Reaction of modified and unmodified tRNATyr substrates with tyrosyl-tRNA synthetase (Bacillus stearothermophilus)

Avis, Johanna M.; Day, Anthony G.; Garcia, George A.; Fersht, Alan R.

doi:10.1021/bi00071a005

Cited by 35 publications

(77 citation statements)

References 49 publications

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“…To determine whether there are mechanistic differences in the way human and bacterial tyrosyl-tRNA synthetases recognize and aminoacylate their cognate tRNA substrates, tRNA Tyr substrates corresponding to tRNA Tyr from B. stearothermophilus (35,53) and human placenta (54) were transcribed in vitro, gel purified, and annealed. These tRNAs were used as substrates for aminoacylation by human and B. stearothermophilus tyrosyl-tRNA synthetase.…”

Section: Resultsmentioning

confidence: 99%

“…In Vitro Transcription and Purification of Human and B. stearothermophilus tRNA Tyr -B. stearothermophilus tRNA Tyr was transcribed in vitro from pGFX-WT, a derivative of the plasmid pGAG2 (35) in which a FokI restriction site replaces the BstNI cleavage site in pGAG2 such that digestion with FokI, and subsequent in vitro transcription of the digested pGFX-WT plasmid by T7 RNA polymerase, produces tRNA Tyr containing an intact -CCA 3Ј terminus. Details of the construction of the pGFX-WT plasmid will be published elsewhere.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Human Tyrosyl-tRNA Synthetase Shares Amino Acid Sequence Homology with a Putative Cytokine

Kleeman¹,

Wei²,

Simpson

et al. 1997

Journal of Biological Chemistry

116

102

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To test the hypothesis that tRNA Tyr recognition differs between bacterial and human tyrosyl-tRNA synthetases, we sequenced several clones identified as human tyrosyl-tRNA synthetase cDNAs by the Human Genome Project. We found that human tyrosyl-tRNA synthetase is composed of three domains: 1) an aminoterminal Rossmann fold domain that is responsible for formation of the activated E⅐Tyr-AMP intermediate and is conserved among bacteria, archeae, and eukaryotes; 2) a tRNA anticodon recognition domain that has not been conserved between bacteria and eukaryotes; and 3) a carboxyl-terminal domain that is unique to the human tyrosyl-tRNA synthetase and whose primary structure is 49% identical to the putative human cytokine endothelial monocyte-activating protein II, 50% identical to the carboxyl-terminal domain of methionyl-tRNA synthetase from Caenorhabditis elegans, and 43% identical to the carboxyl-terminal domain of Arc1p from Saccharomyces cerevisiae. The first two domains of the human tyrosyl-tRNA synthetase are 52, 36, and 16% identical to tyrosyl-tRNA synthetases from S. cerevisiae, Methanococcus jannaschii, and Bacillus stearothermophilus, respectively. Nine of fifteen amino acids known to be involved in the formation of the tyrosyl-adenylate complex in B. stearothermophilus are conserved across all of the organisms, whereas amino acids involved in the recognition of tRNA Tyr are not conserved. Kinetic analyses of recombinant human and B. stearothermophilus tyrosyl-tRNA synthetases expressed in Escherichia coli indicate that human tyrosyl-tRNA synthetase aminoacylates human but not B. stearothermophilus tRNA Tyr , and vice versa, supporting the original hypothesis. It is proposed that like endothelial monocyte-activating protein II and the carboxyl-terminal domain of Arc1p, the carboxyl-terminal domain of human tyrosyltRNA synthetase evolved from gene duplication of the carboxyl-terminal domain of methionyl-tRNA synthetase and may direct tRNA to the active site of the enzyme.Aminoacyl-tRNA synthetases catalyze the aminoacylation of tRNA by their cognate amino acid. For most aminoacyl-tRNA synthetases (E), tRNA aminoacylation can be separated into two steps: formation of a stable enzyme-bound aminoacyladenylate intermediate (E⅐AA-AMP, Equation 1), followed by transfer of the amino acid (AA) from the aminoacyl-adenylate intermediate to the 3Ј end of the tRNA substrate (Equation 2).The 20 different aminoacyl-tRNA synthetases fall into two distinct structural classes (1, 2). Class I aminoacyl-tRNA synthetases (of which tyrosyl-tRNA synthetase is a member) are characterized by a structurally well conserved amino-terminal Rossmann fold domain which contains the signature sequences "HIGH" and "KMSKS" (3, 4). In contrast, the carboxyl-terminal domains of class I aminoacyl-tRNA synthetases are structurally diverse suggesting that the primordial aminoacyl-tRNA synthetase consisted solely of the amino-terminal Rossmann fold (5-8). Both x-ray crystallography and site-directed mutagenesis of class I aminoacyl-tRNA synthet...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Human Tyrosyl-tRNA Synthetase Shares Amino Acid Sequence Homology with a Putative Cytokine

Kleeman¹,

Wei²,

Simpson

et al. 1997

Journal of Biological Chemistry

116

102

View full text Add to dashboard Cite

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“…for the reverse of the chemical step is assumed negligible for aminoacylation of tRNA) (25,26). The presence of a lag under these conditions demonstrates that tRNA Gln is not in rapid equilibrium with GlnRS.…”

Section: Assay For Steady-state and Single-turnover Aminoacylation Kimentioning

confidence: 94%

“…However, early studies of MetRS, PheRS, TyrRS, ArgRS, and TrpRS used chemical-quench flow to examine aminoacylation with 14 C-labeled amino acid (35)(36)(37)(38)(39). More recently, intrinsic protein tryptophan fluorescence was used as a probe to examine aminoacyl-tRNA synthesis by TyrRS and TrpRS (26,(40)(41)(42). Among these studies, rate constants for the isolated second step by TrpRS, ArgRS, and TyrRS are in the range of 25-45 sec Ϫ1 , similar to the k chem value of 28 sec Ϫ1 measured here for the two-step reaction by GlnRS (37,39,40,42), whereas the experiments with MetRS and PheRS returned lower values of 2-6 sec Ϫ1 for the transfer step (35)(36).…”

Section: Assay For Steady-state and Single-turnover Aminoacylation Kimentioning

confidence: 99%

Long-range intramolecular signaling in a tRNA synthetase complex revealed by pre-steady-state kinetics

Uter

Perona

2004

Proc. Natl. Acad. Sci. U.S.A.

107

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Pre-steady-state kinetic studies of Escherichia coli glutaminyl-tRNA synthetase conclusively demonstrate the existence of long-distance pathways of communication through the protein-RNA complex. Measurements of aminoacyl-tRNA synthesis reveal a rapid burst of product formation followed by a slower linear increase corresponding to k cat. Thus, a step after chemistry but before regeneration of active enzyme is rate-limiting for synthesis of Gln-tRNA Gln . Single-turnover kinetics validates these observations, confirming that the rate of the chemical step for tRNA aminoacylation (k chem) exceeds the steady-state rate by nearly 10-fold. The concentration dependence of the single-turnover reaction further reveals that the glutamine Kd is significantly higher than the steady-state K m value. The separation of binding from catalytic events by transient kinetics now allows precise interpretation of how alterations in tRNA structure affect the aminoacylation reaction. Mutation of U35 in the tRNA anticodon loop decreases k chem by 30-fold and weakens glutamine binding affinity by 20-fold, demonstrating that the active-site configuration depends on enzyme-tRNA contacts some 40 Å distant. By contrast, mutation of the adjacent G36 has very small effects on k chem and Kd for glutamine. Together with x-ray crystallographic data, these findings allow a comparative evaluation of alternative long-range signaling pathways and lay the groundwork for systematic exploration of how induced-fit conformational transitions may control substrate selection in this model enzyme-RNA complex.R ecognition between proteins and RNA generally occurs by an induced-fit mechanism, in which the protein, the RNA, or both undergo conformational changes en route to the final bound complex (1, 2). Although induced fit necessarily incurs an entropic penalty compared with a preformed and rigid interaction, the built-in flexibility nonetheless may help to increase the kinetic association rate, thus lowering the free-energy barrier for complex formation. In enzymatic reactions, it is also established that induced fit can provide a means to increase substrate specificity: Noncognate substrates may induce partial or incorrect rearrangements, leading to misalignment of reactive moieties along the pathway to the transition state (3). Thus, enzymes that modify RNA may employ induced fit for substrate discrimination at both binding and catalytic steps of the reaction.Aminoacyl-tRNA synthetases are superb model systems for investigating the operation of induced fit in enzyme-RNA complexes. In all organisms, these enzymes maintain fidelity of the genetic code by catalyzing the synthesis of cognate aminoacyl-tRNAs for use in protein synthesis (4). This synthesis occurs by means of a two-step reaction in which the amino acid is first activated to form an aminoacyl adenylate intermediate with release of pyrophosphate (PP i ). In the second step, the nucleophilic oxygen from the 2Ј-OH or 3Ј-OH group on the 3Ј-terminal tRNA ribose sugar attacks the mixed anhydride interm...

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“…An analogous situation exists for the discrimination between tyrosine and phenylalanine by tyrosyl-tRNA synthetase (TyrRS) where it is apparent that the WT enzyme has not reached the optimal level of discrimination between the two amino acids (47). In addition, E. coli glutaminyl-tRNA synthetase balances substrate specificity with catalytic efficiency (48), where the overall rate of aminoacylation is optimized by compromising between the various steps in the reaction pathway (48)(49)(50). It is clear that these biological catalysts have undergone optimization that values efficient and accurate aminoacylation that can only be achieved in some cases by trading off specificity and efficiency.…”

Section: Discussionmentioning

confidence: 99%

A Single Residue in Leucyl-tRNA Synthetase Affecting Amino Acid Specificity and tRNA Aminoacylation

Lue

2007

Biochemistry

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Human mitochondrial leucyl-tRNA synthetase (hs mt LeuRS) achieves high aminoacylation fidelity without a functional editing active site, representing a rare example of a class I aminoacyl-tRNA synthetase (aaRS) that does not proofread its products. Previous studies demonstrated that the enzyme achieves high selectivity by using a more specific synthetic active site that is not prone to errors under physiological conditions. Interestingly, the synthetic active site of hs mt LeuRS displays a high degree of homology with prokaryotic, lower eukaryotic and other mitochondrial LeuRSs that are less specific. However, there is one residue that differs between hs mt and Escherichia coli (E. coli) LeuRSs located on a flexible closing loop near the signature KMSKS motif. Here we describe studies indicating that this particular residue (K600 in hs mt LeuRS and L570 in E. coli LeuRS) strongly impacts aminoacylation in two ways -it affects both amino acid discrimination and transfer RNA (tRNA) binding. While this residue may not be in direct contact with the amino acid or tRNA substrate, substitutions of this position in both enzymes leads to altered catalytic efficiency and perturbations to the discrimination of leucine and isoleucine. In addition, tRNA recognition and aminoacylation is affected. These findings indicate that the conformation of the synthetic active site -modulated by this residue -may be coupled to specificity and provide new insights into the origins of selectivity without editing.Aminoacyl-tRNA synthetases (aaRSs) 1 are important translational factors. They catalyze the covalent attachment of amino acids to their cognate tRNAs, an essential step in the translation of the genetic code (1-3). The fidelity of protein synthesis is dependent upon the accuracy with which aaRSs discriminate between cognate and noncognate amino acids and numerous cellular tRNAs.To explain the high fidelity for cognate amino acids exhibited by aaRSs, a "double sieve" model has been proposed. This model, relevant mainly to class I aaRSs, relies on the use of two functionally independent active sites to achieve amino acid selectivity (4,5). In the first synthetic active site, amino acids are recognized, activated with ATP, converted to aminoacyl adenylates, and then transferred to tRNA. Amino acids larger than the cognate substrate are excluded from this site by sterics; smaller amino acids present a more significant problem as they can be bound, misactivated, and used erroneously to acylate tRNA. To resolve these errors, some aaRSs utilize a second editing active site that proofreads the products made by the activation site and hydrolytically cleaves substrates containing non-cognate amino acids. Both pre-transfer editing of misactivated aminoacyl adenylates and post-transfer editing of misacylated tRNA can occur at this second active site. Many systems are severely affected by *To whom correspondence should be addressed: Phone: 617-552-3121, Fax: 617-552-2705, E-mail: shana.kelley@bc.edu. Many previous studies have focused ...

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Reaction of modified and unmodified tRNATyr substrates with tyrosyl-tRNA synthetase (Bacillus stearothermophilus)

Cited by 35 publications

References 49 publications

Human Tyrosyl-tRNA Synthetase Shares Amino Acid Sequence Homology with a Putative Cytokine

Human Tyrosyl-tRNA Synthetase Shares Amino Acid Sequence Homology with a Putative Cytokine

Long-range intramolecular signaling in a tRNA synthetase complex revealed by pre-steady-state kinetics

A Single Residue in Leucyl-tRNA Synthetase Affecting Amino Acid Specificity and tRNA Aminoacylation

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