight into tRNA recognition by a radical SAM methylating enzyme. PMID:27902775

  18. Methylation of tRNA by normal mouse liver and Erhlich ascites cell extracts

    SciTech Connect

    Smith, S.D.; Sitz, T.O.

    1986-05-01

    When Erhlich ascites tumor and normal mouse liver extracts were incubated with (/sup 3/H-CH/sub 3/)-S-adenosylmethionine and E. coli tRNA, the tumor extracts had a ten-fold higher methyltransferase activity than mouse liver extracts. However, only a two-fold higher methyltransferase activity was observed when methyl deficient mouse liver tRNA was used as a substrate. DEAE-Sephadex chromatography of alkaline hydrolysates of methyl deficient mouse liver tRNA (tRNA isolated from mice treated with ethionine) showed 70% of the /sup 3/H-methyl groups in the mononucleotide peak and 30% in alkaline stable di-, tri- and oligonucleotide peaks demonstrating methylation of both the base and ribose moieties. Perchloric acid digest of material isolated from the di-, tri- and oligonucleotide peaks confirm that the /sup 3/H-methyl groups are on the ribose moieties. In hydrolysates of E. coli tRNA /sup 3/H-methyl groups appeared in only the mononucleotide peak, i.e. only base methylation. These data, from Erhlich ascites cells, suggest that ribose methyltransferases may have greater RNA substrate specificity than the base methyltransferases and may act processively to produce alkaline stable oligonucleotides.

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

    PubMed

    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.

  20. tRNA integrity is a prerequisite for rapid CCA addition: implication for quality control.

    PubMed

    Dupasquier, Marcel; Kim, Sangbumn; Halkidis, Konstantine; Gamper, Howard; Hou, Ya-Ming

    2008-06-06

    CCA addition to the 3' end is an essential step in tRNA maturation. High-resolution crystal structures of the CCA enzymes reveal primary enzyme contact with the tRNA minihelix domain, consisting of the acceptor stem and T stem-loop. RNA and DNA minihelices are efficient substrates for CCA addition in steady-state kinetics. However, in contrast to structural models and steady-state experiments, we show here by single-turnover kinetics that minihelices are insufficient substrates for the Escherichia coli CCA enzyme and that only the full-length tRNA is kinetically competent. Even a nick in the full-length tRNA backbone in the T loop, or as far away from the minihelix domain as in the anticodon loop, prevents efficient CCA addition. These results suggest a kinetic quality control provided by the CCA enzyme to inspect the integrity of the tRNA molecule and to discriminate against nicked or damaged species from further maturation.

  1. The CCA-adding enzyme: A central scrutinizer in tRNA quality control.

    PubMed

    Betat, Heike; Mörl, Mario

    2015-09-01

    tRNA nucleotidyltransferase adds the invariant CCA-terminus to the tRNA 3'-end, a central step in tRNA maturation. This CCA-adding enzyme is a specialized RNA polymerase that synthesizes the CCA sequence at high fidelity in all kingdoms of life. Recently, an additional function of this enzyme was identified, where it generates a specific degradation tag on structurally unstable tRNAs. This tag consists of an additional repeat of the CCA triplet, leading to a 3'-terminal CCACCA sequence. In order to explain how the enzyme catalyzes this extended polymerization reaction, Kuhn et al. solved a series of co-crystal structures of the CCA-adding enzyme from Archaeoglobus fulgidus in complex with different tRNA substrates. They show that the enzyme forces a bound unstable tRNA to refold the acceptor stem for a second round of CCA-addition, while stable transcripts are robust enough to resist this isomerization. In this review, we discuss how the CCA-adding enzyme uses a simple yet very elegant way to scrutinize its substrates for sufficient structural stability and, consequently, functionality.

  2. The tRNA recognition mechanism of the minimalist SPOUT methyltransferase, TrmL

    PubMed Central

    Liu, Ru-Juan; Zhou, Mi; Fang, Zhi-Peng; Wang, Meng; Zhou, Xiao-Long; Wang, En-Duo

    2013-01-01

    Unlike other transfer RNAs (tRNA)-modifying enzymes from the SPOUT methyltransferase superfamily, the tRNA (Um34/Cm34) methyltransferase TrmL lacks the usual extension domain for tRNA binding and consists only of a SPOUT domain. Both the catalytic and tRNA recognition mechanisms of this enzyme remain elusive. By using tRNAs purified from an Escherichia coli strain with the TrmL gene deleted, we found that TrmL can independently catalyze the methyl transfer from S-adenosyl-L-methionine to and isoacceptors without the involvement of other tRNA-binding proteins. We have solved the crystal structures of TrmL in apo form and in complex with S-adenosyl-homocysteine and identified the cofactor binding site and a possible active site. Methyltransferase activity and tRNA-binding affinity of TrmL mutants were measured to identify residues important for tRNA binding of TrmL. Our results suggest that TrmL functions as a homodimer by using the conserved C-terminal half of the SPOUT domain for catalysis, whereas residues from the less-conserved N-terminal half of the other subunit participate in tRNA recognition. PMID:23804755

  3. T box RNA decodes both the information content and geometry of tRNA to affect gene expression.

    PubMed

    Grigg, Jason C; Chen, Yujie; Grundy, Frank J; Henkin, Tina M; Pollack, Lois; Ke, Ailong

    2013-04-30

    The T box leader sequence is an RNA element that controls gene expression by binding directly to a specific tRNA and sensing its aminoacylation state. This interaction controls expression of amino acid-related genes in a negative feedback loop. The T box RNA structure is highly conserved, but its tRNA binding mechanism is only partially understood. Known sequence elements are the specifier sequence, which recognizes the tRNA anticodon, and the antiterminator bulge, which base pairs with the tRNA acceptor end. Here, we reveal the crucial function of the highly conserved stem I distal region in tRNA recognition and report its 2.65-Å crystal structure. The apex of this region contains an intricately woven loop-loop interaction between two conserved motifs, the Adenine-guanine (AG) bulge and the distal loop. This loop-loop structure presents a base triple on its surface that is optimally positioned for base-stacking interactions. Mutagenesis, cross-linking, and small-angle X-ray scattering data demonstrate that the apical base triple serves as a binding platform to dock the tRNA D- and T-loops. Strikingly, the binding platform strongly resembles the D- and T-loop binding elements from RNase P and the ribosome exit site, suggesting that this loop-loop structure may represent a widespread tRNA recognition platform. We propose a two-checkpoint molecular ruler model for tRNA decoding in which the information content of tRNA is first examined through specifier sequence-anticodon interaction, and the length of the tRNA anticodon arm is then measured by the distal loop-loop platform. When both conditions are met, tRNA is secured, and its aminoacylation state is sensed.

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

    PubMed

    Bernard, Stéphanie M; Habash, Dimah Z

    2009-01-01

    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.

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

    SciTech Connect

    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.

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

    PubMed

    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

    2016-04-01

    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.

  7. A mitochondrial tRNA aspartate mutation causing isolated mitochondrial myopathy.

    PubMed

    Seneca, Sara; Goemans, Nathalie; Van Coster, Rudy; Givron, Patrice; Reybrouck, Tony; Sciot, Raf; Meulemans, Ann; Smet, Joel; Van Hove, Johan L K

    2005-08-30

    Several mutations in mitochondrial transfer RNA (tRNA) genes can cause mitochondrial myopathy. We describe a young girl who presented with pronounced exercise intolerance. The anaerobic threshold and the maximal oxygen consumption were decreased. She had decreased complex I and IV enzyme activity and ragged red fibers on muscle biopsy. An A to G transition at nucleotide position 7526 in tRNA Aspartate (tRNA(Asp)) gene was heteroplasmic in several of the patient's tissues. We were unable to detect the mutation in muscle tissue from the patient's mother. This case adds a new genetic etiology for mitochondrial myopathy. It also illustrates for patients with combined deficiency of the complex I and IV enzyme activity the value of sequencing in the affected tissue muscle, and not only in blood, all mitochondrial tRNA genes including those not commonly affected, such as in this case mt tRNA(Asp).

  8. E. coli initiator tRNA analogs with different nucleotides in the discriminator base position.

    PubMed Central

    Uemura, H; Imai, M; Ohtsuka, E; Ikehara, M; Söll, D

    1982-01-01

    The effect of base changes at the fourth position from the 3'-terminus of Escherichia coli initiator tRNAMet has been studied to test the 'discriminator hypothesis' which proposed that the nucleotide in this position might have a role in the specificity of the aminoacylation reaction. E. coli initiator tRNA lacking the 3'-terminal tetranucleotide was prepared by partial digestion with S1 nuclease. To construct tRNA analogs with different bases in the fourth position this truncated tRNA was joined by RNA ligase to each of four chemically synthesized 2',3'-ethoxy-methylidene tetranucleotides pACCA(em), pCCCA(em), pGCCA(em), and pUCCA(em). In vitro aminoacylation studies showed that all four molecules accepted methionine, albeit with different Vmax values. Images PMID:6294608

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

    PubMed Central

    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

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

    PubMed Central

    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

  11. Rp-phosphorothioate modifications in RNase P RNA that interfere with tRNA binding.

    PubMed Central

    Hardt, W D; Warnecke, J M; Erdmann, V A; Hartmann, R K

    1995-01-01

    We have used Rp-phosphorothioate modifications and a binding interference assay to analyse the role of phosphate oxygens in tRNA recognition by Escherichia coli ribonuclease P (RNase P) RNA. Total (100%) Rp-phosphorothioate modification at A, C or G positions of RNase P RNA strongly impaired tRNA binding and pre-tRNA processing, while effects were less pronounced at U positions. Partially modified E. coli RNase P RNAs were separated into tRNA binding and non-binding fractions by gel retardation. Rp-phosphorothioate modifications that interfered with tRNA binding were found 5' of nucleotides A67, G68, U69, C70, C71, G72, A130, A132, A248, A249, G300, A317, A330, A352, C353 and C354. Manganese rescue at positions U69, C70, A130 and A132 identified, for the first time, sites of direct metal ion coordination in RNase P RNA. Most sites of interference are at strongly conserved nucleotides and nine reside within a long-range base-pairing interaction present in all known RNase P RNAs. In contrast to RNase P RNA, 100% Rp-phosphorothioate substitutions in tRNA showed only moderate effects on binding to RNase P RNAs from E. coli, Bacillus subtilis and Chromatium vinosum, suggesting that pro-Rp phosphate oxygens of mature tRNA contribute relatively little to the formation of the tRNA-RNase P RNA complex. Images PMID:7540978

  12. A voltage-gated pore for translocation of tRNA

    SciTech Connect

    Koley, Sandip; Adhya, Samit

    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.

  13. Clone and functional analysis of Seryl-tRNA synthetase and Tyrosyl-tRNA synthetase from silkworm, Bombyx mori

    PubMed Central

    Hu, Jingsheng; Tian, Jianghai; Li, Fanchi; Xue, Bin; Hu, Jiahuan; Cheng, Xiaoyu; Li, Jinxin; Shen, Weide; Li, Bing

    2017-01-01

    Aminoacyl-tRNA synthetases are the key enzymes for protein synthesis. Glycine, alanine, serine and tyrosine are the major amino acids composing fibroin of silkworm. Among them, the genes of alanyl-tRNA synthetase (AlaRS) and glycyl-tRNA synthetase (GlyRS) have been cloned. In this study, the seryl-tRNA synthetase (SerRS) and tyrosyl-tRNA synthetase (TyrRS) genes from silkworm were cloned. Their full length are 1709 bp and 1868 bp and contain open reading frame (ORF) of 1485 bp and 1575 bp, respectively. RT-PCR examination showed that the transcription levels of SerRS, TyrRS, AlaRS and GlyRS are significantly higher in silk gland than in other tissues. In addition, their transcription levels are much higher in middle and posterior silk gland than in anterior silk gland. Moreover, treatment of silkworms with phoxim, an inhibitor of silk protein synthesis, but not TiO2 NP, an enhancer of silk protein synthesis, significantly reduced the transcription levels of aaRS and content of free amino acids in posterior silk gland, therefore affecting silk protein synthesis, which may be the mechanism of phoxim-silking disorders. Furthermore, low concentration of TiO2 NPs showed no effect on the transcription of aaRS and content of free amino acids, suggesting that TiO2 NPs promotes silk protein synthesis possibly by increasing the activity of fibroin synthase in silkworm. PMID:28134300

  14. Kinetic abnormalities of carbamyl phosphate synthetase-I in a case of congenital hyperammonaemia.

    PubMed

    Qureshi, I A; Letarte, J; Ouellet, R; Lemieux, B; Cathelineau, L

    1986-01-01

    A sensitive direct colourimetric method has been employed to measure kinetic parameters and pH dependence of carbamyl phosphate synthetase-I, in a liver sample from a 2 1/2-month-old girl, who died from complications of a late-developing congenital hyperammonaemia. The residual activity of carbamyl phosphate synthetase-I was 25%, whereas other urea cycle enzymes were within normal range. Apparent Km for ammonium ion (0.73 mmol/L) was significantly increased (normal range 0.24-0.51). Km for bicarbonate ion was normal, while Km for NAG showed a slight variation from normal. The pH dependence curve of the patient's enzyme was flat, as compared to two controls showing pH optima at 7.8. Radial immunodiffusion (Mancini) of the abnormal enzyme against human enzyme antiserum gave a cross-reacting material of 10-20%. The methodological approach presented can be used to characterize abnormal enzymes in cases of partial deficiency with only 100-200 mg of liver tissue.

  15. Crystal structures of trypanosomal histidyl-tRNA synthetase illuminate differences between eukaryotic and prokaryotic homologs.

    PubMed

    Merritt, Ethan A; Arakaki, Tracy L; Gillespie, J Robert; Larson, Eric T; Kelley, Angela; Mueller, Natascha; Napuli, Alberto J; Kim, Jessica; Zhang, Li; Verlinde, Christophe L M J; Fan, Erkang; Zucker, Frank; Buckner, Frederick S; van Voorhis, Wesley C; Hol, Wim G J

    2010-03-26

    Crystal structures of histidyl-tRNA synthetase (HisRS) from the eukaryotic parasites Trypanosoma brucei and Trypanosoma cruzi provide a first structural view of a eukaryotic form of this enzyme and reveal differences from bacterial homologs. HisRSs in general contain an extra domain inserted between conserved motifs 2 and 3 of the Class II aminoacyl-tRNA synthetase catalytic core. The current structures show that the three-dimensional topology of this domain is very different in bacterial and archaeal/eukaryotic forms of the enzyme. Comparison of apo and histidine-bound trypanosomal structures indicates substantial active-site rearrangement upon histidine binding but relatively little subsequent rearrangement after reaction of histidine with ATP to form the enzyme's first reaction product, histidyladenylate. The specific residues involved in forming the binding pocket for the adenine moiety differ substantially both from the previously characterized binding site in bacterial structures and from the homologous residues in human HisRSs. The essentiality of the single HisRS gene in T. brucei is shown by a severe depression of parasite growth rate that results from even partial suppression of expression by RNA interference.

  16. Mutation and Selection on the Wobble Nucleotide in tRNA Anticodons in Marine Bivalve Mitochondrial Genomes

    PubMed Central

    Yu, Hong; Li, Qi

    2011-01-01

    Background Animal mitochondrial genomes typically encode one tRNA for each synonymous codon family, so that each tRNA anticodon essentially has to wobble to recognize two or four synonymous codons. Several factors have been hypothesized to determine the nucleotide at the wobble site of a tRNA anticodon in mitochondrial genomes, such as the codon-anticodon adaptation hypothesis, the wobble versatility hypothesis, the translation initiation and elongation conflict hypothesis, and the wobble cost hypothesis. Principal Findings In this study, we analyzed codon usage and tRNA anticodon wobble sites of 29 marine bivalve mitochondrial genomes to evaluate features of the wobble nucleotides in tRNA anticodons. The strand-specific mutation bias favors G and T on the H strand in all the 29 marine bivalve mitochondrial genomes. A bias favoring G and T is also visible in the third codon positions of protein-coding genes and the wobble sites of anticodons, rejecting that codon usage bias drives the wobble sites of tRNA anticodons or tRNA anticodon bias drives the evolution of codon usage. Almost all codon families (98.9%) from marine bivalve mitogenomes support the wobble versatility hypothesis. There are a few interesting exceptions involving tRNATrp with an anticodon CCA fixed in Pectinoida species, tRNASer with a GCU anticodon fixed in Mytiloida mitogenomes, and the uniform anticodon CAU of tRNAMet translating the AUR codon family. Conclusions/Significance These results demonstrate that most of the nucleotides at the wobble sites of tRNA anticodons in marine bivalve mitogenomes are determined by wobble versatility. Other factors such as the translation initiation and elongation conflict, and the cost of wobble translation may contribute to the determination of the wobble nucleotide in tRNA anticodons. The finding presented here provides valuable insights into the previous hypotheses of the wobble nucleotide in tRNA anticodons by adding some new evidence. PMID:21267462

  17. The Methyl Group of the N6-Methyl-N6-Threonylcarbamoyladenosine in tRNA of Escherichia coli Modestly Improves the Efficiency of the tRNA

    PubMed Central

    Qian, Qiang; Curran, James F.; Björk, Glenn R.

    1998-01-01

    tRNA species that read codons starting with adenosine (A) contain N6-threonylcarbamoyladenosine (t6A) derivatives adjacent to and 3′ of the anticodons from all organisms. In Escherichia coli there are 12 such tRNA species of which two (tRNAGGUThr1 and tRNAGGUThr3) have the t6A derivative N6-methyl-N6-threonylcarbamoyladenosine (m6t6A37). We have isolated a mutant of E. coli that lacks the m6t6A37 in these two tRNAGGUThr species. These tRNA species in the mutant are likely to have t6A37 instead of m6t6A37. We show that the methyl group of m6t6A37 originates from S-adenosyl-l-methionine and that the gene (tsaA) which most likely encodes tRNA(m6t6A37)methyltransferase is located at min 4.6 on the E. coli chromosomal map. The growth rate of the cell, the polypeptide chain elongation rate, and the selection of Thr-tRNAGGUThr to the ribosomal A site programmed with either of the cognate codons ACC and ACU were the same for the tsaA1 mutant as for the congenic wild-type strain. The expression of the threonine operon is regulated by an attenuator which contains in its leader mRNA seven ACC codons that are read by these two m6t6A37-containing tRNAGGUThr species. We show that the tsaA1 mutation resulted in a twofold derepression of this operon, suggesting that the lack of the methyl group of m6t6A37 in tRNAGGUThr slightly reduces the efficiency of this tRNA to read cognate codon ACC. PMID:9537379

  18. Post-transfer editing by a eukaryotic leucyl-tRNA synthetase resistant to the broad-spectrum drug AN2690.

    PubMed

    Zhou, Xiao-Long; Tan, Min; Wang, Meng; Chen, Xin; Wang, En-Duo

    2010-09-01

    Some aaRSs (aminoacyl-tRNA synthetases) develop editing mechanisms to correct mis-charged tRNA. The CP1 (connective peptide 1) domain of LeuRS (leucyl-tRNA synthetase) contains the editing active site, which is the proven target for the broad-spectrum drug AN2690 (5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole). The ESI (eukarya-specific insertion 1) in the CP1 domain of GlLeuRS (Giardia lamblia LeuRS) has been identified. Similar substitution with the ESI from HsLeuRS (Homo sapiens LeuRS) impeded the leucine activation, aminoacylation and post-transfer editing of the enzyme, but had no effect on the editing specificity toward non-specific amino acids. Thr341 in GlLeuRS served as a specificity discriminator, as found in other LeuRS systems, although its substitution with an alanine residue did not destroy Leu-tRNALeu synthesis in vitro and in vivo. The Arg338 was crucial for tRNALeu charging and the Asp440 was crucial for leucine activation and aminoacylation. The post-transfer editing required the CTD (C-terminal domain), Arg338 and Asp440 of GlLeuRS. Interestingly, GlLeuRS was completely resistant to the AN2690, which is an inhibitor of various LeuRSs. The universally conserved aspartate residue in the LeuRS CP1 domains was responsible for the resistance of GlLeuRS and another recently reported AN2690-resistant AaLeuRS (Aquifex aeolicus LeuRS). Our results indicate the functional divergence of some absolutely conserved sites, improve the understanding of the editing function of eukaryotic/archaeal LeuRSs and shed light on the development of a GlLeuRS-specific inhibitor for the treatment of giardiasis.

  19. Idiosyncrasy and identity in the prokaryotic phe-system: Crystal structure of E. coli phenylalanyl-tRNA synthetase complexed with phenylalanine and AMP

    PubMed Central

    Mermershtain, Inbal; Finarov, Igal; Klipcan, Liron; Kessler, Naama; Rozenberg, Haim; Safro, Mark G

    2011-01-01

    The crystal structure of Phenylalanyl-tRNA synthetase from E. coli (EcPheRS), a class II aminoacyl-tRNA synthetase, complexed with phenylalanine and AMP was determined at 3.05 Å resolution. EcPheRS is a (αβ)2 heterotetramer: the αβ heterodimer of EcPheRS consists of 11 structural domains. Three of them: the N-terminus, A1 and A2 belong to the α-subunit and B1-B8 domains to the β subunit. The structure of EcPheRS revealed that architecture of four helix-bundle interface, characteristic of class IIc heterotetrameric aaRSs, is changed: each of the two long helices belonging to CLM transformed into the coil-short helix structural fragments. The N-terminal domain of the α-subunit in EcPheRS forms compact triple helix domain. This observation is contradictory to the structure of the apo form of TtPheRS, where N-terminal domain was not detected in the electron density map. Comparison of EcPheRS structure with TtPheRS has uncovered significant rearrangements of the structural domains involved in tRNAPhe binding/translocation. As it follows from modeling experiments, to achieve a tighter fit with anticodon loop of tRNA, a shift of ∼5 Å is required for C-terminal domain B8, and of ∼6 to 7 Å for the whole N terminus. EcPheRSs have emerged as an important target for the incorporation of novel amino acids into genetic code. Further progress in design of novel compounds is anticipated based on the structural data of EcPheRS. PMID:21082706

  20. Inhibition of recombinant Pneumocystis carinii dihydropteroate synthetase by sulfa drugs.

    PubMed

    Hong, Y L; Hossler, P A; Calhoun, D H; Meshnick, S R

    1995-08-01

    Forty-four sulfa drugs were screened against crude preparations of recombinant Pneumocystis carinii dihydropteroate synthetase. The apparent Michaelis-Menten constants (Km) for p-aminobenzoic acid and 7,8-dihydro-6-hydroxymethylpterin pyrophosphate were 0.34 +/- 0.02 and 2.50 +/- 0.71 microM, respectively. Several sulfa drugs, including sulfathiazole, sulfachlorpyridazine, sulfamethoxypyridazine, and sulfathiourea, inhibited dihydropteroate synthetase approximately as well as sulfamethoxazole, as determined by the concentrations which cause 50% inhibition and/or by Ki. For all sulfones and sulfonamides tested, unsubstituted p-amino groups were necessary for activity, and sulfonamides containing an N1-heterocyclic substituent were found to be the most effective inhibitors. Folate biosynthesis in isolated intact P. carinii was approximately equally sensitive to inhibition by sulfamethoxazole, sulfachlorpyridazine, sulfamethoxypyridazine, sulfisoxazole, and sulfathiazole. Two of these drugs, sulfamethoxypyridazine and sulfisoxazole, are known to be less toxic than sulfamethoxazole and should be further evaluated for the treatment of P. carinii pneumonia.

  1. Aminoacyl-tRNA Synthetase Complexes in Evolution

    PubMed Central

    Havrylenko, Svitlana; Mirande, Marc

    2015-01-01

    Aminoacyl-tRNA synthetases are essential enzymes for interpreting the genetic code. They are responsible for the proper pairing of codons on mRNA with amino acids. In addition to this canonical, translational function, they are also involved in the control of many cellular pathways essential for the maintenance of cellular homeostasis. Association of several of these enzymes within supramolecular assemblies is a key feature of organization of the translation apparatus in eukaryotes. It could be a means to control their oscillation between translational functions, when associated within a multi-aminoacyl-tRNA synthetase complex (MARS), and nontranslational functions, after dissociation from the MARS and association with other partners. In this review, we summarize the composition of the different MARS described from archaea to mammals, the mode of assembly of these complexes, and their roles in maintenance of cellular homeostasis. PMID:25807264

  2. Biochemical and inhibition studies of glutamine synthetase from Leishmania donovani.

    PubMed

    Kumar, Vinay; Yadav, Shailendra; Soumya, Neelagiri; Kumar, Rohit; Babu, Neerupudi Kishore; Singh, Sushma

    2017-03-25

    Leishmaniasis is a group of tropical diseases caused by protozoan parasites of the genus Leishmania. Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis, a fatal disease if left untreated. Chemotherapy for leishmaniasis is problematic as the available drugs are toxic, costly and shows drug resistance, hence, there is a necessity to look out for the novel drug targets, chemical entities and vaccine. Glutamine synthetase (GS) catalyzes the synthesis of glutamine from glutamate and ammonia. In the present study, we have identified and characterized GS from L. donovani. The nucleotide sequence encoding putative glutamine synthetase like sequence from L. donovani (LdGS, LDBPK_060370) was cloned. A 43.5 kDa protein with 6X-His tag at the C-terminal end was obtained by overexpression of LdGS in Escherichia coli BL21 (DE3) strain. Expression of native LdGS in promastigotes and recombinant L. donovani glutamine synthetase (rLdGS) was confirmed by western blot analysis. An increase in expression of GS was observed at different phases of growth of the parasite. Expression of LdGS in promastigote and amastigote was confirmed by western blot analysis. Immunofluorescence studies of both the promastigote and amastigote stages of the parasite revealed the presence of LdGS in cytoplasm. GS exists as a single copy gene in parasite genome. Kinetic analysis of GS enzyme revealed Km value of 26.3 ± 0.4 mM for l- glutamate and Vmax value of 2.15 ± 0.07 U mg(-1). Present study confirms the presence of glutamine synthetase in L. donovani and provides comprehensive overview of LdGS for further validating it as a potential drug target.

  3. Mutational Analysis of Mitochondrial tRNA Genes in Patients with Lung Cancer

    PubMed Central

    He, ZF; Zheng, LC; Xie, DY; Yu, SS; Zhao, J

    2016-01-01

    Abstract Mutations in mitochondrial tRNA (mt-tRNA) genes have been found to be associated with various diseases including lung cancer. To understand the possible relationship between mtRNA mutations and lung cancer, we sequenced the 22 mt-tRNA genes from 200 lung cancer blood samples, as well as 100 healthy subjects. As a result, five mutations were identified including the tRNAAla T5655C, tRNAArg T10454C, tRNALeu(CUN) A12330G, tRNASer(UCN) T7505C and tRNAThr G15927A. These mutations were absent in the healthy subjects. These mutations and polymorphisms were localized at the highly conserved nucleotides of the corresponding mitochondrial tRNAs, which are critical for the tRNA steady state level and may result in failure in the tRNA metabolism. Moreover, through the application of the pathogenicity scoring system, we found that only the T10454C mutation should be classified as a “neutral polymorphism,” while the other mutations were regarded as “definitely pathogenic.” Taken together, our data indicate that tRNA genes are the hot-spots for pathogenic mutations associated with lung cancer. Our findings may provide valuable information for pathophysiology, management and genetic counseling of lung cancer. PMID:28289588

  4. Evolution of a tRNA operon in gamma purple bacteria.

    PubMed Central

    Giroux, S; Cedergren, R

    1989-01-01

    Genomic DNA from eubacteria belonging to the gamma-3 subdivision of purple bacteria, as classified by Woese (C.R. Woese, Microbiol. Rev. 51:221-271, 1987), were probed with the argT operon of Escherichia coli encoding 5'-tRNA(Arg)-tRNA(His)-tRNA(Leu)-tRNA(Pro)-3'. The homologous operon from Vibrio harveyi was isolated and sequenced. Comparison of the five available sequences of this tRNA cluster from members of the families Enterobacteriaceae, Aeromonadaceae, and Vibrionaceae led to the conclusion that variations in different versions of this operon arose not only by point mutations but also by duplication and addition-deletion of entire tRNA genes. This data base permitted the formulation of a proposal dealing with the evolutionary history of this operon and suggested that DNA regions containing tRNA genes are active centers (hot spots) of recombination. Finally, since the operon from V. harveyi was not highly repetitive and did not contain tRNA pseudogenes, as in the Photobacterium phosphoreum operon, hybridization of genomic DNAs from different photobacterial strains with probes specific for the repeated pseudogene element was performed. We conclude that the phylogenetic distribution of the repetitive DNA is restricted to strains of P. phosphoreum. Images PMID:2687235

  5. An evaluation of mitochondrial tRNA gene evolution and its relation to the genetic code.

    PubMed

    Cedergren, R J

    1982-04-01

    Extensive sequence data on mitochondrial (mt) tRNAs give for the first time an opportunity to evaluate tRNA gene evolution in this organelle. Deductions from these gene structures relate to the evolution of tRNA genes in other cellular systems and to the origin of the genetic code. Mt tRNAs, in contrast to the prokaryotic nature of chloroplastic tRNA structure, can not at the present time be definitely related to either prokaryotic or eukaryotic tRNAs, probably because of a higher mutation rate in mitochondria. Fungal mt tRNAs having the same anticodon and function are generally similar enough to be considered homologous. Comparisons af all mt tRNA sequences contained in the same mitochondrion indicate that some tRNAs originated by duplication of a prototypic gene which, after divergence, led to tRNAs having different amino acid specificities. The deviant mt genetic code, although admittedly permitting a simpler decoding mechanism, is not useful in determining whether the origin of mitochondria had preceded or was derived from prokaryotes or eukaryotes, since the genetic code is variable even among mitochondria. Variants of the mt genetic code lead to speculation on the nature of the primordial code and its relation to the present "universal" code.

  6. A correlation between N2-dimethylguanosine presence and alternate tRNA conformers.

    PubMed

    Steinberg, S; Cedergren, R

    1995-11-01

    Even though the evolutionary conservation of the cloverleaf model is strongly suggestive of powerful constraints on the secondary structure of functional tRNAs, some mitochondrial tRNAs cannot be folded into this form. From the optimal base pairing pattern of these recalcitrant tRNAs, structural correlations between the length of the anticodon stem and the lengths of connector regions between the two helical domains, formed by the coaxial stacking of the anticodon and D-stems and the acceptor and T-stems, have been derived and used to scan the tRNA and tRNA gene database. We show here that some cytosolic tRNA gene sequences that are compatible with the cloverleaf model can also be folded into patterns proposed for the unusual mitochondrial tRNAs. Furthermore, the ability to be folded into these atypical structures correlates in the mature RNA sequences with the presence of dimethylguanosine, whose role may be to prevent the unusual mitochondrial tRNA pattern folding.

  7. Biochemical and Structures Studies of tRNA Modificaton and Repair Enzymes

    ERIC Educational Resources Information Center

    Zhou, Chun

    2009-01-01

    RNA hypermodifications near the anticodon of tRNA are fundamental for the efficiency and fidelity of protein synthesis. Dimethylallyltransferase (DMATase) catalyzes transfer of a dimethylallyl moiety from dimethylallyl pyrophosphate to N6 of A37 in certain tRNAs. We first determined the crystal structures of "Pseudomonas aeruginosa" DMATase.…

  8. A yeast glutamine tRNA signals nitrogen status for regulation of dimorphic growth and sporulation

    PubMed Central

    Murray, L. E.; Rowley, N.; Dawes, I. W.; Johnston, G. C.; Singer, R. A.

    1998-01-01

    Dimorphic growth of the budding yeast Saccharomyces cerevisiae is regulated by the quality of the nitrogen supply. On a preferred nitrogen source diploid cells grow as ellipsoidal cells by using a bipolar pattern of budding, whereas on a poor nitrogen source a unipolar pattern of budding is adopted, resulting in extended pseudohyphal chains of filamentous cells. Here we report that the quality of the nitrogen source is signaled by the glutamine tRNA isoform with a 5′-CUG anticodon (tRNACUG). Mutations that alter this tRNA impair assessment of the nitrogen supply without measurably affecting protein synthesis, so that mutant cells display pseudohyphal growth even on a preferred nitrogen source. The nitrogen status for other nitrogen-responsive processes such as catabolic gene expression and sporulation also is signaled by this tRNA: mutant cells inappropriately induce the nitrogen-repressed gene CAR1 and undergo precocious sporulation in nitrogen-rich media. Therefore, in addition to its role in mRNA translation, this tRNA also transduces nitrogen signals that regulate development. PMID:9671727

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

    PubMed Central

    Adir, Idan; Dahan, Orna; Broday, Limor; Pilpel, Yitzhak; Rechavi, Oded

    2016-01-01

    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

  10. N6-methyladenosine in mRNA disrupts tRNA selection and translation elongation dynamics

    PubMed Central

    Choi, Junhong; Ieong, Ka-Weng; Demirci, Hasan; Chen, Jin; Petrov, Alexey; Prabhakar, Arjun; O'Leary, Seán E.; Dominissini, Dan; Rechavi, Gideon; Soltis, S. Michael; Ehrenberg, Måns

    2016-01-01

    N6-methylation of adenosine (m6A) is the most abundant post-transcriptional modification within the coding region of mRNA, but its role during translation remains unknown. Here, we used bulk kinetic and single-molecule methods to probe the effect of m6A in mRNA decoding. Although m6A base pairs with uridine during decoding as shown by x-ray crystallographic analyses of Thermus thermophilus ribosomal complexes, our measurements employing an Escherichia coli translation system revealed that m6A modification of mRNA can act as a barrier to tRNA accommodation and translation elongation. The interaction between an m6A-modified codon and cognate tRNA echoes the interaction between a near-cognate codon and tRNA, as delay in tRNA accommodation depends on the position and context of m6A within codons and on the accuracy level of translation. Overall, our results demonstrate that chemical modification of mRNA can change translational dynamics. PMID:26751643

  11. Site-selective hydrolysis of tRNA by lanthanide metal complexes

    SciTech Connect

    Hayashi, Nobuhiro ); Takeda, Naoya; Yashiro, Morio; Watanabe, Kimitsuna; Komiyama, Makoto ); Shiiba, Tetsuro )

    1993-12-22

    tRNA[sup Phe] is site-selectively hydrolyzed by lanthanide metal complexes (Ce(III), Eu(III), La(III)) of hexaimine macrocyclic ligands. The selectivities of the complexes are much higher than those of the metal ions and are strongly dependent on the ligand structure. The tertiary structure of tRNA is essential for the site-selective scission.

  12. Identification of the glutamine synthetase adenylyltransferase of Azospirillum brasilense.

    PubMed

    Van Dommelen, Anne; Spaepen, Stijn; Vanderleyden, Jozef

    2009-04-01

    Glutamine synthetase, a key enzyme in nitrogen metabolism of both prokaryotes and eukaryotes, is strictly regulated. One means of regulation is the modulation of activity through adenylylation catalyzed by adenylyltransferases. Using PCR primers based on conserved sequences in glutamine synthetase adenylyltransferases, we amplified part of the glnE gene of Azospirillum brasilense Sp7. The complete glnE sequence of A. brasilense Sp245 was retrieved from the draft genome sequence of this organism (http://genomics.ornl.gov/research/azo/). Adenylyltransferase is a bifunctional enzyme consisting of an N-terminal domain responsible for deadenylylation activity and a C-terminal domain responsible for adenylylation activity. Both domains are partially homologous to each other. Residues important for catalytic activity were present in the deduced amino acid sequence of the A. brasilense Sp245 glnE sequence. A glnE mutant was constructed in A. brasilense Sp7 by inserting a kanamycin resistance cassette between the two active domains of the enzyme. The resulting mutant was unable to adenylylate the glutamine synthetase enzyme and was impaired in growth when shifted from nitrogen-poor to nitrogen-rich medium.

  13. Expression of glutamine synthetase in balloon cells: a basis of their antiepileptic role?

    PubMed

    Buccoliero, Anna Maria; Barba, Carmen; Giordano, Flavio; Baroni, Gianna; Genitori, Lorenzo; Guerrini, Renzo; Taddei, Gian Luigi

    2015-01-01

    Glutamine synthetase is an enzyme involved in the clearance of glutamate, the most potent excitatory neurotransmitter. We studied the immunohistochemical expression of glutamine synthetase in neocortical samples from 5 children who underwent surgery for pharmacoresistant epilepsy and a histological diagnosis of focal cortical dysplasia IIb. In all cases, balloon cells, but not dysmorphic neurons, were immunopositive for glutamine synthetase. This finding suggests that balloon cells can be involved in the neutralization of glutamate and play a protective anti-seizure role.

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

    PubMed Central

    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 E cleavage site, the AU-rich sequence element (AUE), in the 3′ trailer. This suggests that RNase E cleavage and exonucleolytic trimming is a general pathway for tRNA maturation in this organism. Remarkably, the AUE immediately downstream of each tRNA is selectively conserved in bacteria having RNase E and tRNA-specific exoribonucleases, suggesting that this pathway for tRNA processing is also commonly used in these bacteria. Two types of RNase E-like proteins are identified in actinobacteria and the α-subdivision of proteobacteria. The tRNA 3′ proximal AUE is conserved in bacteria with only one type of E-like protein. Selective conservation of the AUE is usually not observed in bacteria without RNase E. These results demonstrate a novel example of co-evolution of RNA sequences with processing activities. PMID:15811923

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

    SciTech Connect

    Ogawa, Tetsuhiro Shimizu, Ayano; Takahashi, Kazutoshi; Hidaka, Makoto; Masaki, Haruhiko

    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.

  16. Yeast tRNA3Leu gene transcribed and spliced in a HeLa cell extract.

    PubMed Central

    Standring, D N; Venegas, A; Rutter, W J

    1981-01-01

    A cloned yeast tRNA3Leu gene containing a 33-base intervening sequence (IVS) is selectively transcribed by a soluble extract from HeLa cells. The 130-nucleotide tRNA3Leu precursor RNA formed is colinear with the gene and contains approximately 4 leader nucleotides and up to 9 trailer nucleotides. The IVS is accurately and efficiently removed by an endogenous HeLa excision-ligase activity to yield the spliced tRNA, the free IVS, and the half-tRNA intermediates. The splicing reaction occurs without prior 5' and 3' maturation of the precursor but, with this exception, this pattern of synthesis and subsequent maturation of the tRNA3Leu precursor conforms to the scheme for tRNA biosynthesis deduced for the xenopus system. Indeed, the two systems utilize similar or identical tRNA3Leu precursors. Our results stress the extraordinary conservation of tRNA biosynthesis in eukaryotes and demonstrate that a HeLa extract provides a useful system for investigating this process. Images PMID:6796956

  17. Structural rules and conformational compensations in the tRNA L-form.

    PubMed

    Steinberg, S; Leclerc, F; Cedergren, R

    1997-02-21

    The mitochondrial tRNAs (mtRNA) of five distinct, secondary structure types have been identified in the tRNA sequence compilation, and the three-dimensional modeling for representative sequences of these types has been carried out using a new criterion for the lengths of the helical domains and connector regions in a full-sized tRNA conformation. This criterion has been derived from the analysis of the known structures of cytosolic tRNAs and states that in the tRNA structure nucleotide 59 of the T-loop should stack onto Domain 1. To ensure this, Domain 1 must have 12 layers of stacked nucleotides, and in the case of a deletion of a base-pair in the T-stem, an additional 13th layer is required. Although a number of mitochondrial tRNAs harbored deficiencies in this criterion and, therefore, could not be modeled directly, this disability could be corrected and modeling accomplished by invoking structural compensations derived from one of two unusual aspects of these tRNAs. One class of these tRNAs contained an unpaired nucleotide in their anticodon stem, and their three-dimensional structure was successfully modeled when the unpaired nucleotide was intercalated into the helical domain of the stem. The second class contained more than the required number of nucleotides connecting the tRNA helical domains; the conformational flexibility of these nucleotides allowed them to take the place of the absent layers. The conformational compensation that we report rationalizes disparate features of these tRNAs and suggests that the stacking of nucleotide 59 on Domain 1 is an essential feature of the three-dimensional L-form of tRNA.

  18. Binding of tRNA nucleotidyltransferase to Affi-Gel Blue: rapid purification of the enzyme and binding studies.

    PubMed

    Deutscher, M P; Masiakowski, P

    1978-06-01

    Rabbit liver tRNA nucleotidyldransferase bound to columns of Affi-Gel Blue and could be specifically eluted with tRNA. This observation led to development of a rapid purification procedure for the enzyme. The adsorbent was also used to assess interaction of tRNA nucleotidyltransferase with various polynucleotides and substrates. The enzyme could be efficiently desorbed from Affi-Gel Blue by low concentrations of tRNA-C-C, less well by tRNA-C-C-A, and not at all by poly(A), poly(C), ATP or CTP.

  19. Adenovirus vector-based incorporation of a photo-cross-linkable amino acid into proteins in human primary cells and cancerous cell lines

    PubMed Central

    Kita, Ayami; Hino, Nobumasa; Higashi, Sakiko; Hirota, Kohji; Narumi, Ryohei; Adachi, Jun; Takafuji, Kazuaki; Ishimoto, Kenji; Okada, Yoshiaki; Sakamoto, Kensaku; Tomonaga, Takeshi; Takashima, Seiji; Mizuguchi, Hiroyuki; Doi, Takefumi

    2016-01-01

    The site-specific incorporation of cross-linkable designer amino acids into proteins is useful for covalently bonding protein complexes upon exposure to light. This technology can be used to study networks of protein-protein interactions in living cells; however, to date it has only been applicable for use with a narrow range of cell types, due to the limited availability of plasmid-based transfection protocols. In the present study, we achieved adenovirus-based expression of a variant of an archaeal pyrrolysyl-tRNA synthetase and UAG-recognising tRNA pair, which was used to incorporate unnatural amino acids into proteins at sites defined by in-frame UAG codons within genes. As such, the site-specific photo-cross-linking method is now applicable to a wide variety of mammalian cells. In addition, we repositioned the reactive substituent of a useful photo-cross-linker, Nε-(para-trifluoromethyl-diazirinyl-benzyloxycarbonyl)-l-lysine (pTmdZLys), to the meta position, which improved its availability at low concentration. Finally, we successfully applied this system to analyse the formation of a protein complex in response to a growth signal in human cancerous cells and human umbilical vein endothelial cells. This adenovirus-based system, together with the newly designed cross-linkable amino acid, will facilitate studies on molecular interactions in various cell lines of medical interest. PMID:27833131

  20. Mutation of the mitochondrial tyrosyl-tRNA synthetase gene, YARS2, causes myopathy, lactic acidosis, and sideroblastic anemia--MLASA syndrome.

    PubMed

    Riley, Lisa G; Cooper, Sandra; Hickey, Peter; Rudinger-Thirion, Joëlle; McKenzie, Matthew; Compton, Alison; Lim, Sze Chern; Thorburn, David; Ryan, Michael T; Giegé, Richard; Bahlo, Melanie; Christodoulou, John

    2010-07-09

    Mitochondrial respiratory chain disorders are a heterogeneous group of disorders in which the underlying genetic defect is often unknown. We have identified a pathogenic mutation (c.156C>G [p.F52L]) in YARS2, located at chromosome 12p11.21, by using genome-wide SNP-based homozygosity analysis of a family with affected members displaying myopathy, lactic acidosis, and sideroblastic anemia (MLASA). We subsequently identified the same mutation in another unrelated MLASA patient. The YARS2 gene product, mitochondrial tyrosyl-tRNA synthetase (YARS2), was present at lower levels in skeletal muscle whereas fibroblasts were relatively normal. Complex I, III, and IV were dysfunctional as indicated by enzyme analysis, immunoblotting, and immunohistochemistry. A mitochondrial protein-synthesis assay showed reduced levels of respiratory chain subunits in myotubes generated from patient cell lines. A tRNA aminoacylation assay revealed that mutant YARS2 was still active; however, enzyme kinetics were abnormal compared to the wild-type protein. We propose that the reduced aminoacylation activity of mutant YARS2 enzyme leads to decreased mitochondrial protein synthesis, resulting in mitochondrial respiratory chain dysfunction. MLASA has previously been associated with PUS1 mutations; hence, the YARS2 mutation reported here is an alternative cause of MLASA.

  1. Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of NAD synthetase from methicillin-resistant Staphylococcus aureus.

    PubMed

    Arbade, Gajanan Kashinathrao; Srivastava, Sandeep Kumar

    2015-06-01

    Staphylococcus aureus is an important human and animal pathogen that causes a wide range of infections. The prevalence of multidrug-resistant S. aureus strains in both hospital and community settings makes it imperative to characterize new drug targets to combat S. aureus infections. In this context, enzymes involved in NAD metabolism and synthesis are significant drug targets as NAD is a central player in several cellular processes. NAD synthetase catalyzes the last step in the biosynthesis of nicotinamide adenine dinucleotide, making it a crucial intermediate enzyme linked to the biosynthesis of several amino acids, purine and pyrimidine nucleotides, coenzymes and antibiotics.

  2. The m1A(58) modification in eubacterial tRNA: An overview of tRNA recognition and mechanism of catalysis by TrmI.

    PubMed

    Dégut, Clément; Ponchon, Luc; Folly-Klan, Marcia; Barraud, Pierre; Tisné, Carine

    2016-03-01

    The enzymes of the TrmI family catalyze the formation of the m(1)A58 modification in tRNA. We previously solved the crystal structure of the Thermus thermophilus enzyme and conducted a biophysical study to characterize the interaction between TrmI and tRNA. TrmI enzymes are active as a tetramer and up to two tRNAs can bind to TrmI simultaneously. In this paper, we present the structures of two TrmI mutants (D170A and Y78A). These residues are conserved in the active site of TrmIs and their mutations result in a dramatic alteration of TrmI activity. Both structures of TrmI mutants revealed the flexibility of the N-terminal domain that is probably important to bind tRNA. The structure of TrmI Y78A catalytic domain is unmodified regarding the binding of the SAM co-factor and the conformation of residues potentially interacting with the substrate adenine. This structure reinforces the previously proposed role of Y78, i.e. stabilize the conformation of the A58 ribose needed to hold the adenosine in the active site. The structure of the D170A mutant shows a flexible active site with one loop occupying in part the place of the co-factor and the second loop moving at the entrance to the active site. This structure and recent data confirms the central role of D170 residue binding the amino moiety of SAM and the exocyclic amino group of adenine. Possible mechanisms for methyl transfer are then discussed.

  3. Enhanced dynamics of hydrated tRNA on nanodiamond surfaces: A combined neutron scattering and MD simulation study

    DOE PAGES

    Dhindsa, Gurpreet K.; Bhowmik, Debsindhu; Goswami, Monojoy; ...

    2016-09-01

    Nontoxic, biocompatible nanodiamonds (ND) have recently been implemented in rational, systematic design of optimal therapeutic use in nanomedicines. However, hydrophilicity of the ND surface strongly influences structure and dynamics of biomolecules that restrict in situ applications of ND. Therefore, fundamental understanding of the impact of hydrophilic ND surface on biomolecules at the molecular level is essential. For tRNA, we observe an enhancement of dynamical behavior in the presence of ND contrary to generally observed slow motion at strongly interacting interfaces. We took advantage of neutron scattering experiments and computer simulations to demonstrate this atypical faster dynamics of tRNA on NDmore » surface. The strong attractive interactions between ND, tRNA, and water give rise to unlike dynamical behavior and structural changes of tRNA in front of ND compared to without ND. As a result, our new findings may provide new design principles for safer, improved drug delivery platforms.« less

  4. Enhanced dynamics of hydrated tRNA on nanodiamond surfaces: A combined neutron scattering and MD simulation study

    SciTech Connect

    Dhindsa, Gurpreet K.; Bhowmik, Debsindhu; Goswami, Monojoy; O’Neill, Hugh; Mamontov, Eugene; Sumpter, Bobby G.; Hong, Liang; Ganesh, Panchapakesan; Chu, Xiang -qiang

    2016-09-01

    Nontoxic, biocompatible nanodiamonds (ND) have recently been implemented in rational, systematic design of optimal therapeutic use in nanomedicines. However, hydrophilicity of the ND surface strongly influences structure and dynamics of biomolecules that restrict in situ applications of ND. Therefore, fundamental understanding of the impact of hydrophilic ND surface on biomolecules at the molecular level is essential. For tRNA, we observe an enhancement of dynamical behavior in the presence of ND contrary to generally observed slow motion at strongly interacting interfaces. We took advantage of neutron scattering experiments and computer simulations to demonstrate this atypical faster dynamics of tRNA on ND surface. The strong attractive interactions between ND, tRNA, and water give rise to unlike dynamical behavior and structural changes of tRNA in front of ND compared to without ND. As a result, our new findings may provide new design principles for safer, improved drug delivery platforms.

  5. Empower multiplex cell and tissue-specific CRISPR-mediated gene manipulation with self-cleaving ribozymes and tRNA.

    PubMed

    Xu, Li; Zhao, Lixia; Gao, Yandi; Xu, Jing; Han, Renzhi

    2016-10-30

    Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system has emerged in recent years as a highly efficient RNA-guided gene manipulation platform. Simultaneous editing or transcriptional activation/suppression of different genes becomes feasible with the co-delivery of multiple guide RNAs (gRNAs). Here, we report that multiple gRNAs linked with self-cleaving ribozymes and/or tRNA could be simultaneously expressed from a single U6 promoter to exert genome editing of dystrophin and myosin binding protein C3 in human and mouse cells. Moreover, this strategy allows the expression of multiple gRNAs for synergistic transcription activation of follistatin when used with catalytically inactive dCas9-VP64 or dCas9-p300(core) fusions. Finally, the gRNAs linked by the self-cleaving ribozymes and tRNA could be expressed from RNA polymerase type II (pol II) promoters such as generic CMV and muscle/heart-specific MHCK7. This is particularly useful for in vivo applications when the packaging capacity of recombinant adeno-associated virus is limited while tissue-specific delivery of gRNAs and Cas9 is desired. Taken together, this study provides a novel strategy to enable tissue-specific expression of more than one gRNAs for multiplex gene editing from a single pol II promoter.

  6. 2'-5'-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1.

    PubMed

    Dhar, Jayeeta; Cuevas, Rolando A; Goswami, Ramansu; Zhu, Jianzhong; Sarkar, Saumendra N; Barik, Sailen

    2015-10-01

    2'-5'-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth.

  7. Binding of DNA-binding alkaloids berberine and palmatine to tRNA and comparison to ethidium: Spectroscopic and molecular modeling studies

    NASA Astrophysics Data System (ADS)

    Islam, Md. Maidul; Pandya, Prateek; Chowdhury, Sebanti Roy; Kumar, Surat; Kumar, Gopinatha Suresh

    2008-11-01

    The interaction of two natural protoberberine plant alkaloids berberine and palmatine with tRNA phe was studied using various biophysical techniques and molecular modeling and the data were compared with the binding of the classical DNA intercalator, ethidium. Circular dichroic studies revealed that the tRNA conformation was moderately perturbed on binding of the alkaloids. The cooperative binding of both the alkaloids and ethidium to tRNA was revealed from absorbance and fluorescence studies. Fluorescence quenching studies advanced a conclusion that while berberine and palmatine are partially intercalated, ethidium is fully intercalated on the tRNA molecule. The binding of the alkaloids as well as ethidium stabilized the tRNA melting, and the binding constant evaluated from the averaged optical melting temperature data was in agreement with fluorescence spectral-binding data. Differential scanning calorimetry revealed that the tRNA melting showed three close transitions that were affected on binding of these small molecules. Molecular docking calculations performed showed the preferred regions of binding of these small molecules on the tRNA. Taken together, the results suggest that the binding of the alkaloids berberine and palmatine on the tRNA structure appears to be mostly by partial intercalation while ethidium intercalates fully on the tRNA. These results further advance our knowledge on the molecular aspects on the interaction of these alkaloids to tRNA.

  8. Mitochondrial 16S rRNA Is Methylated by tRNA Methyltransferase TRMT61B in All Vertebrates

    PubMed Central

    Bar-Yaacov, Dan; Frumkin, Idan; Yashiro, Yuka; Schlesinger, Orr; Bieri, Philipp; Greber, Basil; Ban, Nenad; Zarivach, Raz; Alfonta, Lital; Pilpel, Yitzhak; Suzuki, Tsutomu; Mishmar, Dan

    2016-01-01

    The mitochondrial ribosome, which translates all mitochondrial DNA (mtDNA)-encoded proteins, should be tightly regulated pre- and post-transcriptionally. Recently, we found RNA-DNA differences (RDDs) at human mitochondrial 16S (large) rRNA position 947 that were indicative of post-transcriptional modification. Here, we show that these 16S rRNA RDDs result from a 1-methyladenosine (m1A) modification introduced by TRMT61B, thus being the first vertebrate methyltransferase that modifies both tRNA and rRNAs. m1A947 is conserved in humans and all vertebrates having adenine at the corresponding mtDNA position (90% of vertebrates). However, this mtDNA base is a thymine in 10% of the vertebrates and a guanine in the 23S rRNA of 95% of bacteria, suggesting alternative evolutionary solutions. m1A, uridine, or guanine may stabilize the local structure of mitochondrial and bacterial ribosomes. Experimental assessment of genome-edited Escherichia coli showed that unmodified adenine caused impaired protein synthesis and growth. Our findings revealed a conserved mechanism of rRNA modification that has been selected instead of DNA mutations to enable proper mitochondrial ribosome function. PMID:27631568

  9. Structural analysis of FAD synthetase from Corynebacterium ammoniagenes

    PubMed Central

    Frago, Susana; Martínez-Júlvez, Marta; Serrano, Ana; Medina, Milagros

    2008-01-01

    Background The prokaryotic FAD synthetase family – a group of bifunctional enzymes that catalyse riboflavin phosphorylation and FMN adenylylation within a single polypeptide chain- was analysed in terms of sequence and structure. Results Sequences of nearly 800 prokaryotic species were aligned. Those related with bifunctional FAD synthetase activities showed conservation of several consensus regions and highly conserved residues. A 3D model for the FAD synthetase from Corynebacterium ammoniagenes (CaFADS) was generated. This model confirms that the N-terminal and C-terminal domains are related to nucleotydyltransferases and riboflavin kinases, respectively. Models for the interaction of CaFADS with its substrates were also produced, allowing location of all the protein substrates in their putative binding pockets. These include two independent flavin binding sites for each CaFADS activity. Conclusion For the first time, the putative presence of a flavin binding site for the adenylylation activity, independent from that related with the phosphorylation activity, is shown. Additionally, these models suggest the functional relevance of some residues putatively involved in the catalytic processes. Their relevant roles were analysed by site-directed mutagenesis. A role was confirmed for H28, H31, S164 and T165 in the stabilisation of the P groups and the adenine moiety of ATP and, the P of FMN for the adenylylation. Similarly, T208, N210 and E268 appear critical for accommodation of the P groups of ATP and the ribityl end of RF in the active site for the phosphorylation process. Finally, the C-terminal domain was shown to catalyse the phosphorylation process on its own, but no reaction at all was observed with the individually expressed N-terminal domain. PMID:18811972

  10. Inactivation and covalent modification of CTP synthetase by thiourea dioxide.

    PubMed Central

    Robertson, J. G.; Sparvero, L. J.; Villafranca, J. J.

    1992-01-01

    Thiourea dioxide was used in chemical modification studies to identify functionally important amino acids in Escherichia coli CTP synthetase. Incubation at pH 8.0 in the absence of substrates led to rapid, time dependent, and irreversible inactivation of the enzyme. The second-order rate constant for inactivation was 0.18 M-1 s-1. Inactivation also occurred in the absence of oxygen and in the presence of catalase, thereby ruling out mixed-function oxidation/reduction as the mode of amino acid modification. Saturating concentrations of the substrates ATP and UTP, and the allosteric activator GTP prevented inactivation by thiourea dioxide, whereas saturating concentrations of glutamine (a substrate) did not. The concentration dependence of nucleotide protection revealed cooperative behavior with respect to individual nucleotides and with respect to various combinations of nucleotides. Mixtures of nucleotides afforded greater protection against inactivation than single nucleotides alone, and a combination of the substrates ATP and UTP provided the most protection. The Hill coefficient for nucleotide protection was approximately 2 for ATP, UTP, and GTP. In the presence of 1:1 ratios of ATP:UTP, ATP:GTP, and UTP:GTP, the Hill coefficient was approximately 4 in each case. Fluorescence and circular dichroism measurements indicated that modification by thiourea dioxide causes detectable changes in the structure of the protein. Modification with [14C]thiourea dioxide demonstrated that complete inactivation correlates with incorporation of 3 mol of [14C]thiourea dioxide per mole of CTP synthetase monomer. The specificity of thiourea dioxide for lysine residues indicates that one or more lysines are most likely involved in CTP synthetase activity. The data further indicate that nucleotide binding prevents access to these functionally important residues. PMID:1303749

  11. Protein Synthesis in E. coli: Dependence of Codon-Specific Elongation on tRNA Concentration and Codon Usage.

    PubMed

    Rudorf, Sophia; Lipowsky, Reinhard

    2015-01-01

    To synthesize a protein, a ribosome moves along a messenger RNA (mRNA), reads it codon by codon, and takes up the corresponding ternary complexes which consist of aminoacylated transfer RNAs (aa-tRNAs), elongation factor Tu (EF-Tu), and GTP. During this process of translation elongation, the ribosome proceeds with a codon-specific rate. Here, we present a general theoretical framework to calculate codon-specific elongation rates and error frequencies based on tRNA concentrations and codon usages. Our theory takes three important aspects of in-vivo translation elongation into account. First, non-cognate, near-cognate and cognate ternary complexes compete for the binding sites on the ribosomes. Second, the corresponding binding rates are determined by the concentrations of free ternary complexes, which must be distinguished from the total tRNA concentrations as measured in vivo. Third, for each tRNA species, the difference between total tRNA and ternary complex concentration depends on the codon usages of the corresponding cognate and near-cognate codons. Furthermore, we apply our theory to two alternative pathways for tRNA release from the ribosomal E site and show how the mechanism of tRNA release influences the concentrations of free ternary complexes and thus the codon-specific elongation rates. Using a recently introduced method to determine kinetic rates of in-vivo translation from in-vitro data, we compute elongation rates for all codons in Escherichia coli. We show that for some tRNA species only a few tRNA molecules are part of ternary complexes and, thus, available for the translating ribosomes. In addition, we find that codon-specific elongation rates strongly depend on the overall codon usage in the cell, which could be altered experimentally by overexpression of individual genes.

  12. Stability of Rat Brain Glutamine Synthetase to Oxygen Toxicity (Oxygen at High Pressure).

    DTIC Science & Technology

    1983-07-01

    Enzyme assays using the gamma-glutamyl transferase method provided estimates of glutamine synthetase activity in rat brain homogenates subjected to a...supports the lack of any connection between convulsions caused by in vivo inhibition of glutamine synthetase and convulsions caused by oxygen toxicity (oxygen at high pressure). (Author)

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

    PubMed

    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.

  14. Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals

    PubMed Central

    Shea, Jeremy M.; Boskovic, Ana; Derr, Alan G.; Bing, Xin Y.; Belleannee, Clemence; Kucukural, Alper; Serra, Ryan W.; Sun, Fengyun; Song, Lina; Carone, Benjamin R.; Ricci, Emiliano P.; Li, Xin Z.; Fauquier, Lucas; Moore, Melissa J.; Sullivan, Robert; Mello, Craig C.; Garber, Manuel; Rando, Oliver J.

    2016-01-01

    Several recent studies link parental environments to phenotypes in subsequent generations. Here, we investigate the mechanism by which paternal diet affects offspring metabolism. Protein restriction in mice affects small RNA levels in mature sperm, with decreased let-7 levels and increased levels of 5’ fragments of glycine tRNAs. tRNA fragments are scarce in testicular sperm, but are gained as sperm mature in the epididymis. Epididymosomes – vesicles that fuse with sperm during epididymal transit – carry RNA payloads matching those of mature sperm, and deliver RNAs to immature sperm in vitro. Functionally, tRNA-Gly-GCC fragments repress genes associated with the endogenous retroelement MERVL, both in ES cells and embryos. Our results shed light on small RNA biogenesis, and its dietary regulation, during post-testicular sperm maturation, and link tRNA fragments to regulation of endogenous retroelements active in the preimplantation embryo. PMID:26721685

  15. Dyskerin, tRNA genes, and condensin tether pericentric chromatin to the spindle axis in mitosis

    PubMed Central

    Snider, Chloe E.; Stephens, Andrew D.; Kirkland, Jacob G.; Hamdani, Omar; Kamakaka, Rohinton T.

    2014-01-01

    Condensin is enriched in the pericentromere of budding yeast chromosomes where it is constrained to the spindle axis in metaphase. Pericentric condensin contributes to chromatin compaction, resistance to microtubule-based spindle forces, and spindle length and variance regulation. Condensin is clustered along the spindle axis in a heterogeneous fashion. We demonstrate that pericentric enrichment of condensin is mediated by interactions with transfer ribonucleic acid (tRNA) genes and their regulatory factors. This recruitment is important for generating axial tension on the pericentromere and coordinating movement between pericentromeres from different chromosomes. The interaction between condensin and tRNA genes in the pericentromere reveals a feature of yeast centromeres that has profound implications for the function and evolution of mitotic segregation mechanisms. PMID:25332162

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

    PubMed

    Abe, Takashi; Inokuchi, Hachiro; Yamada, Yuko; Muto, Akira; Iwasaki, Yuki; Ikemura, Toshimichi

    2014-01-01

    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 (ISG) 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 batch-learning self-organizing-map with oligonucleotide composition is useful for efficient knowledge discovery from big sequence data.

  17. Hydrophobic Properties of tRNA with Varied Conformations Evaluated by an Aqueous Two-Phase System

    PubMed Central

    Suga, Keishi; Tomita, Hibiki; Tanaka, Seishiro; Umakoshi, Hiroshi

    2012-01-01

    The surface properties of transfer RNA (tRNA) were analyzed using a poly(ethylene glycol)/dextran aqueous two-phase system (ATPS), where the surface net hydrophobicity (HFS) and the local hydrophobicity (LH) were evaluated based on the partition coefficient of tRNA in the ATPS. According to the evaluated HFS values, the surface of the tRNA molecule was hydrophilic at 20° -40 °C, and it became hydrophobic at 50° -80 °C because of the exposure of the intrinsic nucleobases of tRNA. In contrast, the LH values were found to be maximal at 20° -40 °C. The conformation of tRNA was investigated by Raman and circular dichroism (CD) spectroscopies, corroborating the results with the calculated prediction of its secondary structure (Mfold). It was shown that 66% of A-form structure existed at room temperature; the base stacking (θ265) was gradually decreased, and the A-form structure (θ208) was denatured along with a sigmoid curve against the temperature increase; the denatured secondary structures were observed above 50° C by Mfold prediction. The HFS value of the DNA duplex was found to be hydrophilic, compared to that of the single-stranded DNA, indicating that the exposure of nucleobases is a key factor of the hydrophobic properties of nucleotides. We conclude that the hydrophobic property of the tRNA surface was directly affected by its conformational transition. PMID:23091416

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

    PubMed

    Andersen, Kasper L; 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 Tetrahymena thermophila. We constructed strains lacking individual or combinations of these RNT2 genes that were viable but had distinct cellular and molecular phenotypes. In strains lacking only one Rnt2 protein or lacking a subfamily of three catalytically inactive Rnt2 proteins, starvation-induced tRNA fragments continued to accumulate, with only a minor change in fragment profile in one strain. We therefore generated strains lacking pairwise combinations of the top three candidates for Rnt2 tRNases. Each of these strains showed a distinct starvation-specific profile of tRNA and rRNA fragment accumulation. These results, the delineation of a broadened range of conditions that induce the accumulation of tRNA halves, and the demonstration of a predominantly ribonucleoprotein-free state of tRNA halves in cell extract suggest that ciliate tRNA halves are degradation intermediates in an autophagy pathway induced by growth arrest that functions to recycle idle protein synthesis machinery.

  19. Mapping hidden potential identity elements by computing the average discriminating power of individual tRNA positions.

    PubMed

    Szenes, Aron; Pál, Gábor

    2012-06-01

    The recently published discrete mathematical method, extended consensus partition (ECP), identifies nucleotide types at each position that are strictly absent from a given sequence set, while occur in other sets. These are defined as discriminating elements (DEs). In this study using the ECP approach, we mapped potential hidden identity elements that discriminate the 20 different tRNA identities. We filtered the tDNA data set for the obligatory presence of well-established tRNA features, and then separately for each identity set, the presence of already experimentally identified strictly present identity elements. The analysis was performed on the three kingdoms of life. We determined the number of DE, e.g. the number of sets discriminated by the given position, for each tRNA position of each tRNA identity set. Then, from the positional DE numbers obtained from the 380 pairwise comparisons of the 20 identity sets, we calculated the average excluding value (AEV) for each tRNA position. The AEV provides a measure on the overall discriminating power of each position. Using a statistical analysis, we show that positional AEVs correlate with the number of already identified identity elements. Positions having high AEV but lacking published identity elements predict hitherto undiscovered tRNA identity elements.

  20. Plant-Specific Preprotein and Amino Acid Transporter Proteins Are Required for tRNA Import into Mitochondria1[OPEN

    PubMed Central

    Kubiszewski-Jakubiak, Szymon; Teixeira, Pedro F.; Narsai, Reena; Ivanova, Aneta; Megel, Cyrille; Schock, Annette; Kraus, Sabrina; Glaser, Elzbieta; Philippar, Katrin; Maréchal-Drouard, Laurence; Soll, Jürgen

    2016-01-01

    A variety of eukaryotes, in particular plants, do not contain the required number of tRNAs to support the translation of mitochondria-encoded genes and thus need to import tRNAs from the cytosol. This study identified two Arabidopsis (Arabidopsis thaliana) proteins, Tric1 and Tric2 (for tRNA import component), which on simultaneous inactivation by T-DNA insertion lines displayed a severely delayed and chlorotic growth phenotype and significantly reduced tRNA import capacity into isolated mitochondria. The predicted tRNA-binding domain of Tric1 and Tric2, a sterile-α-motif at the C-terminal end of the protein, was required to restore tRNA uptake ability in mitochondria of complemented plants. The purified predicted tRNA-binding domain binds the T-arm of the tRNA for alanine with conserved lysine residues required for binding. T-DNA inactivation of both Tric proteins further resulted in an increase in the in vitro rate of in organello protein synthesis, which was mediated by a reorganization of the nuclear transcriptome, in particular of genes encoding a variety of proteins required for mitochondrial gene expression at both the transcriptional and translational levels. The characterization of Tric1/2 provides mechanistic insight into the process of tRNA import into mitochondria and supports the theory that the tRNA import pathway resulted from the repurposing of a preexisting protein import apparatus. PMID:27789739

  1. Mapping Hidden Potential Identity Elements by Computing the Average Discriminating Power of Individual tRNA Positions

    PubMed Central

    Szenes, Áron; Pál, Gábor

    2012-01-01

    The recently published discrete mathematical method, extended consensus partition (ECP), identifies nucleotide types at each position that are strictly absent from a given sequence set, while occur in other sets. These are defined as discriminating elements (DEs). In this study using the ECP approach, we mapped potential hidden identity elements that discriminate the 20 different tRNA identities. We filtered the tDNA data set for the obligatory presence of well-established tRNA features, and then separately for each identity set, the presence of already experimentally identified strictly present identity elements. The analysis was performed on the three kingdoms of life. We determined the number of DE, e.g. the number of sets discriminated by the given position, for each tRNA position of each tRNA identity set. Then, from the positional DE numbers obtained from the 380 pairwise comparisons of the 20 identity sets, we calculated the average excluding value (AEV) for each tRNA position. The AEV provides a measure on the overall discriminating power of each position. Using a statistical analysis, we show that positional AEVs correlate with the number of already identified identity elements. Positions having high AEV but lacking published identity elements predict hitherto undiscovered tRNA identity elements. PMID:22378766

  2. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences

    PubMed Central

    Laslett, Dean; Canback, Bjorn

    2004-01-01

    A computer program, ARAGORN, identifies tRNA and tmRNA genes. The program employs heuristic algorithms to predict tRNA secondary structure, based on homology with recognized tRNA consensus sequences and ability to form a base-paired cloverleaf. tmRNA genes are identified using a modified version of the BRUCE program. ARAGORN achieves a detection sensitivity of 99% from a set of 1290 eubacterial, eukaryotic and archaeal tRNA genes and detects all complete tmRNA sequences in the tmRNA database, improving on the performance of the BRUCE program. Recently discovered tmRNA genes in the chloroplasts of two species from the ‘green’ algae lineage are detected. The output of the program reports the proposed tRNA secondary structure and, for tmRNA genes, the secondary structure of the tRNA domain, the tmRNA gene sequence, the tag peptide and a list of organisms with matching tmRNA peptide tags. PMID:14704338

  3. Peptide Mapping of Aminoacyl-tRNA Synthetases: Evidence for Internal Sequence Homology in Escherichia coli Leucyl-tRNA Synthetase

    PubMed Central

    Waterson, Robert M.; Konigsberg, William H.

    1974-01-01

    Most aminoacyl-tRNA synthetases contain polypeptide chains of about either 50,000 or 100,000 daltons. Peptide mapping of tryptic, chymotryptic, or Staphylococcus aureus acid protease digests of seryl-tRNA synthetase (100,000, dimer) and leucyl-tRNA synthetase (100,000, monomer) from E. coli was done after selective modification of lysine residues with [14C]succinic anhydride or of methionine residues with [14C]iodoacetate. By use of thin-layer electrophoresis and chromatography on silicagel or cellulose plates followed by radioautography it was possible, depending upon the specific activity of the reagent used, to detect radioactive peptides obtained from as little as l μg of protein. Seryl-tRNA synthetase gave the correct number of tryptic peptides expected for a dimer of identical subunits. Leucyl-tRNA synthetase, on the other hand, gave roughly half the number of radioactive tryptic, chymotryptic, and acid protease peptides expected from the lysine, arginine, and methionine content of the 100,000 monomer. We have interpreted these results as indicating that extensive internal homology exists among lysine- and methionine-containing peptides within the leucyl-tRNA synthetase. The simplest conclusion that can be drawn from these observations is that the NH2- and COOH-terminal halves of leucyl-tRNA synthetase and perhaps other synthetases of 100,000 molecular weight may have evolved through a process of gene duplication and fusion, followed by limited diversification by way of amino-acid substitutions accumulating during evolution. Images PMID:4592690

  4. Inhibition of rabbit gastric glucosamine synthetase activity by Cu2+, Zn2+ and Se4+.

    PubMed

    Fujita, T; Sakuma, S; Takahashi, K; Bohtani, Y; Nishida, H; Fujimoto, Y

    1997-05-01

    The effects of Fe2+, Cu2+, Zn2+ and Se4+ on the activity of glucosamine synthetase, the rate-limiting enzyme of mucus synthesis, in rabbit gastric corporal mucosa were examined. Cu2+, Zn2+ and Se4+ inhibited the glucosamine synthetase activity at concentrations ranging from 1 to 10 microM (Cu2+, 8-98% inhibition; Zn2+, 10-99% inhibition; Se4+, 32-89% inhibition). The inhibitory effects of these three ions were much stronger than that of UDP-N-acetylglúcosamine known as a representative inhibitor of the glucosamine synthetase activity (10 microM, 52% inhibition). Fe2+ had no significant effect on the glucosamine synthetase activity up to 100 microM. These results suggest that Cu2+, Zn2+ and Se4+ can be potent inhibitors of gastric glucosamine synthetase activity.

  5. Circumstantial evidence for a role of glutamine-synthetase in suicide.

    PubMed

    Kalkman, Hans O

    2011-06-01

    Suicide occurs during depression, schizophrenia, diabetes and epilepsy. A common denominator of these disorders is the presence of inflammation. Inflammatory cytokines affect function and expression of the glial enzyme glutamine synthetase and post mortem studies indicate that brain glutamine synthetase function is suppressed in mood disorders and epilepsy. In a study of schizophrenia brains, the expression of glutamine synthetase was reduced in those cases where the cause of death was suicide. The glycogen synthase kinase 3 (GSK3) inhibitor, lithium, which has a proven efficacy against suicide, increased in an animal experiment the expression of glutamine synthetase. Based on these data one could reason that suicide may be prevented by centrally acting GSK3 inhibitors. However, since inhibition of glutamine synthetase may lead to a deficit in glutamine and as consequence a GABA and glutamate deficit, even simple food supplementation with glutamine might help to reduce suicide.

  6. Purification, Structure and Properties of Escherichia coli tRNA Pseudouridine Synthase 1.

    DTIC Science & Technology

    1987-01-01

    RD-8193 9" PURIFICATION STRUCTURE AMD PROPERTIES OF ESCNERICHIA 11 COLI TRt4A PSEUDOURIDINE SYNTHASE 1(U) CALIFORNIA UNY OAKLAND NAVAL BIOSCIENCES...Keywo rd S: tN Pseudou ridine Synthase 1, Escherichia Cal i, 03 Plasmid, 19. ABSTRACT (Continue on reverse if necessary and identify by block number...The RNA modification enzyme, tRNA pseudouridine synthase I (PSUI) has been isolated in 95% purity from an Escherichia coli strain harboring a

  7. Dynamics of RNA modification by a multi-site-specific tRNA methyltransferase

    PubMed Central

    Hamdane, Djemel; Guelorget, Amandine; Guérineau, Vincent; Golinelli-Pimpaneau, Béatrice

    2014-01-01

    In most organisms, the widely conserved 1-methyl-adenosine58 (m1A58) tRNA modification is catalyzed by an S-adenosyl-L-methionine (SAM)-dependent, site-specific enzyme TrmI. In archaea, TrmI also methylates the adjacent adenine 57, m1A57 being an obligatory intermediate of 1-methyl-inosine57 formation. To study this multi-site specificity, we used three oligoribonucleotide substrates of Pyrococcus abyssi TrmI (PabTrmI) containing a fluorescent 2-aminopurine (2-AP) at the two target positions and followed the RNA binding kinetics and methylation reactions by stopped-flow and mass spectrometry. PabTrmI did not modify 2-AP but methylated the adjacent target adenine. 2-AP seriously impaired the methylation of A57 but not A58, confirming that PabTrmI methylates efficiently the first adenine of the A57A58A59 sequence. PabTrmI binding provoked a rapid increase of fluorescence, attributed to base unstacking in the environment of 2-AP. Then, a slow decrease was observed only with 2-AP at position 57 and SAM, suggesting that m1A58 formation triggers RNA release. A model of the protein–tRNA complex shows both target adenines in proximity of SAM and emphasizes no major tRNA conformational change except base flipping during the reaction. The solvent accessibility of the SAM pocket is not affected by the tRNA, thereby enabling S-adenosyl-L-homocysteine to be replaced by SAM without prior release of monomethylated tRNA. PMID:25217588

  8. Nucleoside modifications in the regulation of gene expression: focus on tRNA.

    PubMed

    Duechler, Markus; Leszczyńska, Grażyna; Sochacka, Elzbieta; Nawrot, Barbara

    2016-08-01

    Both, DNA and RNA nucleoside modifications contribute to the complex multi-level regulation of gene expression. Modified bases in tRNAs modulate protein translation rates in a highly dynamic manner. Synonymous codons, which differ by the third nucleoside in the triplet but code for the same amino acid, may be utilized at different rates according to codon-anticodon affinity. Nucleoside modifications in the tRNA anticodon loop can favor the interaction with selected codons by stabilizing specific base pairs. Similarly, weakening of base pairing can discriminate against binding to near-cognate codons. mRNAs enriched in favored codons are translated in higher rates constituting a fine-tuning mechanism for protein synthesis. This so-called codon bias establishes a basic protein level, but sometimes it is necessary to further adjust the production rate of a particular protein to actual requirements, brought by, e.g., stages in circadian rhythms, cell cycle progression or exposure to stress. Such an adjustment is realized by the dynamic change of tRNA modifications resulting in the preferential translation of mRNAs coding for example for stress proteins to facilitate cell survival. Furthermore, tRNAs contribute in an entirely different way to another, less specific stress response consisting in modification-dependent tRNA cleavage that contributes to the general down-regulation of protein synthesis. In this review, we summarize control functions of nucleoside modifications in gene regulation with a focus on recent findings on protein synthesis control by tRNA base modifications.

  9. Replacement of the folC gene, encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli, with genes mutagenized in vitro.

    PubMed Central

    Pyne, C; Bognar, A L

    1992-01-01

    The folylpolyglutamate synthetase-dihydrofolate synthetase gene (folC) in Escherichia coli was deleted from the bacterial chromosome and replaced by a selectable Kmr marker. The deletion strain required a complementing gene expressing folylpolyglutamate synthetase encoded on a plasmid for viability, indicating that folC is an essential gene in E. coli. The complementing folC gene was cloned into the vector pPM103 (pSC101, temperature sensitive for replication), which segregated spontaneously at 42 degrees C in the absence of selection. This complementing plasmid was replaced in the folC deletion strain by compatible pUC plasmids containing folC genes with mutations generated in vitro, producing strains which express only mutant folylpolyglutamate synthetase. Mutant folC genes expressing insufficient enzyme activity could not complement the chromosomal deletion, resulting in retention of the pPM103 plasmid. Some mutant genes expressing low levels of enzyme activity replaced the complementing plasmid, but the strains produced were auxotrophic for products of folate-dependent pathways. The folylpolyglutamate synthetase gene from Lactobacillus casei, which may lack dihydrofolate synthetase activity, replaced the complementing plasmid, but the strain was auxotrophic for all folate end products. Images PMID:1548226

  10. Purification and comparison of two forms of S-adenosyl-L-methionine synthetase from rat liver.

    PubMed

    Cabrero, C; Puerta, J; Alemany, S

    1987-12-30

    Only two S-adenosyl-L-methionine synthetase forms exist in rat liver: high-Mr S-adenosyl-L-methionine synthetase and low-Mr S-adenosyl-L-methionine synthetase, which have been purified to apparent homogeneity as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. High-Mr S-adenosyl-L-methionine synthetase had an apparent molecular mass, determined by gel filtration, of 210 kDa and was a tetramer constituted by 48.5-kDa subunits, estimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The apparent molecular mass of low-Mr S-adenosyl-L-methionine synthetase, as estimated by gel filtration, was 110 kDa and was constituted by two subunits of 47 kDa. An antiserum against low-Mr S-adenosyl-L-methionine synthetase cross-reacted with the two forms. Reverse-phase HPLC runs of tryptic digestions of high-Mr and low-Mr S-adenosyl-L-methionine synthetase showed that the peptide maps of the two forms were very similar, if not identical. High-Mr S-adenosyl-L-methionine synthetase activity was inhibited by S-adenosyl-L-methionine and pyrophosphate. Depending on the dose used, S-adenosyl-L-methionine activated or inhibited low-Mr S-adenosyl-L-methionine synthetase and pyrophosphate had no effect on this form. The two synthetases showed a different specific activity at the physiological concentration of methionine. This report shows that even though the two forms are constructed of the same polypeptide chains, they are regulated in a different manner by methionine and by the products of the reaction.

  11. MMB-GUI: a fast morphing method demonstrates a possible ribosomal tRNA translocation trajectory.

    PubMed

    Tek, Alex; Korostelev, Andrei A; Flores, Samuel Coulbourn

    2016-01-08

    Easy-to-use macromolecular viewers, such as UCSF Chimera, are a standard tool in structural biology. They allow rendering and performing geometric operations on large complexes, such as viruses and ribosomes. Dynamical simulation codes enable modeling of conformational changes, but may require considerable time and many CPUs. There is an unmet demand from structural and molecular biologists for software in the middle ground, which would allow visualization combined with quick and interactive modeling of conformational changes, even of large complexes. This motivates MMB-GUI. MMB uses an internal-coordinate, multiscale approach, yielding as much as a 2000-fold speedup over conventional simulation methods. We use Chimera as an interactive graphical interface to control MMB. We show how this can be used for morphing of macromolecules that can be heterogeneous in biopolymer type, sequence, and chain count, accurately recapitulating structural intermediates. We use MMB-GUI to create a possible trajectory of EF-G mediated gate-passing translocation in the ribosome, with all-atom structures. This shows that the GUI makes modeling of large macromolecules accessible to a wide audience. The morph highlights similarities in tRNA conformational changes as tRNA translocates from A to P and from P to E sites and suggests that tRNA flexibility is critical for translocation completion.

  12. Polyadenylation helps regulate functional tRNA levels in Escherichia coli

    PubMed Central

    Mohanty, Bijoy K.; Maples, Valerie F.; Kushner, Sidney R.

    2012-01-01

    Here we demonstrate a new regulatory mechanism for tRNA processing in Escherichia coli whereby RNase T and RNase PH, the two primary 3′ → 5′ exonucleases involved in the final step of 3′-end maturation, compete with poly(A) polymerase I (PAP I) for tRNA precursors in wild-type cells. In the absence of both RNase T and RNase PH, there is a >30-fold increase of PAP I-dependent poly(A) tails that are ≤10 nt in length coupled with a 2.3- to 4.2-fold decrease in the level of aminoacylated tRNAs and a >2-fold decrease in growth rate. Only 7 out of 86 tRNAs are not regulated by this mechanism and are also not substrates for RNase T, RNase PH or PAP I. Surprisingly, neither PNPase nor RNase II has any effect on tRNA poly(A) tail length. Our data suggest that the polyadenylation of tRNAs by PAP I likely proceeds in a distributive fashion unlike what is observed with mRNAs. PMID:22287637

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

    PubMed Central

    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

  14. Rearrangement of mitochondrial tRNA genes in flat bugs (Hemiptera: Aradidae)

    PubMed Central

    Song, Fan; Li, Hu; Shao, Renfu; Shi, Aimin; Bai, Xiaoshuan; Zheng, Xiaorong; Heiss, Ernst; Cai, Wanzhi

    2016-01-01

    The typical insect mitochondrial (mt) genome organization, which contains a single chromosome with 37 genes, was found in the infraorder Pentatomomorpha (suborder Heteroptera). The arrangement of mt genes in these true bugs is usually the same as the ancestral mt gene arrangement of insects. Rearrangement of transfer RNA (tRNA) genes, however, has been found in two subfamilies of flat bugs (Mezirinae and Calisiinae, family Aradidae). In this study, we sequenced the complete mt genomes of four species from three other subfamilies (Aradinae, Carventinae and Aneurinae). We found tRNA gene rearrangement in all of these four species. All of the rearranged tRNA genes are located between the mitochondrial control region and cox1, indicating this region as a hotspot for gene rearrangement in flat bugs; the rearrangement is likely caused by events of tandem duplication and random deletion of genes. Furthermore, our phylogenetic and dating analyses indicated that the swap of positions between trnQ and trnI occurred ~162 million years ago (MYA) in the most recent common ancestor of the five subfamilies of flat bugs investigated to date, whereas the swap of positions between trnC and trnW occurred later in the lineage leading to Calisiinae, and the translocation of trnC and trnY occurred later than 134 MYA in the lineage leading to Aradinae. PMID:27180804

  15. TRNA mutations that affect decoding fidelity deregulate development and the proteostasis network in zebrafish.

    PubMed

    Reverendo, Marisa; Soares, Ana R; Pereira, Patrícia M; Carreto, Laura; Ferreira, Violeta; Gatti, Evelina; Pierre, Philippe; Moura, Gabriela R; Santos, Manuel A

    2014-01-01

    Mutations in genes that encode tRNAs, aminoacyl-tRNA syntheases, tRNA modifying enzymes and other tRNA interacting partners are associated with neuropathies, cancer, type-II diabetes and hearing loss, but how these mutations cause disease is unclear. We have hypothesized that levels of tRNA decoding error (mistranslation) that do not fully impair embryonic development can accelerate cell degeneration through proteome instability and saturation of the proteostasis network. To test this hypothesis we have induced mistranslation in zebrafish embryos using mutant tRNAs that misincorporate Serine (Ser) at various non-cognate codon sites. Embryo viability was affected and malformations were observed, but a significant proportion of embryos survived by activating the unfolded protein response (UPR), the ubiquitin proteasome pathway (UPP) and downregulating protein biosynthesis. Accumulation of reactive oxygen species (ROS), mitochondrial and nuclear DNA damage and disruption of the mitochondrial network, were also observed, suggesting that mistranslation had a strong negative impact on protein synthesis rate, ER and mitochondrial homeostasis. We postulate that mistranslation promotes gradual cellular degeneration and disease through protein aggregation, mitochondrial dysfunction and genome instability.

  16. RNase MRP cleaves pre-tRNASer-Met in the tRNA maturation pathway.

    PubMed

    Saito, Yuichiro; Takeda, Jun; Adachi, Kousuke; Nobe, Yuko; Kobayashi, Junya; Hirota, Kouji; Oliveira, Douglas V; Taoka, Masato; Isobe, Toshiaki

    2014-01-01

    Ribonuclease mitochondrial RNA processing (RNase MRP) is a multifunctional ribonucleoprotein (RNP) complex that is involved in the maturation of various types of RNA including ribosomal RNA. RNase MRP consists of a potential catalytic RNA and several protein components, all of which are required for cell viability. We show here that the temperature-sensitive mutant of rmp1, the gene for a unique protein component of RNase MRP, accumulates the dimeric tRNA precursor, pre-tRNA(Ser-Met). To examine whether RNase MRP mediates tRNA maturation, we purified the RNase MRP holoenzyme from the fission yeast Schizosaccharomyces pombe and found that the enzyme directly and selectively cleaves pre-tRNA(Ser-Met), suggesting that RNase MRP participates in the maturation of specific tRNA in vivo. In addition, mass spectrometry-based ribonucleoproteomic analysis demonstrated that this RNase MRP consists of one RNA molecule and 11 protein components, including a previously unknown component Rpl701. Notably, limited nucleolysis of RNase MRP generated an active catalytic core consisting of partial mrp1 RNA fragments, which constitute "Domain 1" in the secondary structure of RNase MRP, and 8 proteins. Thus, the present study provides new insight into the structure and function of RNase MRP.

  17. Snapshots of Dynamics in Synthesizing N6-Isopentenyladenosine at the tRNA Anticodon

    SciTech Connect

    Chimnaronk, R.; Forouhar, F; Sakai, J; Yao, M; Tron, C; Atta, M; Fontecave, M; Hunt, J; Tanaka, I

    2009-01-01

    Bacterial and eukaryotic tRNAs that decode codons starting with uridine have a hydrophobically hypermodified adenosine at position 37 (A37) adjacent to the 3?-end of the anticodon, which is essential for efficient and highly accurate protein translation by the ribosome. However, it remains unclear as to how the corresponding tRNAs are selected to be modified by alkylation at the correct position of the adenosine base. We have determined a series of crystal structures of bacterial tRNA isopentenyltransferase (MiaA) in apo- and tRNA-bound forms, which completely render snapshots of substrate selections during the modification of RNA. A compact evolutionary inserted domain (herein swinging domain) in MiaA that exhibits as a highly mobile entity moves around the catalytic domain as likely to reach and trap the tRNA substrate. Thereby, MiaA clamps the anticodon stem loop of the tRNA substrate between the catalytic and swinging domains, where the two conserved elongated residues from the swinging domain pinch the two flanking A36 and A38 together to squeeze out A37 into the reaction tunnel. The site-specific isopentenylation of RNA is thus ensured by a characteristic pinch-and-flip mechanism and by a reaction tunnel to confine the substrate selection. Furthermore, combining information from soaking experiments with structural comparisons, we propose a mechanism for the ordered substrate binding of MiaA.

  18. Structure of a bacterial ribonuclease P holoenzyme in complex with tRNA.

    PubMed

    Reiter, Nicholas J; Osterman, Amy; Torres-Larios, Alfredo; Swinger, Kerren K; Pan, Tao; Mondragón, Alfonso

    2010-12-09

    Ribonuclease (RNase) P is the universal ribozyme responsible for 5'-end tRNA processing. We report the crystal structure of the Thermotoga maritima RNase P holoenzyme in complex with tRNA(Phe). The 154 kDa complex consists of a large catalytic RNA (P RNA), a small protein cofactor and a mature tRNA. The structure shows that RNA-RNA recognition occurs through shape complementarity, specific intermolecular contacts and base-pairing interactions. Soaks with a pre-tRNA 5' leader sequence with and without metal help to identify the 5' substrate path and potential catalytic metal ions. The protein binds on top of a universally conserved structural module in P RNA and interacts with the leader, but not with the mature tRNA. The active site is composed of phosphate backbone moieties, a universally conserved uridine nucleobase, and at least two catalytically important metal ions. The active site structure and conserved RNase P-tRNA contacts suggest a universal mechanism of catalysis by RNase P.

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

    PubMed Central

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

    2016-01-01

    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

  20. MMB-GUI: a fast morphing method demonstrates a possible ribosomal tRNA translocation trajectory

    PubMed Central

    Tek, Alex; Korostelev, Andrei A.; Flores, Samuel Coulbourn

    2016-01-01

    Easy-to-use macromolecular viewers, such as UCSF Chimera, are a standard tool in structural biology. They allow rendering and performing geometric operations on large complexes, such as viruses and ribosomes. Dynamical simulation codes enable modeling of conformational changes, but may require considerable time and many CPUs. There is an unmet demand from structural and molecular biologists for software in the middle ground, which would allow visualization combined with quick and interactive modeling of conformational changes, even of large complexes. This motivates MMB-GUI. MMB uses an internal-coordinate, multiscale approach, yielding as much as a 2000-fold speedup over conventional simulation methods. We use Chimera as an interactive graphical interface to control MMB. We show how this can be used for morphing of macromolecules that can be heterogeneous in biopolymer type, sequence, and chain count, accurately recapitulating structural intermediates. We use MMB-GUI to create a possible trajectory of EF-G mediated gate-passing translocation in the ribosome, with all-atom structures. This shows that the GUI makes modeling of large macromolecules accessible to a wide audience. The morph highlights similarities in tRNA conformational changes as tRNA translocates from A to P and from P to E sites and suggests that tRNA flexibility is critical for translocation completion. PMID:26673695

  1. Rearrangement of mitochondrial tRNA genes in flat bugs (Hemiptera: Aradidae).

    PubMed

    Song, Fan; Li, Hu; Shao, Renfu; Shi, Aimin; Bai, Xiaoshuan; Zheng, Xiaorong; Heiss, Ernst; Cai, Wanzhi

    2016-05-16

    The typical insect mitochondrial (mt) genome organization, which contains a single chromosome with 37 genes, was found in the infraorder Pentatomomorpha (suborder Heteroptera). The arrangement of mt genes in these true bugs is usually the same as the ancestral mt gene arrangement of insects. Rearrangement of transfer RNA (tRNA) genes, however, has been found in two subfamilies of flat bugs (Mezirinae and Calisiinae, family Aradidae). In this study, we sequenced the complete mt genomes of four species from three other subfamilies (Aradinae, Carventinae and Aneurinae). We found tRNA gene rearrangement in all of these four species. All of the rearranged tRNA genes are located between the mitochondrial control region and cox1, indicating this region as a hotspot for gene rearrangement in flat bugs; the rearrangement is likely caused by events of tandem duplication and random deletion of genes. Furthermore, our phylogenetic and dating analyses indicated that the swap of positions between trnQ and trnI occurred ~162 million years ago (MYA) in the most recent common ancestor of the five subfamilies of flat bugs investigated to date, whereas the swap of positions between trnC and trnW occurred later in the lineage leading to Calisiinae, and the translocation of trnC and trnY occurred later than 134 MYA in the lineage leading to Aradinae.

  2. Horizontal gene transfer of chlamydial-like tRNA genes into early vascular plant mitochondria.

    PubMed

    Knie, Nils; Polsakiewicz, Monika; Knoop, Volker

    2015-03-01

    Mitochondrial genomes of lycophytes are surprisingly diverse, including strikingly different transfer RNA (tRNA) gene complements: No mitochondrial tRNA genes are present in the spikemoss Selaginella moellendorffii, whereas 26 tRNAs are encoded in the chondrome of the clubmoss Huperzia squarrosa. Reinvestigating the latter we found that trnL(gag) and trnS(gga) had never before been identified in any other land plant mitochondrial DNA. Sensitive sequence comparisons showed these two tRNAs as well as trnN(guu) and trnS(gcu) to be very similar to their respective counterparts in chlamydial bacteria. We identified homologs of these chlamydial-type tRNAs also in other lycophyte, fern, and gymnosperm DNAs, suggesting horizontal gene transfer (HGT) into mitochondria in the early vascular plant stem lineages. These findings extend plant mitochondrial HGT to affect individual tRNA genes, to include bacterial donors, and suggest that Chlamydiae on top of their recently proposed key role in primary chloroplast establishment may also have participated in early tracheophyte genome evolution.

  3. Novel Insights into Regulation of Asparagine Synthetase in Conifers

    PubMed Central

    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 plants. A comparative study of PpAS1 and PpAS2 gene expression profiles showed that PpAS1 gene is highly regulated by developmental and environmental factors, while PpAS2 is expressed constitutively. To determine the molecular mechanisms underpinning the differential expression of PpAS1, the promoter region of the gene was isolated and putative binding sites for MYB transcription factors were identified. Gel mobility shift assays showed that a MYB protein from Pinus taeda (PtMYB1) was able to interact with the promoter region of PpAS1. Furthermore, transient expression analyses in pine cells revealed a negative effect of PtMYB1 on PpAS1 expression. The potential role of MYB factors in the transcriptional regulation of PpAS1 in vascular cells is discussed. PMID:22654888

  4. Biochemical characterization of the Mycobacterium tuberculosis phosphoribosyl-1-pyrophosphate synthetase

    PubMed Central

    Alderwick, Luke J; Lloyd, Georgina S; Lloyd, Adrian J; Lovering, Andrew L; Eggeling, Lothar; Besra, Gurdyal S

    2011-01-01

    Mycobacterium tuberculosis arabinogalactan (AG) is an essential cell wall component. It provides a molecular framework serving to connect peptidoglycan to the outer mycolic acid layer. The biosynthesis of the arabinan domains of AG and lipoarabinomannan (LAM) occurs via a combination of membrane bound arabinofuranosyltransferases, all of which utilize decaprenol-1-monophosphorabinose as a substrate. The source of arabinose ultimately destined for deposition into cell wall AG or LAM originates exclusively from phosphoribosyl-1-pyrophosphate (pRpp), a central metabolite which is also required for other essential metabolic processes, such as de novo purine and pyrimidine biosyntheses. In M. tuberculosis, a single pRpp synthetase enzyme (Mt-PrsA) is solely responsible for the generation of pRpp, by catalyzing the transfer of pyrophosphate from ATP to the C1 hydroxyl position of ribose-5-phosphate. Here, we report a detailed biochemical and biophysical study of Mt-PrsA, which exhibits the most rapid enzyme kinetics reported for a pRpp synthetase. PMID:21045009

  5. Mammalian folylpoly-. gamma. -glutamate synthetase. 3. Specificity for folate analogues

    SciTech Connect

    George, S.; Cichowicz, D.J.; Shane, B.

    1987-01-27

    A variety of folate analogues were synthesized to explore the specificity of the folate binding site of hog liver folypolyglutamate synthetase and the requirements for catalysis. Modifications of the internal and terminal glutamate moieties of folate cause large drops in on rates and/or affinity for the protein. The only exceptions are glutamine, homocysteate, and ornithine analogues, indicating a less stringent specificity around the delta-carbon of glutamate. It is proposed that initial folate binding to the enzyme involves low-affinity interactions at a pterin and a glutamate site and that the first glutamate bound is the internal residue adjacent to the benzoyl group. Processive movement of the polyglutamate chain through the glutamate site and a possible conformational change in the protein when the terminal residue is bound would result in tight binding and would position the ..gamma..-carboxyl of the terminal glutamate in the correct position for catalysis. The 4-amino substitution of folate increases the on rate for monoglutamate derivatives but severely impairs catalysis with diglutamate derivatives. Pteroylornithine derivatives are the first potent and specific inhibitors of folylpolyglutamate synthetase to be identified and may act as analogues of reaction intermediates. Other folate derivatives with tetrahedral chemistry replacing the peptide bond, such as pteroyl-..gamma..-glutamyl-(psi,CH/sub 2/-NH)-glutamate, retain affinity for the protein but are considerably less effective inhibitors than the ornithine derivatives. Enzyme activity was assayed using (/sup 14/C)glutamate.

  6. Novel insights into regulation of asparagine synthetase in conifers.

    PubMed

    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 plants. A comparative study of PpAS1 and PpAS2 gene expression profiles showed that PpAS1 gene is highly regulated by developmental and environmental factors, while PpAS2 is expressed constitutively. To determine the molecular mechanisms underpinning the differential expression of PpAS1, the promoter region of the gene was isolated and putative binding sites for MYB transcription factors were identified. Gel mobility shift assays showed that a MYB protein from Pinus taeda (PtMYB1) was able to interact with the promoter region of PpAS1. Furthermore, transient expression analyses in pine cells revealed a negative effect of PtMYB1 on PpAS1 expression. The potential role of MYB factors in the transcriptional regulation of PpAS1 in vascular cells is discussed.

  7. Cleavage specificity of chloroplast and nuclear tRNA 3'-processing nucleases.

    PubMed Central

    Oommen, A; Li, X Q; Gegenheimer, P

    1992-01-01

    tRNAs in eukaryotic nuclei and organelles are synthesized as precursors lacking the 3'-terminal CCA sequence and possessing 5' (leader) and 3' (trailer) extensions. Nucleolytic cleavage of the 3' trailer and addition of CCA are therefore required for formation of functional tRNA 3' termini. Many chloroplast tRNA genes encode a C at position 74 which is not removed during processing but which can be incorporated as the first base of the CCAOH terminus. Sequences downstream of nucleotide 74, however, are always removed. Synthetic yeast pre-tRNA(Phe) substrates containing the complete CCA74-76 sequence were processed with crude or partially purified chloroplast enzyme fractions. The 3'-extended substrates (tRNA-CCA-trailer) were cleaved exclusively between nucleotides 74 and 75 to give tRNA-COH, whereas a 3'-mature transcript (tRNA-CCAOH) was not cleaved at all. A 5'-, 3'-extended chloroplast tRNA-CAG-trailer was also processed entirely to tRNA-COH. Furthermore, a 5'-mature, 3'-extended yeast pre-tRNA(Phe) derivative, tRNA-ACA-trailer, in which C74 was replaced by A, was cleaved precisely after A74. In contrast, we found that a partially purified enzyme fraction (a nuclear/cytoplasmic activity) from wheat embryo cleaved the 3'-extended yeast tRNA(Phe) precursors between nucleotides 73 and 74 to give tRNA(OH). This specificity is consistent with that of all previously characterized nuclear enzyme preparations. We conclude that (i) chloroplast tRNA 3'-processing endonuclease cleaves after nucleotide 74 regardless of the nature of the surrounding sequences; (ii) this specificity differs from that of the plant nuclear/cytoplasmic processing nuclease, which cleaves after base 73; and (iii) since 3'-mature tRNA is not a substrate for either activity, these 3' nucleases must require substrates possessing a 3'-terminal extension that extends past nucleotide 76. This substrate specificity may prevent mature tRNA from counterproductive cleavage by the 3' processing system

  8. Evolution of the Glx-tRNA synthetase family: the glutaminyl enzyme as a case of horizontal gene transfer.

    PubMed Central

    Lamour, V; Quevillon, S; Diriong, S; N'Guyen, V C; Lipinski, M; Mirande, M

    1994-01-01

    An important step ensuring the fidelity in protein biosynthesis is the aminoacylation of tRNAs by aminoacyl-tRNA synthetases. The accuracy of this process rests on a family of 20 enzymes, one for each amino acid. One exception is the formation of Gln-tRNA(Gln) that can be accomplished by two different pathways: aminoacylation of tRNA(Gln) with Gln by glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) or transamidation of Glu from Glu-tRNA(Gln) mischarged by glutamyl-tRNA synthetase (GluRS; EC 6.1.1.17). The latter pathway is widespread among bacteria and organelles that, accordingly, lack GlnRS. However, some bacterial species, such as Escherichia coli, do possess a GlnRS activity, which is responsible for Gln-tRNA(Gln) formation. In the cytoplasm of eukaryotic cells, both GluRS and GlnRS activities can be detected. To gain more insight into the evolutionary relationship between GluRS and GlnRS enzyme species, we have now isolated and characterized a human cDNA encoding GlnRS. The deduced amino acid sequence shows a strong similarity with other known GlnRSs and with eukaryotic GluRSs. A molecular phylogenetic analysis was conducted on the 14 GlxRS (GluRS or GlnRS) sequences available to date. Our data suggest that bacterial GlnRS has a eukaryotic origin and was acquired by a mechanism of horizontal gene transfer. Images PMID:8078941

  9. The Bacillus subtilis tyrZ Gene Encodes a Highly Selective Tyrosyl-tRNA Synthetase and Is Regulated by a MarR Regulator and T Box Riboswitch

    PubMed Central

    Williams-Wagner, Rebecca N.; Grundy, Frank J.; Raina, Medha; Ibba, Michael

    2015-01-01

    ABSTRACT Misincorporation of d-tyrosine (d-Tyr) into cellular proteins due to mischarging of tRNATyr with d-Tyr by tyrosyl-tRNA synthetase inhibits growth and biofilm formation of Bacillus subtilis. Furthermore, many B. subtilis strains lack a functional gene encoding d-aminoacyl-tRNA deacylase, which prevents misincorporation of d-Tyr in most organisms. B. subtilis has two genes that encode tyrosyl-tRNA synthetase: tyrS is expressed under normal growth conditions, and tyrZ is known to be expressed only when tyrS is inactivated by mutation. We hypothesized that tyrZ encodes an alternate tyrosyl-tRNA synthetase, expression of which allows the cell to grow when d-Tyr is present. We show that TyrZ is more selective for l-Tyr over d-Tyr than is TyrS; however, TyrZ is less efficient overall. We also show that expression of tyrZ is required for growth and biofilm formation in the presence of d-Tyr. Both tyrS and tyrZ are preceded by a T box riboswitch, but tyrZ is found in an operon with ywaE, which is predicted to encode a MarR family transcriptional regulator. Expression of tyrZ is repressed by YwaE and also is regulated at the level of transcription attenuation by the T box riboswitch. We conclude that expression of tyrZ may allow growth when excess d-Tyr is present. IMPORTANCE Accurate protein synthesis requires correct aminoacylation of each tRNA with the cognate amino acid and discrimination against related compounds. Bacillus subtilis produces d-Tyr, an analog of l-Tyr that is toxic when incorporated into protein, during stationary phase. Most organisms utilize a d-aminoacyl-tRNA deacylase to prevent misincorporation of d-Tyr. This work demonstrates that the increased selectivity of the TyrZ form of tyrosyl-tRNA synthetase may provide a mechanism by which B. subtilis prevents misincorporation of d-Tyr in the absence of a functional d-aminoacyl-tRNA deacylase gene. PMID:25733610

  10. Protein kinase A is part of a mechanism that regulates nuclear reimport of the nuclear tRNA export receptors Los1p and Msn5p.

    PubMed

    Pierce, Jacqueline B; van der Merwe, George; Mangroo, Dev

    2014-02-01

    The two main signal transduction mechanisms that allow eukaryotes to sense and respond to changes in glucose availability in the environment are the cyclic AMP (cAMP)/protein kinase A (PKA) and AMP-activated protein kinase (AMPK)/Snf1 kinase-dependent pathways. Previous studies have shown that the nuclear tRNA export process is inhibited in Saccharomyces cerevisiae deprived of glucose. However, the signal transduction pathway involved and the mechanism by which glucose availability regulates nuclear-cytoplasmic tRNA trafficking are not understood. Here, we show that inhibition of nuclear tRNA export is caused by a block in nuclear reimport of the tRNA export receptors during glucose deprivation. Cytoplasmic accumulation of the tRNA export receptors during glucose deprivation is not caused by activation of Snf1p. Evidence obtained suggests that PKA is part of the mechanism that regulates nuclear reimport of the tRNA export receptors in response to glucose availability. This mechanism does not appear to involve phosphorylation of the nuclear tRNA export receptors by PKA. The block in nuclear reimport of the tRNA export receptors appears to be caused by activation of an unidentified mechanism when PKA is turned off during glucose deprivation. Taken together, the data suggest that PKA facilitates return of the tRNA export receptors to the nucleus by inhibiting an unidentified activity that facilitates cytoplasmic accumulation of the tRNA export receptors when glucose in the environment is limiting. A PKA-independent mechanism was also found to regulate nuclear tRNA export in response to glucose availability. This mechanism, however, does not regulate nuclear reimport of the tRNA export receptors.

  11. ARM-Seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape of modified tRNA fragments

    PubMed Central

    Cozen, Aaron E.; Quartley, Erin; Holmes, Andrew D.; Robinson, Eva H.; Phizicky, Eric M.; Lowe, Todd M.

    2015-01-01

    High throughput RNA sequencing has accelerated discovery of the complex regulatory roles of small RNAs, but RNAs containing modified nucleosides may escape detection when those modifications interfere with reverse transcription during RNA-seq library preparation. Here we describe AlkB-facilitated RNA Methylation sequencing (ARM-Seq) which uses pre-treatment with Escherichia coli AlkB to demethylate 1-methyladenosine, 3-methylcytidine, and 1-methylguanosine, all commonly found in transfer RNAs. Comparative methylation analysis using ARM-Seq provides the first detailed, transcriptome-scale map of these modifications, and reveals an abundance of previously undetected, methylated small RNAs derived from tRNAs. ARM-Seq demonstrates that tRNA-derived small RNAs accurately recapitulate the m1A modification state for well-characterized yeast tRNAs, and generates new predictions for a large number of human tRNAs, including tRNA precursors and mitochondrial tRNAs. Thus, ARM-Seq provides broad utility for identifying previously overlooked methyl-modified RNAs, can efficiently monitor methylation state, and may reveal new roles for tRNA-derived RNAs as biomarkers or signaling molecules. PMID:26237225

  12. Codon-reading specificity of an unmodified form of Escherichia coli tRNA1Ser in cell-free protein synthesis.

    PubMed Central

    Takai, K; Takaku, H; Yokoyama, S

    1996-01-01

    Unmodified tRNA molecules are useful for many purposes in cell-free protein biosynthesis, but there is little information about how the lack of tRNA post-transcriptional modifications affects the coding specificity for synonymous codons. In the present study, we prepared an unmodified form of Escherichia coli tRNA1Ser, which originally has the cmo5UGA anticodon (cmo5U = uridine 5-oxyacetic acid) and recognizes the UCU, UCA and UCG codons. The codon specificity of the unmodified tRNA was tested in a cell-free protein synthesis directed by designed mRNAs under competition conditions with the parent tRNA1Ser. It was found that the unmodified tRNA with the UGA anti-codon recognizes the UCA codon nearly as efficiently as the modified tRNA. The unmodified tRNA recognized the UCU codon with low, but detectable efficiency, whereas no recognition of the UCC and UCG codons was detected. Therefore, the absence of modifications makes this tRNA more specific to the UCA codon by remarkably reducing the efficiencies of wobble reading of other synonymous codons, without a significant decrease in the UCA reading efficiency. PMID:8760870

  13. Overexpression of tnaC of Escherichia coli Inhibits Growth by Depleting tRNA2Pro Availability

    PubMed Central

    Gong, Ming; Gong, Feng; Yanofsky, Charles

    2006-01-01

    Transcription of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. Induction results from ribosome stalling after translation of tnaC, the coding region for a 24-residue leader peptide. The last sense codon of tnaC, proline codon 24 (CCU), is translated by tRNA2Pro. We analyzed the consequences of overexpression of tnaC from a multicopy plasmid and observed that under inducing conditions more than 60% of the tRNA2Pro in the cell was sequestered in ribosomes as TnaC-tRNA2Pro. The half-life of this TnaC-tRNA2Pro was shown to be 10 to 15 min under these conditions. Plasmid-mediated overexpression of tnaC, under inducing conditions, reduced cell growth rate appreciably. Increasing the tRNA2Pro level relieved this growth inhibition, suggesting that depletion of this tRNA was primarily responsible for the growth rate reduction. Growth inhibition was not relieved by overexpression of tRNA1Pro, a tRNAPro that translates CCG, but not CCU. Replacing the Pro24CCU codon of tnaC by Pro24CCG, a Pro codon translated by tRNA1Pro, also led to growth rate reduction, and this reduction was relieved by overexpression of tRNA1Pro. These findings establish that the growth inhibition caused by tnaC overexpression during induction by tryptophan is primarily a consequence of tRNAPro depletion, resulting from TnaC-tRNAPro retention within stalled, translating ribosomes. PMID:16484200

  14. The mammalian 2'-5' oligoadenylate synthetase gene family: evidence for concerted evolution of paralogous Oas1 genes in Rodentia and Artiodactyla.

    PubMed

    Perelygin, Andrey A; Zharkikh, Andrey A; Scherbik, Svetlana V; Brinton, Margo A

    2006-10-01

    Multiple 2'-5' oligoadenylate (2-5A) synthetases are important components of innate immunity in mammals. Gene families encoding these proteins have previously been studied mainly in humans and mice. To reconstruct the evolution of this gene family in mammals, a search for additional 2-5A synthetase genes was performed in rat, cattle, pig, and dog. Twelve 2'-5' oligoadenylate synthetase (Oas) genes were identified in the rat genome, including eight Oas1 genes, two Oas1 pseudogenes, single copies of Oas2 and Oas3, and two Oas-like genes, Oasl1 and Oasl2. Four OAS genes were detected in the pig genome and five OAS genes were found in both the cattle and dog genomes. An OAS3 gene was not found in either the cattle or the pig genome. While two tandemly duplicated OAS-like (OASL) genes were identified in the dog genome, only a single OASL orthologue was found in both the cattle and the pig genomes. The bovine and porcine OASL genes contain premature stop codons and encode truncated proteins, which lack the typical C-terminal double ubiquitin domains. The cDNA sequences of the rat, cattle, pig, and dog OAS genes were amplified, sequenced and compared with each other and with those in the human, mouse, horse, and chicken genomes. Evidence of concerted evolution of paralogous 2'-5' oligoadenylate synthetase 1 genes was obtained in rodents (Rodentia) and even-toed ungulates (Artiodactyla). Calculations using the nonparametric Kolmogorov-Smirnov test suggested that the homogenization of paralogous OAS1 sequences was due to gene conversion rather than stabilizing selection.

  15. Effect of Liver Damage and Hyperbaric Oxygenation on Glutamine Synthetase of Hepatocytes.

    PubMed

    Savilov, P N; Yakovlev, V N

    2016-01-01

    Activity of glutamine synthetase in the hepatocytes of healthy animals and animals with chronic CCl4-induced hepatitis was studied on white mature female rats after liver resection (15-20% of organ weight) and hyperbaric oxygenation (3 atm, 50 min, 3 times). Surgically operated left and non-operated middle lobes of the liver were analyzed on day 3 after liver resection and exposure to hyperbaric oxygenation. On day 65 of CCl4 poisoning, activity of glutamine synthetase decreased in both lobes and did not recover on day 3 after toxin cessation. Liver resection under conditions of CCl4-induced hepatitis restored reduced activity of glutamine synthetase in both liver lobes to the normal level. In healthy rats, the increase in glutamine synthetase activity after liver resection was found only in the middle lobe of the liver. Hyperbaric oxygenation enhanced the stimulatory effect of liver resection on glutamine synthetase activity in hepatocytes during chronic CCl4-induced hepatitis. In healthy animals with liver resection, activity of glutamine synthetase did not change after hyperbaric oxygenation, while normally oxygenation inhibited glutamine synthetase activity.

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

    PubMed

    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.

  17. tRNA N6-adenosine threonylcarbamoyltransferase defect due to KAE1/TCS3 (OSGEP) mutation manifest by neurodegeneration and renal tubulopathy.

    PubMed

    Edvardson, Simon; Prunetti, Laurence; Arraf, Aiman; Haas, Drago; Bacusmo, Jo Marie; Hu, Jennifer F; Ta-Shma, Asas; Dedon, Peter C; de Crécy-Lagard, Valérie; Elpeleg, Orly

    2017-03-08

    Post-transcriptional tRNA modifications are numerous and require a large set of highly conserved enzymes in humans and other organisms. In yeast, the loss of many modifications is tolerated under unstressed conditions; one exception is the N(6)-threonyl-carbamoyl-adenosine (t(6)A) modification, loss of which causes a severe growth phenotype. Here we aimed at a molecular diagnosis in a brother and sister from a consanguineous family who presented with global developmental delay, failure to thrive and a renal defect manifesting in proteinuria and hypomagnesemia. Using exome sequencing, the patients were found to be homozygous for the c.974G>A (p.(Arg325Gln)) variant of the KAE1 gene. KAE1 is a constituent of the KEOPS complex, a five-subunit complex that catalyzes the second biosynthetic step of t(6)A in the cytosol. The yeast KAE1 allele carrying the equivalent mutation did not rescue the t(6)A deficiency of the kae1Δ yeast strain as efficiently as the WT allele; furthermore, t(6)A levels quantified by LC-MS/MS were lower in the kae1Δ strain which was complemented by the mutation than in the kae1Δ strain, which was complemented by the WT allele. We conclude that homozygosity for c.974G>A (p.(Arg325Gln)) in KAE1 likely exerts its pathogenic effect by perturbing t(6)A synthesis, thereby interfering with global protein production. This is the first report of t(6)A biosynthesis defect in human. KAE1 joins the growing list of cytoplasmic tRNA modification enzymes, all associated with severe neurological disorders.European Journal of Human Genetics advance online publication, 8 March 2017; doi:10.1038/ejhg.2017.30.

  18. The MTCY428.08 Gene of Mycobacterium tuberculosis Codes for NAD+ Synthetase

    PubMed Central

    Cantoni, Rita; Branzoni, Manuela; Labò, Monica; Rizzi, Menico; Riccardi, Giovanna

    1998-01-01

    The product of the MTCY428.08 gene of Mycobacterium tuberculosis shows sequence homology with several NAD+ synthetases. The MTCY428.08 gene was cloned into the expression vectors pGEX-4T-1 and pET-15b. Expression in Escherichia coli led to overproduction of glutathione S-transferase fused and His6-tagged gene products, which were enzymatically assayed for NAD synthetase activity. Our results demonstrate that the MTCY428.08 gene of M. tuberculosis is the structural gene for NAD+ synthetase. PMID:9620974

  19. Critical Evaluation of the Changes in Glutamine Synthetase Activity in Models of Cerebral Stroke.

    PubMed

    Jeitner, Thomas M; Battaile, Kevin; Cooper, Arthur J L

    2015-12-01

    The following article addresses some seemingly paradoxical observations concerning cerebral glutamine synthetase in ischemia-reperfusion injury. In the brain, this enzyme is predominantly found in astrocytes and catalyzes part of the glutamine-glutamate cycle. Glutamine synthetase is also thought to be especially sensitive to inactivation by the oxygen- and nitrogen-centered radicals generated during strokes. Despite this apparent sensitivity, glutamine synthetase specific activity is elevated in the affected tissues during reperfusion. Given the central role of the glutamine-glutamate cycle in the brain, we sought to resolve these conflicting observations with the view of providing an alternative perspective for therapeutic intervention in stroke.

  20. Regulation of Angiogenesis by Aminoacyl-tRNA Synthetases

    PubMed Central

    Mirando, Adam C.; Francklyn, Christopher S.; Lounsbury, Karen M.

    2014-01-01

    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. PMID:25535072

  1. Intra-tRNA distance measurements for nucleocapsid proteindependent tRNA unwinding during priming of HIV reverse transcription.

    PubMed

    Chan, B; Weidemaier, K; Yip, W T; Barbara, P F; Musier-Forsyth, K

    1999-01-19

    We report here the direct measurement of intra-tRNA distances during annealing of the tRNA primer to the HIV RNA genome. This key step in the initiation of retroviral reverse transcription involves hybridization of one strand of the acceptor arm of a specific lysine tRNA to the primer binding site on the RNA genome. Although the mechanism of tRNA unwinding and annealing is not known, previous studies have shown that HIV nucleocapsid protein (NC) greatly accelerates primer/template binary complex formation in vitro. An open question is whether NC alone unwinds the primer or whether unwinding by NC requires the RNA genome. We monitored the annealing process in solution by using fluorescence resonance energy transfer (FRET). Distance measurements demonstrate unequivocally that the tRNA acceptor stem is not substantially unwound by NC in the absence of the RNA genome, that is, unwinding is not separable from hybridization. Moreover, FRET measurements show that both heat- and NC-mediated annealing result in an approximately 40-A increase in the separation of the two ends of the tRNA acceptor arm on binding to the template. This large increase in separation of the two ends suggests a complete displacement of the nonhybridized strand of the acceptor stem in the initiation complex.

  2. Large gene overlaps and tRNA processing in the compact mitochondrial genome of the crustacean Armadillidium vulgare

    PubMed Central

    Doublet, Vincent; Ubrig, Elodie; Alioua, Abdelmalek; Bouchon, Didier; Marcadé, Isabelle; Maréchal-Drouard, Laurence

    2015-01-01

    A faithful expression of the mitochondrial DNA is crucial for cell survival. Animal mitochondrial DNA (mtDNA) presents a highly compact gene organization. The typical 16.5 kbp animal mtDNA encodes 13 proteins, 2 rRNAs and 22 tRNAs. In the backyard pillbug Armadillidium vulgare, the rather small 13.9 kbp mtDNA encodes the same set of proteins and rRNAs as compared to animal kingdom mtDNA, but seems to harbor an incomplete set of tRNA genes. Here, we first confirm the expression of 13 tRNA genes in this mtDNA. Then we show the extensive repair of a truncated tRNA, the expression of tRNA involved in large gene overlaps and of tRNA genes partially or fully integrated within protein-coding genes in either direct or opposite orientation. Under selective pressure, overlaps between genes have been likely favored for strong genome size reduction. Our study underlines the existence of unknown biochemical mechanisms for the complete gene expression of A. vulgare mtDNA, and of co-evolutionary processes to keep overlapping genes functional in a compacted mitochondrial genome. PMID:26361137

  3. Large gene overlaps and tRNA processing in the compact mitochondrial genome of the crustacean Armadillidium vulgare.

    PubMed

    Doublet, Vincent; Ubrig, Elodie; Alioua, Abdelmalek; Bouchon, Didier; Marcadé, Isabelle; Maréchal-Drouard, Laurence

    2015-01-01

    A faithful expression of the mitochondrial DNA is crucial for cell survival. Animal mitochondrial DNA (mtDNA) presents a highly compact gene organization. The typical 16.5 kbp animal mtDNA encodes 13 proteins, 2 rRNAs and 22 tRNAs. In the backyard pillbug Armadillidium vulgare, the rather small 13.9 kbp mtDNA encodes the same set of proteins and rRNAs as compared to animal kingdom mtDNA, but seems to harbor an incomplete set of tRNA genes. Here, we first confirm the expression of 13 tRNA genes in this mtDNA. Then we show the extensive repair of a truncated tRNA, the expression of tRNA involved in large gene overlaps and of tRNA genes partially or fully integrated within protein-coding genes in either direct or opposite orientation. Under selective pressure, overlaps between genes have been likely favored for strong genome size reduction. Our study underlines the existence of unknown biochemical mechanisms for the complete gene expression of A. vulgare mtDNA, and of co-evolutionary processes to keep overlapping genes functional in a compacted mitochondrial genome.

  4. Synthetic RNA-cleaving molecules mimicking ribonuclease A active center. Design and cleavage of tRNA transcripts.

    PubMed Central

    Podyminogin, M A; Vlassov, V V; Giegé, R

    1993-01-01

    RNA cleaving molecules were synthesized by conjugating imidazole residues imitating the essential imidazoles in the active center of pancreatic ribonuclease to an intercalating compound, derivative of phenazine capable of binding to the double stranded regions of polynucleotides. Action of the molecules on tRNA was investigated. It was found, that some of the compounds bearing two imidazole residues cleave tRNA under physiological conditions. The cleavage reaction shows a bell-shaped pH dependence with a maximum at pH 7.0 indicating participation of protonated and non-protonated imidazole residues in the process. Under the conditions stabilizing the tRNA structure, a tRNAAsp transcript was cleaved preferentially at the junctions of the stem and loop regions of the cloverleaf tRNA fold, at the five positions C56, C43, C20.1, U13, and U8, with a marked preference for C56. This cleavage pattern is consistent with a hydrolysis mechanism involving non-covalent binding of the compounds to the double-stranded regions of tRNA followed by an attack of the imidazole residues at the juxtaposed flexible single-stranded regions of the molecule. The compounds provide new probes for the investigation of RNA structure in solution and potential reactive groups for antisense oligonucleotide derivatives. Images PMID:7507235

  5. Activity of formylphosphate in the reaction catalyzed by formyltetrahydrofolate synthetase

    SciTech Connect

    Jahansouz, H.; Kofron, J.L.; Smithers, G.W.; Himes, R.H.; Reed, G.H.

    1986-05-01

    Formylphosphate (FP), a putative enzyme-bound intermediate in the reaction catalyzed by N/sup 10/-formylH/sub 4/folate synthetase, was synthesized from formylfluoride and Pi. Measurement of hydrolysis rates by /sup 31/P NMR showed that FP is very unstable with a half-life of 48 min at 20/sup 0/C and pH 7. At pH 7 hydrolysis occurs with O-P bond cleavage as shown by /sup 18/O incorporation from /sup 18/O-H/sub 2/O into Pi. The substrate activity of FP was tested in the reaction catalyzed by N/sup 10/-formylH/sub 4/folate synthetase isolated from Clostridium cylindrosporum. MgATP + H/sub 4/folate + HCOO/sup -/ in equilibrium MgADP + Pi +N/sup 10/-formylH/sub 4/folate FP supports the reaction in both the forward and reverse directions. Thus, N/sup 10/-formylH/sub 4/folate is produced from H/sub 4/-folate and FP but only if ADP is present, and ATP is produced from FP and ADP but only if H/sub 4/folate is present. The requirements for H/sub 4/folate in the synthesis of ATP from ADP and FP and for ADP in the synthesis of N/sup 10/-formylH/sub 4/folate from FP and H/sub 4/folate, are consistent with past kinetic and isotope exchange studies which showed that the reaction proceeds by a sequential mechanism and that all three substrates must be present for any reaction to occur.

  6. Transformation of Bacillus Subtilis with cloned thymidylate synthetases

    SciTech Connect

    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)

  7. Identification of pantoate kinase and phosphopantothenate synthetase from Methanospirillum hungatei.

    PubMed

    Katoh, Hiroki; Tamaki, Hideyuki; Tokutake, Yuka; Hanada, Satoshi; Chohnan, Shigeru

    2013-04-01

    Pantothenate synthetase (PanC) and pantothenate kinase which function in the canonical coenzyme A (CoA) biosynthetic pathway cannot be found in most archaea. COG1829 and COG1701 intrinsic to archaea were proposed as the candidate proteins for producing 4'-phosphopantothenate instead, and the COG1701 protein from Methanosarcina mazei was assigned as PanC. Meanwhile, the Thermococcus kodakarensis COG1829 and COG1701 proteins were biochemically identified as novel enzymes, i.e., pantoate kinase (PoK) and phosphopantothenate synthetase (PPS). In this study, the functions of Mhun_0831 (COG1829) and Mhun_0832 (COG1701) from Methanospirillum hungatei were identified, and the recombinant enzymes were partially characterized. Plasmids simultaneously possessing the two genes encoding Mhun_0831 and Mhun_0832 complemented the poor growth of the temperature-sensitive Escherichia coli pantothenate kinase mutant ts9. The recombinant Mhun_0831 and Mhun_0832 expressed in E. coli cells exhibited PoK and PPS activities, respectively, being in accord with the functions of T. kodakarensis proteins. The PoK activity was most active at pH 8.5 and 40°C, and accepted ATP and UTP as a phosphate donor. Although CoA did not affect the PoK activity, the end product considerably accelerated the PPS activity. The homologs of both proteins are widely conserved in most archaeal genomes. Taken together, our findings indicate that archaea can synthesize CoA through the unique pathway involving PoK and PPS, in addition to the canonical one that the order Thermoplasmatales employs.

  8. Evaluation of Multi-tRNA Synthetase Complex by Multiple Reaction Monitoring Mass Spectrometry Coupled with Size Exclusion Chromatography

    PubMed Central

    Kim, Jun Seok; Lee, Cheolju

    2015-01-01

    Eight aminoacyl-tRNA synthetases (M, K, Q, D, R, I, EP and LARS) and three auxiliary proteins (AIMP1, 2 and 3) are known to form a multi-tRNA synthetase complex (MSC) in mammalian cells. We combined size exclusion chromatography (SEC) with reversed-phase liquid chromatography multiple reaction monitoring mass spectrometry (RPLC-MRM-MS) to characterize MSC components and free ARS proteins in human embryonic kidney (HEK 293T) cells. Crude cell extract and affinity-purified proteins were fractionated by SEC in non-denaturing state and ARSs were monitored in each fraction by MRM-MS. The eleven MSC components appeared mostly in earlier SEC fractions demonstrating their participation in complex formation. TARSL2 and AIMP2-DX2, despite their low abundance, were co-purified with KARS and detected in the SEC fractions, where MSC appeared. Moreover, other large complex-forming ARS proteins, such as VARS and FARS, were detected in earlier fractions. The MRM-MS results were further confirmed by western blot analysis. Our study demonstrates usefulness of combined SEC-MRM analysis for the characterization of protein complexes and in understanding the behavior of minor isoforms or variant proteins. PMID:26544075

  9. TRMT5 Mutations Cause a Defect in Post-transcriptional Modification of Mitochondrial tRNA Associated with Multiple Respiratory-Chain Deficiencies

    PubMed Central

    Powell, Christopher A.; Kopajtich, Robert; D’Souza, Aaron R.; Rorbach, Joanna; Kremer, Laura S.; Husain, Ralf A.; Dallabona, Cristina; Donnini, Claudia; Alston, Charlotte L.; Griffin, Helen; Pyle, Angela; Chinnery, Patrick F.; Strom, Tim M.; Meitinger, Thomas; Rodenburg, Richard J.; Schottmann, Gudrun; Schuelke, Markus; Romain, Nadine; Haller, Ronald G.; Ferrero, Ileana; Haack, Tobias B.; Taylor, Robert W.; Prokisch, Holger; Minczuk, Michal

    2015-01-01

    Deficiencies in respiratory-chain complexes lead to a variety of clinical phenotypes resulting from inadequate energy production by the mitochondrial oxidative phosphorylation system. Defective expression of mtDNA-encoded genes, caused by mutations in either the mitochondrial or nuclear genome, represents a rapidly growing group of human disorders. By whole-exome sequencing, we identified two unrelated individuals carrying compound heterozygous variants in TRMT5 (tRNA methyltransferase 5). TRMT5 encodes a mitochondrial protein with strong homology to members of the class I-like methyltransferase superfamily. Both affected individuals presented with lactic acidosis and evidence of multiple mitochondrial respiratory-chain-complex deficiencies in skeletal muscle, although the clinical presentation of the two affected subjects was remarkably different; one presented in childhood with failure to thrive and hypertrophic cardiomyopathy, and the other was an adult with a life-long history of exercise intolerance. Mutations in TRMT5 were associated with the hypomodification of a guanosine residue at position 37 (G37) of mitochondrial tRNA; this hypomodification was particularly prominent in skeletal muscle. Deficiency of the G37 modification was also detected in human cells subjected to TRMT5 RNAi. The pathogenicity of the detected variants was further confirmed in a heterologous yeast model and by the rescue of the molecular phenotype after re-expression of wild-type TRMT5 cDNA in cells derived from the affected individuals. Our study highlights the importance of post-transcriptional modification of mitochondrial tRNAs for faithful mitochondrial function. PMID:26189817

  10. Identification of acyl-CoA synthetases involved in the mammalian sphingosine 1-phosphate metabolic pathway.

    PubMed

    Ohkuni, Aya; Ohno, Yusuke; Kihara, Akio

    2013-12-13

    Sphingosine 1-phosphate (S1P) plays important roles both as a bioactive lipid molecule and an intermediate of the sphingolipid-to-glycerophospholipid metabolic pathway. To identify human acyl-CoA synthetases (ACSs) involved in S1P metabolism, we cloned all 26 human ACS genes and examined their abilities to restore deficient sphingolipid-to-glycerophospholipid metabolism in a yeast mutant lacking two ACS genes, FAA1 and FAA4. Here, in addition to the previously identified ACSL family members (ACSL1, 3, 4, 5, and 6), we found that ACSVL1, ACSVL4, and ACSBG1 also restored metabolism. All 8 ACSs were localized either exclusively or partly to the endoplasmic reticulum (ER), where S1P metabolism takes place. We previously proposed the entire S1P metabolic pathway from results obtained using yeast cells, i.e., S1P is metabolized to glycerophospholipids via trans-2-hexadecenal, trans-2-hexadecenoic acid, trans-2-hexadecenoyl-CoA, and palmitoyl-CoA. However, as S1P is not a naturally occurring long-chain base 1-phosphate in yeast, the validity of this pathway required further verification using mammalian cells. In the present study, we treated HeLa cells with the ACS inhibitor triacsin C and found that inhibition of ACSs resulted in accumulation of trans-2-hexadecenoic acid as in ACS mutant yeast. From these results, we conclude that S1P is metabolized by a common pathway in eukaryotes.

  11. Dark-induced and organ-specific expression of two asparagine synthetase genes in Pisum sativum.

    PubMed Central

    Tsai, F Y; Coruzzi, G M

    1990-01-01

    Nucleotide sequence analysis of cDNAs for asparagine synthetase (AS) of Pisum sativum has uncovered two distinct AS mRNAs (AS1 and AS2) encoding polypeptides that are highly homologous to the human AS enzyme. The amino-terminal residues of both AS1 and AS2 polypeptides are identical to the glutamine-binding domain of the human AS enzyme, indicating that the full-length AS1 and AS2 cDNAs encode glutamine-dependent AS enzymes. Analysis of nuclear DNA shows that AS1 and AS2 are each encoded by single genes in P.sativum. Gene-specific Northern blot analysis reveals that dark treatment induces high-level accumulation of AS1 mRNA in leaves, while light treatment represses this effect as much as 30-fold. Moreover, the dark-induced accumulation of AS1 mRNA was shown to be a phytochrome-mediated response. Both AS1 and AS2 mRNAs also accumulate to high levels in cotyledons of germinating seedlings and in nitrogen-fixing root nodules. These patterns of AS gene expression correlate well with the physiological role of asparagine as a nitrogen transport amino acid during plant development. Images Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:1968003

  12. IL-1beta stimulates argininosuccinate synthetase gene expression through NF-kappaB in Caco-2 cells.

    PubMed

    Brasse-Lagnel, Carole; Lavoinne, Alain; Fairand, Alain; Vavasseur, Karine; Husson, Annie

    2005-05-01

    Argininosuccinate synthetase (ASS) is limiting the arginine synthesis and can be stimulated by immunostimulants. We previously identified a putative NF-kappaB element in the human ASS gene promoter but its functionality was unknown (Husson et al., Eur. J. Biochem. 270 (2003) 1887). In the present study, using Caco-2 cells, a human enterocyte line, we demonstrate that IL-1beta rapidly induces the expression of the ASS gene at a transcriptional level through NF-kappaB activation. Using gel shift assay and double-strand oligonucleotide sequence of the identified putative NF-kappaB binding site of the ASS promoter, we provide evidence that NF-kappaB may functionally interact with this element.

  13. Genetics, Structure and Functional Features of the RNA Modification Enzyme, tRNA psi Synthase I.

    DTIC Science & Technology

    1985-01-01

    been sequenced. In collaboration with M. Winkler (Northwestern Univ.), it has been found that the hisT gene is part of an operon containing a second...Recently, the hisT operon has been inserted into a runaway replication plasmi, ,BEU50, which can overproduce the enzyme by another 3-5-fold. In this strain...in the hisT operon are tightly linked, their products appear to be function~llyindepBpdent. PSUILin modify all of the hisT isoacceptors of tRNA

  14. sigma, a repetitive element found adjacent to tRNA genes of yeast.

    PubMed Central

    del Rey, F J; Donahue, T F; Fink, G R

    1982-01-01

    sigma is a DNA element of about 340 base pairs (bp) that is repeated many times in the yeast genome. The element has 8-bp inverted repeats at its ends and is flanked by 5-bp direct repeats. The 5-bp repeats are different for each sigma and have no homology with the ends of the sigma sequence. sigma is located 16 or 18 bp from the 5' end of several tRNA genes. Southern analysis of different yeast strains shows that the pattern of hybridization is different even for closely related strains. Images PMID:6287468

  15. Was there a universal tRNA before specialized tRNAs came into existence?

    NASA Technical Reports Server (NTRS)

    Lacey, James C., Jr.; Staves, Mark P.

    1990-01-01

    It is generally true that evolving systems begin simply and become more complex in the evolutionary process. For those who try to understand the origin of a biochemical system, what is required is the development of an idea as to what simpler system preceded the present one. A hypothesis is presented that a universal tRNA molecule, capable of reading many codons, may have preceded the appearance of individual tRNAs. Evidence seems to suggest that this molecule may have been derived from a common ancestor of the contemporary 5S rRNAs and tRNAs.

  16. Optical Kerr effect of tRNA solution induced by femtosecond laser pulses

    NASA Astrophysics Data System (ADS)

    Kucia, Weronika E.; Sharma, Gargi; Joseph, Cecil S.; Sarbak, Szymon; Oliver, Cameron; Dobek, Andrzej; Giles, Robert H.

    2016-10-01

    The optical Kerr effect (OKE) in a transfer ribonucleic acid (tRNA) solution induced by femtosecond pulses of linearly polarized pump light (λi = 800 nm) and sounded by probe light (λp = 800 nm) was studied. The measurements were performed to find nonlinear optical parameters describing a single molecule (molecular Kerr constant K, mean nonlinear third order optical polarizability cpi) and to compare them with our previous OKE results obtained in ns and ps time range. The OKE experiment has proven to be an efficient method to obtain the nonlinear parameters of single molecules in solution, which reflects dynamic structure changes.

  17. Evidence for two immunologically distinct acetyl-coenzyme A synthetases in yeast

    NASA Technical Reports Server (NTRS)

    Satyanarayana, T.; Mandel, A. D.; Klein, H. P.

    1974-01-01

    Evidence is presented that clearly establishes the presence of two acetyl-CoA synthetases in Saccharomyces cerevisiae, one elaborated under 'aerobic' conditions, the other under 'nonaerobic' conditions. The antibody produced by each enzyme is immunologically specific.

  18. Preparation and cross-reactivity of anti-avian glutamine synthetase antibody.

    PubMed

    Smith, D D; Vorhaben, J E; Campbell, J W

    1983-04-01

    Rabbit antibody to chicken liver mitochondrial glutamine synthetase was purified by immunoaffinity chromatography for analysis of the immunological relatedness of vertebrate glutamine synthetases. The antibody cross-reacted with enzymes from representatives of all five vertebrate classes, indicating a high degree of evolutionary conservatism in the structure of the enzymes. A unique aspect of the immunological similarity of these enzymes is that it exists between cytosolic and mitochondrial enzymes which are, in general, immunologically distinct. The antibody did not cross-react with two insect glutamine synthetases. Compositional difference indices, calculated from the amino acid compositions of glutamine synthetases from several species, gave a mean estimate of over 80% sequence homology for the vertebrate enzymes. The avian mitochondrial enzyme gave a mean 78% homology with the mammalian cytosolic enzyme.

  19. A homozygous truncating mutation in PUS3 expands the role of tRNA modification in normal cognition

    PubMed Central

    Shaheen, Ranad; Han, Lu; Faqeih, Eissa; Ewida, Nour; Alobeid, Eman; Phizicky, Eric M.; Alkuraya, Fowzan S.

    2016-01-01

    Intellectual disability is a common and highly heterogeneous disorder etiologically. In a multiplex consanguineous family, we applied autozygosity mapping and exome sequencing and identified a novel homozygous truncating mutation in PUS3 that fully segregates with the intellectual disability phenotype. Consistent with the known role of Pus3 in isomerizing uracil to pseudouridine at positions 38 and 39 in tRNA, we found a significant reduction in this post-transcriptional modification of tRNA in patient cells. Our finding adds to a growing list of intellectual disability disorders that are caused by perturbation of various tRNA modifications, which highlights the sensitivity of the brain to these highly conserved processes. PMID:27055666

  20. tRNA genes of Streptomyces lividans: new sequences and comparison of structure and organization with those of other bacteria.

    PubMed Central

    Sedlmeier, R; Werner, T; Kieser, H M; Hopwood, D A; Schmieger, H

    1994-01-01

    Three closely linked Streptomyces lividans tRNA genes encoding two tRNA(Lys)s and a tRNA(Gly) were cloned and sequences. The structure of tRNA(Gly) is unusual for eubacterial tRNAs. Including those in previous reports (R. Sedlmeier and H. Schmieger, Nucleic Acids Res. 18:4027, 1990, and R. Sedlmeier, G. Linti, K. Gregor, and H. Schmieger, Gene 132:125-130, 1993), 18 S. lividans tRNA genes were physically mapped on the chromosome of the closely related strain Streptomyces coelicolor A3(2). The structure and organization of tRNA genes of S. lividans and S. coelicolor are compared with those of Escherichia coli and Bacillus subtilis. PMID:8071238

  1. Phylogeny from function: evidence from the molecular fossil record that tRNA originated in replication, not translation.

    PubMed

    Maizels, N; Weiner, A M

    1994-07-19

    We propose a phylogeny for the evolution of tRNA that is based on the ubiquity and conservation of tRNA-like structures in the replication of contemporary genomes. This phylogeny is unique in suggesting that the function of tRNA in replication dates back to the very beginnings of life on earth, before the advent of templated protein synthesis. The origin we propose for tRNA has distinct implications for the order in which other components of the modern translational apparatus evolved. We further suggest that the "top half" of modern tRNA-a coaxial stack of the acceptor stem on the T psi C arm--is the ancient structural and functional domain and that the "bottom half" of tRNA--a coaxial stack of the dihydrouracil arm on the anticodon arm--arose later to provide additional specificity.

  2. Phylogeny from function: evidence from the molecular fossil record that tRNA originated in replication, not translation.

    PubMed Central

    Maizels, N; Weiner, A M

    1994-01-01

    We propose a phylogeny for the evolution of tRNA that is based on the ubiquity and conservation of tRNA-like structures in the replication of contemporary genomes. This phylogeny is unique in suggesting that the function of tRNA in replication dates back to the very beginnings of life on earth, before the advent of templated protein synthesis. The origin we propose for tRNA has distinct implications for the order in which other components of the modern translational apparatus evolved. We further suggest that the "top half" of modern tRNA-a coaxial stack of the acceptor stem on the T psi C arm--is the ancient structural and functional domain and that the "bottom half" of tRNA--a coaxial stack of the dihydrouracil arm on the anticodon arm--arose later to provide additional specificity. Images PMID:8041690

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

    PubMed Central

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

    2013-01-01

    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

  4. Diffuse glutamine synthetase overexpression restricted to areas of peliosis in a β-catenin-activated hepatocellular adenoma: a potential pitfall in glutamine synthetase interpretation.

    PubMed

    Berry, Ryan S; Gullapalli, Rama R; Wu, Jin; Morris, Katherine; Hanson, Joshua A

    2014-08-01

    Hepatocellular adenomas have recently been classified into four subtypes based on molecular findings: hepatocyte nuclear factor 1α (HNF1α) inactivated, inflammatory/telangiectatic, β-catenin activated, and unclassifiable. β-catenin-activated adenomas have the potential for malignant transformation and are thus important to recognize. Diffuse glutamine synthetase immunohistochemical positivity has been shown to be a reliable surrogate marker for β-catenin activation, though variations in staining patterns may be difficult to interpret. We report a case of a peliotic adenoma that was morphologically consistent with a β-catenin wild-type hepatocellular adenoma but harbored a β-catenin mutation by molecular analysis. The tumor lacked nuclear β-catenin positivity and demonstrated a hitherto undescribed pattern of glutamine synthetase overexpression restricted to areas of peliosis with mostly negative staining in non-peliotic areas. This pattern was initially interpreted as physiologic and may represent a potential pitfall in glutamine synthetase interpretation.

  5. Effect of intron mutations on processing and function of Saccharomyces cerevisiae SUP53 tRNA in vitro and in vivo.

    PubMed Central

    Strobel, M C; Abelson, J

    1986-01-01

    The Saccharomyces cerevisiae leucine-inserting amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the relationship between precursor tRNA structure and mature tRNA function. This gene encodes a pre-tRNA which contains a 32-base intron. The mature tRNASUP53 contains a 5-methylcytosine modification of the anticodon wobble base. Mutations were made in the SUP53 intron. These mutant genes were transcribed in an S. cerevisiae nuclear extract preparation. In this extract, primary tRNA gene transcripts are end-processed and base modified after addition of cofactors. The base modifications made in vitro were examined, and the mutant pre-tRNAs were analyzed for their ability to serve as substrates for partially purified S. cerevisiae tRNA endonuclease and ligase. Finally, the suppressor function of these mutant tRNA genes was assayed after their integration into the S. cerevisiae genome. Mutant analysis showed that the totally intact precursor tRNA, rather than any specific sequence or structure of the intron, was necessary for efficient nonsense suppression by tRNASUP53. Less efficient suppressor activity correlated with the absence of the 5-methylcytosine modification. Most of the intron-altered precursor tRNAs were successfully spliced in vitro, indicating that modifications are not critical for recognition by the tRNA endonuclease and ligase. Images PMID:3537724

  6. The identification of new cytosolic glutamine synthetase and asparagine synthetase genes in barley (Hordeum vulgare L.), and their expression during leaf senescence.

    PubMed

    Avila-Ospina, Liliana; Marmagne, Anne; Talbotec, Joël; Krupinska, Karin; Masclaux-Daubresse, Céline

    2015-04-01

    Glutamine synthetase and asparagine synthetase are two master enzymes involved in ammonium assimilation in plants. Their roles in nitrogen remobilization and nitrogen use efficiency have been proposed. In this report, the genes coding for the cytosolic glutamine synthetases (HvGS1) and asparagine synthetases (HvASN) in barley were identified. In addition to the three HvGS1 and two HvASN sequences previously reported, two prokaryotic-like HvGS1 and three HvASN cDNA sequences were identified. Gene structures were then characterized, obtaining full genomic sequences. The response of the five HvGS1 and five HvASN genes to leaf senescence was then studied. Developmental senescence was studied using primary and flag leaves. Dark-exposure or low-nitrate conditions were also used to trigger stress-induced senescence. Well-known senescence markers such as the chlorophyll and Rubisco contents were monitored in order to characterize senescence levels in the different leaves. The three eukaryotic-like HvGS1_1, HvGS1_2, and HvGS1_3 sequences showed the typical senescence-induced reduction in gene expression described in many plant species. By contrast, the two prokaryotic-like HvGS1_4 and HvGS1_5 sequences were repressed by leaf senescence, similar to the HvGS2 gene, which encodes the chloroplast glutamine synthetase isoenzyme. There was a greater contrast in the responses of the five HvASN and this suggested that these genes are needed for N remobilization in senescing leaves only when plants are well fertilized with nitrate. Responses of the HvASN sequences to dark-induced senescence showed that there are two categories of asparagine synthetases, one induced in the dark and the other repressed by the same conditions.

  7. Recurrent seizures and brain pathology after inhibition of glutamine synthetase in the hippocampus in rats.

    PubMed

    Eid, Tore; Ghosh, Arko; Wang, Yue; Beckström, Henning; Zaveri, Hitten P; Lee, Tih-Shih W; Lai, James C K; Malthankar-Phatak, Gauri H; de Lanerolle, Nihal C

    2008-08-01

    An excess of extracellular glutamate in the hippocampus has been linked to the generation of recurrent seizures and brain pathology in patients with medically intractable mesial temporal lobe epilepsy (MTLE). However, the mechanism which results in glutamate excess in MTLE remains unknown. We recently reported that the glutamate-metabolizing enzyme glutamine synthetase is deficient in the hippocampus in patients with MTLE, and we postulated that this deficiency is critically involved in the pathophysiology of the disease. To further explore the role of glutamine synthetase in MTLE we created a novel animal model of hippocampal glutamine synthetase deficiency by continuous (approximately 28 days) microinfusion of methionine sulfoximine (MSO: 0.625 to 2.5 microg/h) unilaterally into the hippocampus in rats. This treatment led to a deficiency in hippocampal glutamine synthetase activity by 82-97% versus saline. The majority (>95%) of the MSO-treated animals exhibited recurrent seizures that continued for several weeks. Some of the MSO-treated animals exhibited neuropathological features that were similar to mesial temporal sclerosis, such as hippocampal atrophy and patterned loss of hippocampal neurons. However, many MSO-treated animals displayed only minimal injury to the hippocampus, with no clear evidence of mesial temporal sclerosis. These findings support the hypothesis that a deficiency in hippocampal glutamine synthetase causes recurrent seizures, even in the absence of classical mesial temporal sclerosis, and that restoration of glutamine synthetase may represent a novel approach to therapeutic intervention in this disease.

  8. Structure of tRNA Dimethylallyltransferase: RNA Modification through a Channel

    SciTech Connect

    Xie, Wei; Zhou, Chun; Huang, Raven H.

    2008-09-04

    Dimethylallyltransferase (DMATase) transfers a five-carbon isoprenoid moiety from dimethylallyl pyrophosphate (DMAPP) to the amino group of adenosine at position 37 of certain tRNAs. Reported here are the crystal structures of Pseudomonas aeruginosa DMATase alone and in complex with pyrophosphate at 1.9 {angstrom} resolution. Surprisingly, the enzyme possesses a central channel spanning the entire width of the enzyme. Both the accepting substrate tRNA and the donating substrate DMAPP appear to enter the channel from opposite sides in an ordered sequence, with tRNA first and DMAPP second, and the RNA modification reaction occurs in the middle of the channel once the two substrates have met. The structure of DMATase is homologous to a class of small soluble kinases involved in biosynthesis of nucleotide precursors for nucleic acids, indicating its possibly evolutionary origin. Furthermore, specific recognition of the pyrophosphate by a conserved loop in DMATase, similar to the P-loop commonly seen in diverse nucleotide-binding proteins, demonstrates that DMATase is structurally and mechanistically distinct from farnesyltransferase, another family of prenyltransferases involved in protein modification.

  9. Cross-Talk between Dnmt2-Dependent tRNA Methylation and Queuosine Modification.

    PubMed

    Ehrenhofer-Murray, Ann E

    2017-02-10

    Enzymes of the Dnmt2 family of methyltransferases have yielded a number of unexpected discoveries. The first surprise came more than ten years ago when it was realized that, rather than being DNA methyltransferases, Dnmt2 enzymes actually are transfer RNA (tRNA) methyltransferases for cytosine-5 methylation, foremost C38 (m5C38) of tRNAAsp. The second unanticipated finding was our recent discovery of a nutritional regulation of Dnmt2 in the fission yeast Schizosaccharomyces pombe. Significantly, the presence of the nucleotide queuosine in tRNAAsp strongly stimulates Dnmt2 activity both in vivo and in vitro in S. pombe. Queuine, the respective base, is a hypermodified guanine analog that is synthesized from guanosine-5'-triphosphate (GTP) by bacteria. Interestingly, most eukaryotes have queuosine in their tRNA. However, they cannot synthesize it themselves, but rather salvage it from food or from gut microbes. The queuine obtained from these sources comes from the breakdown of tRNAs, where the queuine ultimately was synthesized by bacteria. Queuine thus has been termed a micronutrient. This review summarizes the current knowledge of Dnmt2 methylation and queuosine modification with respect to translation as well as the organismal consequences of the absence of these modifications. Models for the functional cooperation between these modifications and its wider implications are discussed.

  10. Structural basis for tRNA modification by Elp3 from Dehalococcoides mccartyi.

    PubMed

    Glatt, Sebastian; Zabel, Rene; Kolaj-Robin, Olga; Onuma, Osita F; Baudin, Florence; Graziadei, Andrea; Taverniti, Valerio; Lin, Ting-Yu; Baymann, Frauke; Séraphin, Bertrand; Breunig, Karin D; Müller, Christoph W

    2016-09-01

    During translation elongation, decoding is based on the recognition of codons by corresponding tRNA anticodon triplets. Molecular mechanisms that regulate global protein synthesis via specific base modifications in tRNA anticodons are receiving increasing attention. The conserved eukaryotic Elongator complex specifically modifies uridines located in the wobble base position of tRNAs. Mutations in Elongator subunits are associated with certain neurodegenerative diseases and cancer. Here we present the crystal structure of D. mccartyi Elp3 (DmcElp3) at 2.15-Å resolution. Our results reveal an unexpected arrangement of Elp3 lysine acetyltransferase (KAT) and radical S-adenosyl methionine (SAM) domains, which share a large interface and form a composite active site and tRNA-binding pocket, with an iron-sulfur cluster located in the dimerization interface of two DmcElp3 molecules. Structure-guided mutagenesis studies of yeast Elp3 confirmed the relevance of our findings for eukaryotic Elp3s and should aid in understanding the cellular functions and pathophysiological roles of Elongator.

  11. Nucleotide sequence of a lysine tRNA from Bacillus subtilis.

    PubMed Central

    Yamada, Y; Ishikura, H

    1977-01-01

    A lysine tRNA (tRNA1Lys) was purified from Bacillus subtilis W168 by a consecutive use of several column chromatographic systems. The nucleotide sequence was determined to be pG-A-G-C-C-A-U-U-A-G-C-U-C-A-G-U-D-G-G-D-A-G-A-G-C-A-U-C-U-G-A-C-U-U(U*)-U-U-K-A-psi-C-A-G-A-G-G-m7G(G)-U-C-G-A-A-G-G-T-psi-C-G-A-G-U-C-C-U-U-C-A-U-G-G-C-U-C-A-C-C-AOH, where K and U* are unidentified nucleosides. The nucleosides of U34 and m7G46 were partially substituted with U* and G, respectively. The binding ability of lysyl-tRNA1Lys to Escherichia coli ribosomes was stimulated with ApApA as well as ApApG. PMID:414208

  12. Complete 5' and 3' end maturation of group II intron-containing tRNA precursors.

    PubMed Central

    Vogel, J; Hess, W R

    2001-01-01

    Higher plant chloroplasts provide the only experimentally validated example of functional tRNA genes that are disrupted by group II introns. Here, precursor transcripts for tRNA(Gly)(UCC), tRNA(Val)(UAC), and tRNA(Ala)(UGC) were investigated for processing of 5' leader and 3' trailer sequences in vivo. Use of intron-specific primer pairs and inclusion of a barley chloroplast splicing mutant specifically allowed us to evaluate the potential effect of intervening sequences that disrupt tRNA secondary and tertiary structures. The data suggest that (1) neither integrity of the dihydrouridine nor the anticodon domain is required for the nucleotidyltransferase-mediated addition of 3'-terminal CCA; (2) interruption of these two structural elements by group II introns does not interfere with nucleotide-specific 5' maturation by RNase P; (3) processing intermediates of chloroplast tRNAs can be 3' polyadenylated; and (4) plastid DNA-encoded proteins are not required for 3' and 5' maturation of plastid tRNAs. PMID:11233985

  13. The Levels of a Universally Conserved tRNA Modification Regulate Cell Growth*

    PubMed Central

    Rojas-Benitez, Diego; Thiaville, Patrick C.; de Crécy-Lagard, Valérie; Glavic, Alvaro

    2015-01-01

    N6-Threonylcarbamoyl-adenosine (t6A) is a universal modification occurring at position 37 in nearly all tRNAs that decode A-starting codons, including the eukaryotic initiator tRNA (tRNAiMet). Yeast lacking central components of the t6A synthesis machinery, such as Tcs3p (Kae1p) or Tcs5p (Bud32p), show slow-growth phenotypes. In the present work, we show that loss of the Drosophila tcs3 homolog also leads to a severe reduction in size and demonstrate, for the first time in a non-microbe, that Tcs3 is required for t6A synthesis. In Drosophila and in mammals, tRNAiMet is a limiting factor for cell and animal growth. We report that the t6A-modified form of tRNAiMet is the actual limiting factor. We show that changing the proportion of t6A-modified tRNAiMet, by expression of an un-modifiable tRNAiMet or changing the levels of Tcs3, regulate target of rapamycin (TOR) kinase activity and influences cell and animal growth in vivo. These findings reveal an unprecedented relationship between the translation machinery and TOR, where translation efficiency, limited by the availability of t6A-modified tRNA, determines growth potential in eukaryotic cells. PMID:26063805

  14. A Divalent Metal Ion-Dependent N1-Methyl Transfer to G37-tRNA

    PubMed Central

    Sakaguchi, Reiko; Lahoud, Georges; Christian, Thomas; Gamper, Howard; Hou, Ya-Ming

    2014-01-01

    The catalytic mechanism of the majority of S-adenosyl methionine (AdoMet)-dependent methyl transferases requires no divalent metal ions. Here we report that methyl transfer from AdoMet to N1 of G37-tRNA, catalyzed by the bacterial TrmD enzyme, is strongly dependent on divalent metal ions and that Mg2+ is the most physiologically relevant. Kinetic isotope analysis, metal rescue, and spectroscopic measurements indicate that Mg2+ is not involved in substrate binding, but in promoting methyl transfer. Based on the pH-activity profile indicating one proton transfer during the TrmD reaction, we propose a catalytic mechanism in which the role of Mg2+ is to help to increase the nucleophilicity of N1 of G37 and stabilize the negative developing charge on O6 during attack on the methyl sulfonium of AdoMet. This work demonstrates how Mg2+ contributes to the catalysis of AdoMet-dependent methyl transfer in one of the most crucial post-transcriptional modifications to tRNA. PMID:25219964

  15. From End to End: tRNA Editing at 5'- and 3'-Terminal Positions

    PubMed Central

    Betat, Heike; Long, Yicheng; Jackman, Jane E.; Mörl, Mario

    2014-01-01

    During maturation, tRNA molecules undergo a series of individual processing steps, ranging from exo- and endonucleolytic trimming reactions at their 5'- and 3'-ends, specific base modifications and intron removal to the addition of the conserved 3'-terminal CCA sequence. Especially in mitochondria, this plethora of processing steps is completed by various editing events, where base identities at internal positions are changed and/or nucleotides at 5'- and 3'-ends are replaced or incorporated. In this review, we will focus predominantly on the latter reactions, where a growing number of cases indicate that these editing events represent a rather frequent and widespread phenomenon. While the mechanistic basis for 5'- and 3'-end editing differs dramatically, both reactions represent an absolute requirement for generating a functional tRNA. Current in vivo and in vitro model systems support a scenario in which these highly specific maturation reactions might have evolved out of ancient promiscuous RNA polymerization or quality control systems. PMID:25535083

  16. Distinct kinetic determinants for the stepwise CCA addition to tRNA.

    PubMed

    Kim, Sangbumn; Liu, Cuiping; Halkidis, Konstantine; Gamper, Howard B; Hou, Ya-Ming

    2009-10-01

    The universally conserved CCA sequence is present at the 3' terminal 74-76 positions of all active tRNA molecules as a functional tag to participate in ribosome protein synthesis. The CCA enzyme catalyzes CCA synthesis in three sequential steps of nucleotide addition at rapid and identical rates. However, the kinetic determinant of each addition is unknown, thus limiting the insights into the kinetic basis of CCA addition. Using our recently developed single turnover kinetics of Escherichia coli CCA enzyme as a model, we show here that the identical rate of the stepwise CCA addition is determined by distinct kinetic parameters. Specifically, the kinetics of C74 and C75 addition is controlled by the chemistry of nucleotidyl transfer, whereas the kinetics of A76 addition is controlled by a prechemistry conformational transition of the active site. In multiple turnover condition, all three steps are controlled by slow product release, indicating enzyme processivity from one addition to the next. However, the processivity decreases as the enzyme progresses to complete the CCA synthesis. Together, these results suggest the existence of a network of diverse kinetic parameters that determines the overall rate of CCA addition for tRNA maturation.

  17. Cyclodipeptide synthases, a family of class-I aminoacyl-tRNA synthetase-like enzymes involved in non-ribosomal peptide synthesis.

    PubMed

    Sauguet, Ludovic; Moutiez, Mireille; Li, Yan; Belin, Pascal; Seguin, Jérôme; Le Du, Marie-Hélène; Thai, Robert; Masson, Cédric; Fonvielle, Matthieu; Pernodet, Jean-Luc; Charbonnier, Jean-Baptiste; Gondry, Muriel

    2011-05-01

    Cyclodipeptide synthases (CDPSs) belong to a newly defined family of enzymes that use aminoacyl-tRNAs (aa-tRNAs) as substrates to synthesize the two peptide bonds of various cyclodipeptides, which are the precursors of many natural products with noteworthy biological activities. Here, we describe the crystal structure of AlbC, a CDPS from Streptomyces noursei. The AlbC structure consists of a monomer containing a Rossmann-fold domain. Strikingly, it is highly similar to the catalytic domain of class-I aminoacyl-tRNA synthetases (aaRSs), especially class-Ic TyrRSs and TrpRSs. AlbC contains a deep pocket, highly conserved among CDPSs. Site-directed mutagenesis studies indicate that this pocket accommodates the aminoacyl moiety of the aa-tRNA substrate in a way similar to that used by TyrRSs to recognize their tyrosine substrates. These studies also suggest that the tRNA moiety of the aa-tRNA interacts with AlbC via at least one patch of basic residues, which is conserved among CDPSs but not present in class-Ic aaRSs. AlbC catalyses its two-substrate reaction via a ping-pong mechanism with a covalent intermediate in which L-Phe is shown to be transferred from Phe-tRNA(Phe) to an active serine. These findings provide insight into the molecular bases of the interactions between CDPSs and their aa-tRNAs substrates, and the catalytic mechanism used by CDPSs to achieve the non-ribosomal synthesis of cyclodipeptides.

  18. Cyclodipeptide synthases, a family of class-I aminoacyl-tRNA synthetase-like enzymes involved in non-ribosomal peptide synthesis

    PubMed Central

    Sauguet, Ludovic; Moutiez, Mireille; Li, Yan; Belin, Pascal; Seguin, Jérôme; Le Du, Marie-Hélène; Thai, Robert; Masson, Cédric; Fonvielle, Matthieu; Pernodet, Jean-Luc; Charbonnier, Jean-Baptiste; Gondry, Muriel

    2011-01-01

    Cyclodipeptide synthases (CDPSs) belong to a newly defined family of enzymes that use aminoacyl-tRNAs (aa-tRNAs) as substrates to synthesize the two peptide bonds of various cyclodipeptides, which are the precursors of many natural products with noteworthy biological activities. Here, we describe the crystal structure of AlbC, a CDPS from Streptomyces noursei. The AlbC structure consists of a monomer containing a Rossmann-fold domain. Strikingly, it is highly similar to the catalytic domain of class-I aminoacyl-tRNA synthetases (aaRSs), especially class-Ic TyrRSs and TrpRSs. AlbC contains a deep pocket, highly conserved among CDPSs. Site-directed mutagenesis studies indicate that this pocket accommodates the aminoacyl moiety of the aa-tRNA substrate in a way similar to that used by TyrRSs to recognize their tyrosine substrates. These studies also suggest that the tRNA moiety of the aa-tRNA interacts with AlbC via at least one patch of basic residues, which is conserved among CDPSs but not present in class-Ic aaRSs. AlbC catalyses its two-substrate reaction via a ping-pong mechanism with a covalent intermediate in which l-Phe is shown to be transferred from Phe-tRNAPhe to an active serine. These findings provide insight into the molecular bases of the interactions between CDPSs and their aa-tRNAs substrates, and the catalytic mechanism used by CDPSs to achieve the non-ribosomal synthesis of cyclodipeptides. PMID:21296757

  19. An update to polyketide synthase and non-ribosomal synthetase genes and nomenclature in Fusarium.

    PubMed

    Hansen, Frederik T; Gardiner, Donald M; Lysøe, Erik; Fuertes, Patricia Romans; Tudzynski, Bettina; Wiemann, Philipp; Sondergaard, Teis Esben; Giese, Henriette; Brodersen, Ditlev E; Sørensen, Jens Laurids

    2015-02-01

    Members of the genus Fusarium produce a plethora of bioactive secondary metabolites, which can be harmful to humans and animals or have potential in drug development. In this study we have performed comparative analyses of polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) from ten different Fusarium species including F. graminearum (two strains), F. verticillioides, F. solani, F. culmorum, F. pseudograminearum, F. fujikuroi, F. acuminatum, F. avenaceum, F. equiseti, and F. oxysporum (12 strains). This led to identification of 52 NRPS and 52 PKSs orthology groups, respectively, and although not all PKSs and NRPSs are assumed to be intact or functional, the analyses illustrate the huge secondary metabolite potential in Fusarium. In our analyses we identified a core collection of eight NRPSs (NRPS2-4, 6, 10-13) and two PKSs (PKS3 and PKS7) that are conserved in all strains analyzed in this study. The identified PKSs and NRPSs were named based on a previously developed classification system (www.FusariumNRPSPKS.dk). We suggest this system be used when PKSs and NRPSs have to be classified in future sequenced Fusarium strains. This system will facilitate identification of orthologous and non-orthologous NRPSs and PKSs from newly sequenced Fusarium genomes and will aid the scientific community by providing a common nomenclature for these two groups of genes/enzymes.

  20. Cryptosporidium and Toxoplasma Parasites Are Inhibited by a Benzoxaborole Targeting Leucyl-tRNA Synthetase

    PubMed Central

    Liu, Ru-Juan; Lukarska, Maria; Gut, Jiri; Bougdour, Alexandre; Touquet, Bastien; Wang, En-Duo; Li, Xianfeng; Alley, M. R. K.; Freund, Yvonne R.; Rosenthal, Philip J.; Hakimi, Mohamed-Ali

    2016-01-01

    The apicomplexan parasites Cryptosporidium and Toxoplasma are serious threats to human health. Cryptosporidiosis is a severe diarrheal disease in malnourished children and immunocompromised individuals, with the only FDA-approved drug treatment currently being nitazoxanide. The existing therapies for toxoplasmosis, an important pathology in immunocompromised individuals and pregnant women, also have serious limitations. With the aim of developing alternative therapeutic options to address these health problems, we tested a number of benzoxaboroles, boron-containing compounds shown to be active against various infectious agents, for inhibition of the growth of Cryptosporidium parasites in mammalian cells. A 3-aminomethyl benzoxaborole, AN6426, with activity in the micromolar range and with activity comparable to that of nitazoxanide, was identified and further characterized using biophysical measurements of affinity and crystal structures of complexes with the editing domain of Cryptosporidium leucyl-tRNA synthetase (LeuRS). The same compound was shown to be active against Toxoplasma parasites, with the activity being enhanced in the presence of norvaline, an amino acid that can be mischarged by LeuRS. Our observations are consistent with AN6426 inhibiting protein synthesis in both Cryptosporidium and Toxoplasma by forming a covalent adduct with tRNALeu in the LeuRS editing active site and suggest that further exploitation of the benzoxaborole scaffold is a valid strategy to develop novel, much needed antiparasitic agents. PMID:27431220

  1. Structural characterization of Helicobacter pylori dethiobiotin synthetase reveals differences between family members

    SciTech Connect

    Porebski, Przemyslaw J.; Klimecka, Maria; Chruszcz, Maksymilian; Nicholls, Robert A.; Murzyn, Krzysztof; Cuff, Marianne E.; Xu, Xiaohui; Cymborowski, Marcin; Murshudov, Garib N.; Savchenko, Alexei; Edwards, Aled; Minor, Wladek

    2012-07-11

    Dethiobiotin synthetase (DTBS) is involved in the biosynthesis of biotin in bacteria, fungi, and plants. As humans lack this pathway, DTBS is a promising antimicrobial drug target. We determined structures of DTBS from Helicobacter pylori (hpDTBS) bound with cofactors and a substrate analog, and described its unique characteristics relative to other DTBS proteins. Comparison with bacterial DTBS orthologs revealed considerable structural differences in nucleotide recognition. The C-terminal region of DTBS proteins, which contains two nucleotide-recognition motifs, differs greatly among DTBS proteins from different species. The structure of hpDTBS revealed that this protein is unique and does not contain a C-terminal region containing one of the motifs. The single nucleotide-binding motif in hpDTBS is similar to its counterpart in GTPases; however, isothermal titration calorimetry binding studies showed that hpDTBS has a strong preference for ATP. The structural determinants of ATP specificity were assessed with X-ray crystallographic studies of hpDTBS-ATP and hpDTBS-GTP complexes. The unique mode of nucleotide recognition in hpDTBS makes this protein a good target for H. pylori-specific inhibitors of the biotin synthesis pathway.

  2. Discovery of potent anti-tuberculosis agents targeting leucyl-tRNA synthetase.

    PubMed

    Gudzera, Olga I; Golub, Andriy G; Bdzhola, Volodymyr G; Volynets, Galyna P; Lukashov, Sergiy S; Kovalenko, Oksana P; Kriklivyi, Ivan A; Yaremchuk, Anna D; Starosyla, Sergiy A; Yarmoluk, Sergiy M; Tukalo, Michail A

    2016-03-01

    Tuberculosis is a serious infectious disease caused by human pathogen bacteria Mycobacterium tuberculosis. Bacterial drug resistance is a very significant medical problem nowadays and development of novel antibiotics with different mechanisms of action is an important goal of modern medical science. Leucyl-tRNA synthetase (LeuRS) has been recently clinically validated as antimicrobial target. Here we report the discovery of small-molecule inhibitors of M. tuberculosis LeuRS. Using receptor-based virtual screening we have identified six inhibitors of M. tuberculosis LeuRS from two different chemical classes. The most active compound 4-{[4-(4-Bromo-phenyl)-thiazol-2-yl]hydrazonomethyl}-2-methoxy-6-nitro-phenol (1) inhibits LeuRS with IC50 of 6μM. A series of derivatives has been synthesized and evaluated in vitro toward M. tuberculosis LeuRS. It was revealed that the most active compound 2,6-Dibromo-4-{[4-(4-nitro-phenyl)-thiazol-2-yl]-hydrazonomethyl}-phenol inhibits LeuRS with IC50 of 2.27μM. All active compounds were tested for antimicrobial effect against M. tuberculosis H37Rv. The compound 1 seems to have the best cell permeability and inhibits growth of pathogenic bacteria with IC50=10.01μM and IC90=13.53μM.

  3. Structure of equine 2'-5'oligoadenylate synthetase (OAS) gene family and FISH mapping of OAS genes to ECA8p15-->p14 and BTA17q24-->q25.

    PubMed

    Perelygin, A A; Lear, T L; Zharkikh, A A; Brinton, M A

    2005-01-01

    Mammalian 2'-5' oligoadenylate (2-5A) synthetases are important mediators of the antiviral activity of interferons. Both human and mouse 2-5A synthetase gene families encode four forms of enzymes: small, medium, large and ubiquitin-like. In this study, the structures of four equine OAS genes were determined using DNA sequences derived from fifteen cDNA and four BAC clones. Composition of the equine OAS gene family is more similar to that of the human OAS family than the mouse Oas family. Two OAS-containing bovine BAC clones were identified in GenBank. Both equine and bovine BAC clones were physically assigned by FISH to horse and cattle chromosomes, ECA8p15-->p14 and BTA17q24--> q25, respectively. The comparative mapping data confirm conservation of synteny between ungulates, humans and rodents.

  4. Actinobacterial Acyl Coenzyme A Synthetases Involved in Steroid Side-Chain Catabolism

    PubMed Central

    Casabon, Israël; Swain, Kendra; Crowe, Adam M.

    2014-01-01

    Bacterial steroid catabolism is an important component of the global carbon cycle and has applications in drug synthesis. Pathways for this catabolism involve multiple acyl coenzyme A (CoA) synthetases, which activate alkanoate substituents for β-oxidation. The functions of these synthetases are poorly understood. We enzymatically characterized four distinct acyl-CoA synthetases from the cholate catabolic pathway of Rhodococcus jostii RHA1 and the cholesterol catabolic pathway of Mycobacterium tuberculosis. Phylogenetic analysis of 70 acyl-CoA synthetases predicted to be involved in steroid metabolism revealed that the characterized synthetases each represent an orthologous class with a distinct function in steroid side-chain degradation. The synthetases were specific for the length of alkanoate substituent. FadD19 from M. tuberculosis H37Rv (FadD19Mtb) transformed 3-oxo-4-cholesten-26-oate (kcat/Km = 0.33 × 105 ± 0.03 × 105 M−1 s−1) and represents orthologs that activate the C8 side chain of cholesterol. Both CasGRHA1 and FadD17Mtb are steroid-24-oyl-CoA synthetases. CasG and its orthologs activate the C5 side chain of cholate, while FadD17 and its orthologs appear to activate the C5 side chain of one or more cholesterol metabolites. CasIRHA1 is a steroid-22-oyl-CoA synthetase, representing orthologs that activate metabolites with a C3 side chain, which accumulate during cholate catabolism. CasI had similar apparent specificities for substrates with intact or extensively degraded steroid nuclei, exemplified by 3-oxo-23,24-bisnorchol-4-en-22-oate and 1β(2′-propanoate)-3aα-H-4α(3″-propanoate)-7aβ-methylhexahydro-5-indanone (kcat/Km = 2.4 × 105 ± 0.1 × 105 M−1 s−1 and 3.2 × 105 ± 0.3 × 105 M−1 s−1, respectively). Acyl-CoA synthetase classes involved in cholate catabolism were found in both Actinobacteria and Proteobacteria. Overall, this study provides insight into the physiological roles of acyl-CoA synthetases in steroid catabolism and

  5. Spectrophotometric studies of acyl-coenzyme A synthetases of rat liver mitochondria

    PubMed Central

    Garland, P. B.; Yates, D. W.; Haddock, B. A.

    1970-01-01

    1. Deca-2,4,6,8-tetraenoic acid is a substrate for both ATP-specific (EC 6.2.1.2 or 3) and GTP-specific (EC 6.2.1.–) acyl-CoA synthetases of rat liver mitochondria. The enzymic synthesis of decatetraenoyl-CoA results in new spectral characteristics. The difference spectrum for the acyl-CoA minus free acid has a maximum at 376nm with εmM 34. Isosbestic points are at 345nm and 440nm. 2. The acylation of CoA by decatetraenoate in mitochondrial suspensions can be continuously measured with a dual-wavelength spectrophotometer. 3. By using this technique, three distinct types of acyl-CoA synthetase activity were demonstrated in rat liver mitochondria. One of these utilized added CoA and ATP, required added Mg2+ and corresponded to a previously described `external' acyl-CoA synthetase. The other two acyl-CoA synthetase activities utilized intramitochondrial CoA and did not require added Mg2+. Of these two `internal' acyl-CoA synthetases, one was insensitive to uncoupling agents, was inhibited by phosphate or arsenate, and corresponded to the GTP-specific enzyme. The other corresponded to the ATP-specific enzyme. 4. Atractylate inhibited the activity of the two internal acyl-CoA synthetases only when the energy source was added ATP. 5. The amount of intramitochondrial CoA acylated by decatetraenoate was independent of whether the internal ATP-specific or GTP-specific acyl-CoA synthetase was active. It is concluded that these two internal acyl-CoA synthetases have access to the same intramitochondrial pool of CoA. 6. The amount of intramitochondrial CoA that could be acylated with decatetraenoate was decreased by the addition of palmitoyl-dl-carnitine, 2-oxoglutarate, or pyruvate. These observations indicated that pyruvate dehydrogenase (EC 1.2.4.1), oxoglutarate dehydrogenase (EC 1.2.4.2), carnitine palmitoyltransferase (EC 2.3.1.–), citrate synthase (EC 4.1.3.7), and succinyl-CoA synthetase (EC 6.2.1.4) all have access to the same intramitochondrial pool of CoA as do

  6. A human and a plant intron-containing tRNATyr gene are both transcribed in a HeLa cell extract but spliced along different pathways.

    PubMed Central

    van Tol, H; Stange, N; Gross, H J; Beier, H

    1987-01-01

    tRNA splicing enzymes had been identified in mammalian and plant cells long before homologous intron-containing tRNA genes were detected. The tRNATyr gene presented here is the first intron-containing, human tRNA gene for which transcription and pre-tRNA maturation has been studied in a homologous system. This gene is disrupted by a 20-bp long intron and encodes one of the two major human tRNAsTyr which have been purified and sequenced. A tRNATyr gene recently isolated from Nicotiana also contains an intron and codes for a functional, major cytoplasmic tRNATyr. Both tRNA genes are efficiently transcribed in a HeLa cell nuclear extract. Each of them produces two independent primary transcripts because of two initiation and termination sites, respectively. The maturation of the tRNATyr precursors proceeds along different pathways. The intervening sequence of the human pre-tRNATyr is excised first, followed by ligation of the tRNA halves and maturation of the flanks, as has been shown for all intron-containing tRNA genes transcribed in HeLa extract. The order of maturation steps is reversed for the plant pre-tRNATyr: processing of the flanking sequences precedes intron excision. This maturation pathway corresponds to that observed in vivo for tRNA biosynthesis in Xenopus oocytes and yeast. Images Fig. 1. Fig. 4. Fig. 5. Fig. 6. PMID:3502708

  7. Expression of glutamine synthetase in the mouse kidney: localization in multiple epithelial cell types and differential regulation by hypokalemia.

    PubMed

    Verlander, Jill W; Chu, Diana; Lee, Hyun-Wook; Handlogten, Mary E; Weiner, I David

    2013-09-01

    Renal glutamine synthetase catalyzes the reaction of NH4+ with glutamate, forming glutamine and decreasing the ammonia available for net acid excretion. The purpose of the present study was to determine glutamine synthetase's specific cellular expression in the mouse kidney and its regulation by hypokalemia, a common cause of altered renal ammonia metabolism. Glutamine synthetase mRNA and protein were present in the renal cortex and in both the outer and inner stripes of the outer medulla. Immunohistochemistry showed glutamine synthetase expression throughout the entire proximal tubule and in nonproximal tubule cells. Double immunolabel with cell-specific markers demonstrated glutamine synthetase expression in type A intercalated cells, non-A, non-B intercalated cells, and distal convoluted tubule cells, but not in principal cells, type B intercalated cells, or connecting segment cells. Hypokalemia induced by feeding a nominally K+ -free diet for 12 days decreased glutamine synthetase expression throughout the entire proximal tubule and in the distal convoluted tubule and simultaneously increased glutamine synthetase expression in type A intercalated cells in both the cortical and outer medullary collecting duct. We conclude that glutamine synthetase is widely and specifically expressed in renal epithelial cells and that the regulation of expression differs in specific cell populations. Glutamine synthetase is likely to mediate an important role in renal ammonia metabolism.

  8. Investigating arsenic susceptibility from a genetic perspective in Drosophila reveals a key role for glutathione synthetase.

    PubMed

    Ortiz, Jorge G Muñiz; Opoka, Robert; Kane, Daniel; Cartwright, Iain L

    2009-02-01

    Chronic exposure to arsenic-contaminated drinking water can lead to a variety of serious pathological outcomes. However, differential responsiveness within human populations suggests that interindividual genetic variation plays an important role. We are using Drosophila to study toxic metal response pathways because of unrivalled access to varied genetic approaches and significant demonstrable overlap with many aspects of mammalian physiology and disease phenotypes. Genetic analysis (via chromosomal segregation and microsatellite marker-based recombination) of various wild-type strains exhibiting relative susceptibility or tolerance to the lethal toxic effects of arsenite identified a limited X-chromosomal region (16D-F) able to confer a differential response phenotype. Using an FRT-based recombination approach, we created lines harboring small, overlapping deficiencies within this region and found that relative arsenite sensitivity arose when the dose of the glutathione synthetase (GS) gene (located at 16F1) was reduced by half. Knockdown of GS expression by RNA interference (RNAi) in cultured S2 cells led to enhanced arsenite sensitivity, while GS RNAi applied to intact organisms dramatically reduced the concentration of food-borne arsenite compatible with successful growth and development. Our analyses, initially guided by observations on naturally occurring variants, provide genetic proof that an optimally functioning two-step glutathione (GSH) biosynthetic pathway is required in vivo for a robust defense against arsenite; the enzymatic implications of this are discussed in the context of GSH supply and demand under arsenite-induced stress. Given an identical pathway for human GSH biosynthesis, we suggest that polymorphisms in GSH biosynthetic genes may be an important contributor to differential arsenic sensitivity and exposure risk in human populations.

  9. Biochemical parameters of glutamine synthetase from Klebsiella aerogenes.

    PubMed Central

    Bender, R A; Janssen, K A; Resnick, A D; Blumenberg, M; Foor, F; Magasanik, B

    1977-01-01

    The glutamine synthetase (GS) from Klebsiella aerogenes is similar to that from Escherichia coli in several respects: (i) it is repressed by high levels of ammonia in the growth medium; (ii) its biosynthetic activity is greatly reduced by adenylylation; and (iii) adenylylation lowers the pH optimum and alters the response of the enzymes to various inhibitors in the gamma-glutamyl transferase (gammaGT) assay. There are, however, several important differences: (i) the isoactivity point for the adenylylated and non-adenylylated forms in the gammaGT assay occurs at pH 7.55 in K. aerogenes and at pH 7.15 in E. coli; (ii) the non-adenylylated form of the GS from K. aerogenes is stimulated by 60 mM MgCl2 in the gammaGT assay at pH 7.15. A biosynthetic reaction assay that correlates well with number of non-adenylylated enzyme subunits, as determined by the method of Mg2+ inhibition of the gammaGT assay, is described. Finally, we have found that it is necessary to use special methods to harvest growing cells to prevent changes in the adenylylation state of GS from occurring during harvesting. PMID:14104

  10. Cloning, expression, and purification of glutamine synthetase from Clostridum acetobutylicum

    SciTech Connect

    Usdin, K.P.; Zappe, H.; Jones, D.T.; Woods, D.R.

    1986-09-01

    A glutamine synthetase (GS) gene, glnA, from the gram-positive obligate anaerobe Clostridium acetobutylicum was cloned on recombinant plasmid pHZ200 and enabled Escherichia coli glnA deletion mutants to utilize (NH/sub 4/)/sub 2/ as a sole source of nitrogen. The cloned C. acetobutylicum gene was expressed from a regulatory region contained within the cloned DNA fragment. glnA expression was subject to nitrogen regulation in E. coli. This cloned glnA DNA did not enable an E. coli glnA ntrB ntrC deletion mutant to utilize arginine or low levels of glutamine as sole nitrogen sources, and failed to activate histidase activity in this strain which contained the Klebsiella aerogenes hut operon. The GS produced by pHZ200 was purified and had an apparent subunit molecular weight of approximately 59,000. There was no DNA or protein homology between the cloned C. acetobutylicum glnA gene and GS and the corresponding gene and GS from E. coli. The C. acetobutylicum GS was inhibited by Mg/sup 2 +/ in the ..gamma..-glutamyl transferase assay, but there was no evidence that the GS was adenylylated.

  11. Insights into an Unusual Nonribosomal Peptide Synthetase Biosynthesis

    PubMed Central

    Binz, Tina M.; Maffioli, Sonia I.; Sosio, Margherita; Donadio, Stefano; Müller, Rolf

    2010-01-01

    The GE81112 tetrapeptides (1–3) represent a structurally unique class of antibiotics, acting as specific inhibitors of prokaryotic protein synthesis. Here we report the cloning and sequencing of the GE81112 biosynthetic gene cluster from Streptomyces sp. L-49973 and the development of a genetic manipulation system for Streptomyces sp. L-49973. The biosynthetic gene cluster for the tetrapeptide antibiotic GE81112 (getA-N) was identified within a 61.7-kb region comprising 29 open reading frames (open reading frames), 14 of which were assigned to the biosynthetic gene cluster. Sequence analysis revealed the GE81112 cluster to consist of six nonribosomal peptide synthetase (NRPS) genes encoding incomplete di-domain NRPS modules and a single free standing NRPS domain as well as genes encoding other biosynthetic and modifying proteins. The involvement of the cloned gene cluster in GE81112 biosynthesis was confirmed by inactivating the NRPS gene getE resulting in a GE81112 production abolished mutant. In addition, we characterized the NRPS A-domains from the pathway by expression in Escherichia coli and in vitro enzymatic assays. The previously unknown stereochemistry of most chiral centers in GE81112 was established from a combined chemical and biosynthetic approach. Taken together, these findings have allowed us to propose a rational model for GE81112 biosynthesis. The results further open the door to developing new derivatives of these promising antibiotic compounds by genetic engineering. PMID:20710026

  12. Structural Biology of Non-Ribosomal Peptide Synthetases

    PubMed Central

    Miller, Bradley R.; Gulick, Andrew M.

    2016-01-01

    Summary The non-ribosomal peptide synthetases are modular enzymes that catalyze synthesis of important peptide products from a variety of standard and non-proteinogenic amino acid substrates. Within a single module are multiple catalytic domains that are responsible for incorporation of a single residue. After the amino acid is activated and covalently attached to an integrated carrier protein domain, the substrates and intermediates are delivered to neighboring catalytic domains for peptide bond formation or, in some modules, chemical modification. In the final module, the peptide is delivered to a terminal thioesterase domain that catalyzes release of the peptide product. This multi-domain modular architecture raises questions about the structural features that enable this assembly line synthesis in an efficient manner. The structures of the core component domains have been determined and demonstrate insights into the catalytic activity. More recently, multi-domain structures have been determined and are providing clues to the features of these enzyme systems that govern the functional interaction between multiple domains. This chapter describes the structures of NRPS proteins and the strategies that are being used to assist structural studies of these dynamic proteins, including careful consideration of domain boundaries for generation of truncated proteins and the use of mechanism-based inhibitors that trap interactions between the catalytic and carrier protein domains. PMID:26831698

  13. In situ autoradiographic detection of folylpolyglutamate synthetase activity

    SciTech Connect

    Sussman, D.J.; Milman, G.; Osborne, C.; Shane, B.

    1986-11-01

    The enzyme folylpolyglutamate synthetase (FPGS) catalyzes the conversion of folate (pteroylmonoglutamate) to the polyglutamate forms (pteroylpolyglutamates) that are required for folate retention by mammalian cells. A rapid in situ autoradiographic assay for FPGS was developed which is based on the folate cofactor requirement of thymidylate synthase. Chinese hamster AUX B1 mutant cells lack FPGS activity and are unable to accumulate folate. As a result, the conversion of (6-/sup 3/H)deoxyuridine to thymidine via the thymidylate synthase reaction is impaired in AUX B1 cells and no detectable label is incorporated into DNA. In contrast, FPGS in wild-type Chinese hamster CHO cells causes folate retention and enables the incorporation of (6-/sup 3/H)deoxyuridine into DNA. Incorporation may be detected by autoradiography of monolayer cultures or of colonies replica plated onto polyester discs. Introduction of Escherichia coli FPGS into AUX B1 cells restores the activity of the thymidylate synthase pathway and demonstrates that the E. coli FPGS enzyme can provide pteroylpolyglutamates which functions in mammalian cells.

  14. The plastidial folylpolyglutamate synthetase and root apical meristem maintenance

    PubMed Central

    Srivastava, Avinash C; Tang, Yuhong; Díaz de la Garza, Rocío I

    2011-01-01

    Folylpolyglutamate synthetase (FPGS) catalyzes the attachment of glutamate residues to the folate molecule in plants. Three isoforms of FPGS have been identified in Arabidopsis and these are localized in the plastid (AtDFB), mitochondria (AtDFC) and cytosol (AtDFD). We recently determined that mutants in the AtDFB (At5G05980) gene disrupt primary root development in Arabidopsis thaliana seedlings. Transient expression of AtDFB-green fluorescent protein (GFP) fusion under the control of the native AtDFB promoter in Nicotiana tabacum leaf epidermal cells verified the plastid localization of AtDFB. Furthermore, low concentrations of methotrexate (MTX), a compound commonly used as a folate antagonist in plant and mammalian cells induced primary root defects in wild type seedlings that were similar to atdfb. In addition, atdfb seedlings were more sensitive to MTX when compared to wild type. Quantitative (q) RT-PCR showed lower transcript levels of the mitochondrial and cytosolic FPGS in roots of 7-day-old atdfb seedling suggesting feedback regulation of AtDFB on the expression of other FPGS isoforms during early seedling development. The primary root defects of atdfb, which can be traced in part to altered quiescent center (QC) identity, pave the way for future studies that could link cell type specific folate and FPGS isoform requirements to whole organ development. PMID:21502816

  15. How the CCA-Adding Enzyme Selects Adenine over Cytosine at Position 76 of tRNA

    SciTech Connect

    Pan, Baocheng; Xiong, Yong; Steitz, Thomas A.

    2010-11-22

    CCA-adding enzymes [ATP(CTP):tRNA nucleotidyltransferases] add CCA onto the 3{prime} end of transfer RNA (tRNA) precursors without using a nucleic acid template. Although the mechanism by which cytosine (C) is selected at position 75 of tRNA has been established, the mechanism by which adenine (A) is selected at position 76 remains elusive. Here, we report five cocrystal structures of the enzyme complexed with both a tRNA mimic and nucleoside triphosphates under catalytically active conditions. These structures suggest that adenosine 5{prime}-monophosphate is incorporated onto the A76 position of the tRNA via a carboxylate-assisted, one-metal-ion mechanism with aspartate 110 functioning as a general base. The discrimination against incorporation of cytidine 5{prime}-triphosphate (CTP) at position 76 arises from improper placement of the {alpha} phosphate of the incoming CTP, which results from the interaction of C with arginine 224 and prevents the nucleophilic attack by the 3{prime} hydroxyl group of cytidine75.

  16. How the CCA-Adding Enzyme Selects Adenine over Cytosine at Position 76 of tRNA

    SciTech Connect

    B Pan; Y Xiong; T Steitz

    2011-12-31

    CCA-adding enzymes [ATP(CTP):tRNA nucleotidyltransferases] add CCA onto the 3' end of transfer RNA (tRNA) precursors without using a nucleic acid template. Although the mechanism by which cytosine (C) is selected at position 75 of tRNA has been established, the mechanism by which adenine (A) is selected at position 76 remains elusive. Here, we report five cocrystal structures of the enzyme complexed with both a tRNA mimic and nucleoside triphosphates under catalytically active conditions. These structures suggest that adenosine 5'-monophosphate is incorporated onto the A76 position of the tRNA via a carboxylate-assisted, one-metal-ion mechanism with aspartate 110 functioning as a general base. The discrimination against incorporation of cytidine 5'-triphosphate (CTP) at position 76 arises from improper placement of the {alpha} phosphate of the incoming CTP, which results from the interaction of C with arginine 224 and prevents the nucleophilic attack by the 3' hydroxyl group of cytidine75.

  17. The 51-63 base pair of tRNA confers specificity for binding by EF-Tu.

    PubMed

    Sanderson, Lee E; Uhlenbeck, Olke C

    2007-06-01

    Elongation factor Tu (EF-Tu) exhibits significant specificity for the different elongator tRNA bodies in order to offset its variable affinity to the esterified amino acid. Three X-ray cocrystal structures reveal that while most of the contacts with the protein involve the phosphodiester backbone of tRNA, a single hydrogen bond is observed between the Glu390 and the amino group of a guanine in the 51-63 base pair in the T-stem of tRNA. Here we show that the Glu390Ala mutation of Thermus thermophilus EF-Tu selectively destabilizes binding of those tRNAs containing a guanine at either position 51 or 63 and that mutagenesis of the 51-63 base pair in several tRNAs modulates their binding affinities to EF-Tu. A comparison of Escherichia coli