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At least 487 records · Page 27Linked to original sources
PubMed1998

Covalent complex of phenylalanyl-tRNA synthetase with 4-thiouridine-substituted tRNA(Phe) gene transcript retains aminoacylation activity.

s4U-containing transcripts of tRNA(Phe) gene have been prepared by complete substitution of 16 U residues or by random incorporation of s4U residues followed by affinity electrophoresis isolation of s4U-monosubstituted tRNA transcripts. Both analogs have been cross-linked to Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS) and the specificity of the cross-linking has been demonstrated. Functional activity of the covalent complex of PheRS with the s4U-monosubstituted transcript has been shown by aminoacylation of 60% of the enzyme-cross-linked tRNA. This is the first instance in which biological activity of aminoacyl-tRNA synthetase and cross-linked tRNA in a specific complex has been revealed.

Acylation↗
PubMed2003

Conformational movements and cooperativity upon amino acid, ATP and tRNA binding in threonyl-tRNA synthetase.

The crystal structures of threonyl-tRNA synthetase (ThrRS) from Staphylococcus aureus, with ATP and an analogue of threonyl adenylate, are described. Together with the previously determined structures of Escherichia coli ThrRS with different substrates, they allow a comprehensive analysis of the effect of binding of all the substrates: threonine, ATP and tRNA. The tRNA, by inserting its acceptor arm between the N-terminal domain and the catalytic domain, causes a large rotation of the former. Within the catalytic domain, four regions surrounding the active site display significant conformational changes upon binding of the different substrates. The binding of threonine induces the movement of as much as 50 consecutive amino acid residues. The binding of ATP triggers a displacement, as large as 8A at some C(alpha) positions, of a strand-loop-strand region of the core beta-sheet. Two other regions move in a cooperative way upon binding of threonine or ATP: the motif 2 loop, which plays an essential role in the first step of the aminoacylation reaction, and the ordering loop, which closes on the active site cavity when the substrates are in place. The tRNA interacts with all four mobile regions, several residues initially bound to threonine or ATP switching to a position in which they can contact the tRNA. Three such conformational switches could be identified, each of them in a different mobile region. The structural analysis suggests that, while the small substrates can bind in any order, they must be in place before productive tRNA binding can occur.

Adenosine Triphosphate↗
PubMed2000

Uncharged tRNA activates GCN2 by displacing the protein kinase moiety from a bipartite tRNA-binding domain.

Protein kinase GCN2 regulates translation in amino acid-starved cells by phosphorylating elF2. GCN2 contains a regulatory domain related to histidyl-tRNA synthetase (HisRS) postulated to bind multiple deacylated tRNAs as a general sensor of starvation. In accordance with this model, GCN2 bound several deacylated tRNAs with similar affinities, and aminoacylation of tRNAphe weakened its interaction with GCN2. Unexpectedly, the C-terminal ribosome binding segment of GCN2 (C-term) was required in addition to the HisRS domain for strong tRNA binding. A combined HisRS+ C-term segment bound to the isolated protein kinase (PK) domain in vitro, and tRNA impeded this interaction. An activating mutation (GCN2c-E803V) that weakens PK-C-term association greatly enhanced tRNA binding by GCN2. These results provide strong evidence that tRNA stimulates the GCN2 kinase moiety by preventing an inhibitory interaction with the bipartite tRNA binding domain.

Binding Sites↗
PubMed1993

Recognition of tRNA(Cys) by Escherichia coli cysteinyl-tRNA synthetase.

A study of the recognition of tRNA(Cys) by Escherichia coli cysteinyl-tRNA synthetase using in vivo and in vitro methods was performed. All three anticodon nucleotides, the discriminator nucleotide (73), and some elements within the tertiary domain (the D stem/loop, the T psi C stem/loop, and the variable loop) are important for recognition; the anticodon stem and acceptor stem appear to contain no essential elements. A T7 RNA polymerase transcript corresponding to tRNA(Cys) is only a 5.5-fold worse substrate than native tRNA(Cys) (in terms of the specificity constant, kcat/Km), mainly due to an increase in the value of Km for the transcript. The greatest loss of specificity caused by mutation of a single nucleotide occurs when the discriminator U73 is changed; kcat/Km declines 3-4 orders of magnitude depending on the substitution. Mutations in the wobble nucleotide of the anticodon also cause reductions in the specificity constant of 3 orders of magnitude, while mutations in the other anticodon nucleotides caused lesser effects. Interestingly, a C35A mutation (with the phenylalanine anticodon GAA) had no effect on aminoacylation by the cysteinyl-tRNA synthetase. Several amber suppressor tRNAs were constructed whose in vivo identity did not correlate with their in vitro specificity, indicating the need for both types of experiments to understand the factors which maintain tRNA specificity.

Acylation↗
PubMed1996

Aminoacyl-tRNA synthetases optimize both cognate tRNA recognition and discrimination against noncognate tRNAs.

Specific protein--nucleic acid interactions are usually the product of sequence-dependent hydrogen bonding. However, in the crystal structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) in complex with tRNAGln, leucine 136 (Leu136) stabilizes the disruption of the weak first (U1-A72) base pair in tRNAGln by stacking between A72 and G2. We have demonstrated, by a combined in vivo and in vitro mutational analysis, that Leu136 is important for tRNA specificity despite making no hydrogen bonds with tRNAGln. Both more (L136F) and less (L136V, L136M, L136A, and L136T) mischarging mutants of GlnRS have been identified. GlnRS(L136F) is more mischarging and less specific than wild-type GlnRS in vivo, due not to an increased affinity for the noncognate tRNAs but to a decreased affinity for tRNAGln. Also, unlike other mischarging mutants of GlnRS that have been characterized, it does not exhibit generally relaxed tRNA specificity in vivo and mischarges only a subset of the tRNAs tested. A possible sequence preference for a Py1-Pu72/Pu2-Py71 combination is suggested. The L136A/M/T/V mutants are the first GlnRS variants, including wild-type, expressed on pBR322 which no longer mischarge tyrT(UAG) in vivo. We have shown that, while the L136A mutant is less mischarging than wild-type both in vivo and in vitro, it is not more specific as it also exhibits reduced affinity for its cognate glutamine tRNA. On the basis of these results, we suggest that the aminoacyl-tRNA synthetases have evolved to balance cognate tRNA recognition and discrimination against noncognate tRNAs.

Base Sequence↗
PubMed2003

Structural basis for orthogonal tRNA specificities of tyrosyl-tRNA synthetases for genetic code expansion.

The archaeal/eukaryotic tyrosyl-tRNA synthetase (TyrRS)-tRNA(Tyr) pairs do not cross-react with their bacterial counterparts. This 'orthogonal' condition is essential for using the archaeal pair to expand the bacterial genetic code. In this study, the structure of the Methanococcus jannaschii TyrRS-tRNA(Tyr)-L-tyrosine complex, solved at a resolution of 1.95 A, reveals that this archaeal TyrRS strictly recognizes the C1-G72 base pair, whereas the bacterial TyrRS recognizes the G1-C72 in a different manner using different residues. These diverse tRNA recognition modes form the basis for the orthogonality. The common tRNA(Tyr) identity determinants (the discriminator, A73 and the anticodon residues) are also recognized in manners different from those of the bacterial TyrRS. Based on this finding, we created a mutant TyrRS that aminoacylates the amber suppressor tRNA with C34 65 times more efficiently than does the wild-type enzyme.

Amino Acid Sequence↗
PubMed2001

Structural and mutational studies of the recognition of the arginine tRNA-specific major identity element, A20, by arginyl-tRNA synthetase.

Arginyl-tRNA synthetase (ArgRS) recognizes two major identity elements of tRNA(Arg): A20, located at the outside corner of the L-shaped tRNA, and C35, the second letter of the anticodon. Only a few exceptional organisms, such as the yeast Saccharomyces cerevisiae, lack A20 in tRNA(Arg). In the present study, we solved the crystal structure of a typical A20-recognizing ArgRS from Thermus thermophilus at 2.3 A resolution. The structure of the T. thermophilus ArgRS was found to be similar to that of the previously reported S. cerevisiae ArgRS, except for short insertions and a concomitant conformational change in the N-terminal domain. The structure of the yeast ArgRS.tRNA(Arg) complex suggested that two residues in the unique N-terminal domain, Tyr(77) and Asn(79), which are phylogenetically invariant in the ArgRSs from all organisms with A20 in tRNA(Arg)s, are involved in A20 recognition. However, in a docking model constructed based on the yeast ArgRS.tRNA(Arg) and T. thermophilus ArgRS structures, Tyr(77) and Asn(79) are not close enough to make direct contact with A20, because of the conformational change in the N-terminal domain. Nevertheless, the replacement of Tyr(77) or Asn(79) by Ala severely reduced the arginylation efficiency. Therefore, some conformational change around A20 is necessary for the recognition. Surprisingly, the N79D mutant equally recognized A20 and G20, with only a slight reduction in the arginylation efficiency as compared with the wild-type enzyme. Other mutants of Asn(79) also exhibited broader specificity for the nucleotide at position 20 of tRNA(Arg). We propose a model of A20 recognition by the ArgRS that is consistent with the present results of the mutational analyses.

Amino Acids↗
PubMed1992

Determinant nucleotides of yeast tRNA(Asp) interact directly with aspartyl-tRNA synthetase.

The interaction of wild-type and mutant yeast tRNA(Asp) transcripts with yeast aspartyl-tRNA synthetase (AspRS; EC 6.1.1.12) has been probed by using iodine cleavage of phosphorothioate-substituted transcripts. AspRS protects phosphates in the anticodon (G34, U35), D-stem (U25), and acceptor end (G73) that correspond to determinant nucleotides for aspartylation. This protection, as well as that in anticodon stem (C29, U40, G41) and D-stem (U11 to U13), is consistent with direct interaction of AspRS at these phosphates. Other protection, in the variable loop (G45), D-loop (G18, G19), and T-stem and loop (G53, U54, U55), as well as enhanced reactivity at G37, may result from conformational changes of the transcript upon binding to AspRS. Transcripts mutated at determinant positions showed a loss of phosphate protection in the region of the mutation while maintaining the global protection pattern. The ensemble of results suggests that aspartylation specificity arises from both protein-base and protein-phosphate contacts and that different regions of tRNA(Asp) interact independently with AspRS. A mutant transcript of yeast tRNA(Phe) that contains the set of identity nucleotides for specific aspartylation gave a phosphate protection pattern strikingly similar to that of wild-type tRNA(Asp). This confirms that a small number of nucleotides within a different tRNA sequence context can direct specific interaction with synthetase.

Aspartate-tRNA Ligase↗
PubMed1994

Functional communication in the recognition of tRNA by Escherichia coli glutaminyl-tRNA synthetase.

Wild-type Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) poorly aminoacylates opal suppressors (GLN) derived from tRNA(Gln). Mutations in glnS (the gene encoding GlnRS) that compensate for impaired aminoacylation were isolated by genetic selection. Two glnS mutants were obtained by using opal suppressors differing in the nucleotides composing the base pair at 3.70: glnS113 with an Asp-235-->Asn change selected with GLNA3U70 (GLN carrying G3-->A and C70-->U changes), and glnS114 with a Gln-318-->Arg change selected with GLNU70 (GLN carrying a C70-->U change). The Asp-235-->Asn change was identified previously by genetic selection. Additional mutants were isolated by site-directed mutagenesis followed by genetic selection; the mutant enzymes have single amino acid changes (Lys-317-->Arg and Gln-318-->Lys). A number of mutants with no phenotype also were obtained randomly. In vitro aminoacylation of a tRNA(Gln) transcript by GlnRS enzymes with Lys-317-->Arg, Gln-318-->Lys, or Gln-318-->Arg changes shows that the enzyme's kinetic parameters are not greatly affected by the mutations. However, aminoacylation of a tRNA(Gln) transcript with an opal (UCA) anticodon shows that the specificity constants (kcat/Km) for the mutant enzymes were 5-10 times above that of the wild-type GlnRS. Interactions between Lys-317 and Gln-318 with the inside of the L-shaped tRNA and with the side chain of Gln-234 provide a connection between the acceptor end-binding and anticodon-binding domains of GlnRS. The GlnRS mutants isolated suggest that perturbation of the interactions with the inside of the tRNA L shape results in relaxed anticodon recognition.

Amino Acid Sequence↗
PubMed2005

A three-dimensional working model of the multienzyme complex of aminoacyl-tRNA synthetases based on electron microscopic placements of tRNA and proteins.

It has become evident that the process of protein synthesis is performed by many cellular polypeptides acting in concert within the structural confines of protein complexes. In multicellular eukaryotes, one of these assemblies is a multienzyme complex composed of eight proteins that have aminoacyl-tRNA synthetase activities as well as three non-synthetase proteins (p43, p38, and p18) with diverse functions. This study uses electron microscopy and three-dimensional reconstruction to explore the arrangement of proteins and tRNA substrates within this "core" multisynthetase complex. Binding of unfractionated tRNA establishes that these molecules are widely distributed on the exterior of the structure. Binding of gold-labeled tRNA(Leu) places leucyl-tRNA synthetase and the bifunctional glutamyl-/prolyl-tRNA synthetase at the base of this asymmetric "V"-shaped particle. A stable cell line has been produced that incorporates hexahistidine-labeled p43 into the multisynthetase complex. Using a gold-labeled nickel-nitrilotriacetic acid probe, the polypeptides of the p43 dimer have been located along one face of the particle. The results of this and previous studies are combined into an initial three-dimensional working model of the multisynthetase complex. This is the first conceptualization of how the protein constituents and tRNA substrates are arrayed within the structural confines of this multiprotein assembly.

Amino Acyl-tRNA Synthetases↗
PubMed1997

Role of zinc in tRNA-acceptor stem binding by glutamyl-tRNA synthetase from E.coli: a molecular modeling study.

A model of the N-terminal half of glutamyl-tRNA synthetase from E. coli was constructed on the basis of similarity in sequence and function of Glutaminyl- and Glutamyl-tRNA synthetases. The glutaminyl-tRNA synthetase does not contain any zinc atom, but glutamyl-tRNA synthetase from E. coli contains one atom of zinc. The specific role of zinc is not yet known. In this article, molecular modeling is employed to show that the zinc atom is well outside the contact region of the acceptor stem of tRNA. The placement of a zinc atom at a significant distance from the tRNA acceptor stem indicates that the role of zinc is likely to be indirect and structural.

Amino Acid Sequence↗
PubMed2002

Class I tyrosyl-tRNA synthetase has a class II mode of cognate tRNA recognition.

Bacterial tyrosyl-tRNA synthetases (TyrRS) possess a flexibly linked C-terminal domain of approximately 80 residues, which has hitherto been disordered in crystal structures of the enzyme. We have determined the structure of Thermus thermophilus TyrRS at 2.0 A resolution in a crystal form in which the C-terminal domain is ordered, and confirm that the fold is similar to part of the C-terminal domain of ribosomal protein S4. We have also determined the structure at 2.9 A resolution of the complex of T.thermophilus TyrRS with cognate tRNA(tyr)(G Psi A). In this structure, the C-terminal domain binds between the characteristic long variable arm of the tRNA and the anti-codon stem, thus recognizing the unique shape of the tRNA. The anticodon bases have a novel conformation with A-36 stacked on G-34, and both G-34 and Psi-35 are base-specifically recognized. The tRNA binds across the two subunits of the dimeric enzyme and, remarkably, the mode of recognition of the class I TyrRS for its cognate tRNA resembles that of a class II synthetase in being from the major groove side of the acceptor stem.

Amino Acid Sequence↗
PubMed1983

Isolation and nucleotide sequence of a plant tRNA gene: petunia asparagine tRNA.

A 14.3 kb petunia genomic DNA fragment was isolated and found to contain a single tRNA gene coding for asparagine tRNA. The nucleotide sequence of the asparagine tRNA gene and its flanking regions has been determined. This gene does not contain intervening sequences nor the 3'-end CCA sequence of the mature tRNA and presents a similar overall sequence homology (70%) to both E. coli and mammalian asparagine tRNA. As in other eukaryotic tRNA genes the 5'-flanking region does not seem to contain any special sequence that could function as a regulatory element and the 3'-end is followed by a short cluster of T that may function as the transcription termination site.

Aspartate-tRNA Ligase↗
PubMed1992

Aminoacyl-tRNA synthetase-induced cleavage of tRNA.

Aminoacyl-tRNA synthetases interact with their cognate tRNAs in a highly specific fashion. We have examined the phenomenon that upon complex formation E. coli glutaminyl-tRNA synthetase destabilizes tRNA(Gln) causing chain scissions in the presence of Mg2+ ions. The phosphodiester bond cleavage produces 3'-phosphate and 5'-hydroxyl ends. This kind of experiment is useful for detecting conformational changes in tRNA. Our results show that the cleavage is synthetase-specific, that mutant and wild-type tRNA(Gln) species can assume a different conformation, and that modified nucleosides in tRNA enhance the structural stability of the molecule.

Amino Acyl-tRNA Synthetases↗
PubMed1993

Influence of tRNA tertiary structure and stability on aminoacylation by yeast aspartyl-tRNA synthetase.

Mutations have been designed that disrupt the tertiary structure of yeast tRNA(Asp). The effects of these mutations on both tRNA structure and specific aspartylation by yeast aspartyl-tRNA synthetase were assayed. Mutations that disrupt tertiary interactions involving the D-stem or D-loop result in destabilization of the base-pairing in the D-stem, as monitored by nuclease digestion and chemical modification studies. These mutations also decrease the specificity constant (kcat/Km) for aspartylation by aspartyl-tRNA synthetase up to 10(3)-10(4) fold. The size of the T-loop also influences tRNA(Asp) structure and function; change of its T-loop to a tetraloop (-UUCG-) sequence results in a denatured D-stem and an almost 10(4) fold decrease of kcat/Km for aspartylation. The negative effects of these mutations on aspartylation activity are significantly alleviated by additional mutations that stabilize the D-stem. These results indicate that a critical role of tertiary structure in tRNA(Asp) for aspartylation is the maintenance of a base-paired D-stem.

Acylation↗
PubMed1999

tRNA recognition and evolution of determinants in seryl-tRNA synthesis.

We have analyzed the evolution of recognition of tRNAsSerby seryl-tRNA synthetases, and compared it to other type 2 tRNAs, which contain a long extra arm. In Eubacteria and chloroplasts this type of tRNA is restricted to three families: tRNALeu, tRNASer and tRNATyr. tRNALeuand tRNASer also carry a long extra arm in Archaea, Eukarya and all organelles with the exception of animal mitochondria. In contrast, the long extra arm of tRNATyr is far less conserved: it was drastically shortened after the separation of Archaea and Eukarya from Eubacteria, and it is also truncated in animal mitochondria. The high degree of phylo-genetic divergence in the length of tRNA variable arms, which are recognized by both class I and class II aminoacyl-tRNA synthetases, makes type 2 tRNA recognition an ideal system with which to study how tRNA discrimination may have evolved in tandem with the evolution of other components of the translation machinery.

Acylation↗
PubMed1978

Interaction of Escherichia coli glutaminyl-tRNA synthesis with noncognate tRNA's.

Several noncognate tRNA's from Escherichia coli were mischarged with glutamine by E. coli glutaminyl-tRNA synthetase if dimethylsulfoxide was present in the reaction mixture. Kinetic analysis of the mischarging revealed that dimethyl sulfoxide stimulated the misacylation by affecting the maximum velocity. Several noncognate tRNA's were shown to interact with glutaminyl-tRNA synthetase as measured by their ability to protect the enzyme against thermal inactivation or to replace cognate tRNA in stimulating glutamine-dependent ATP-PPi exchange reaction. These tRNA's, however, did not coincide with those which were mischargeable with glutamine.

Amino Acids↗
PubMed2006

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

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 alpha-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 alpha-proteobacterial tRNA(His) 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 alpha-proteobacteria followed by in situ adaptation of the bacterial tDNA(His) and identity rule divergence. Our results demonstrate that TFAM is an effective tool for the bioinformatics, comparative genomics and evolutionary study of tRNA identity.

Alphaproteobacteria↗