Aminoacyl-tRNA Synthetases: General Features and Recognition of Transfer RNAs

The accuracy of protein synthesis relies on the ability of aminoacyl-tRNA synthetases (aaRSs) to discriminate among true and near cognate substrates. To date, analysis of aaRSs function, including identification of residues of aaRS participating in amino acid and tRNA discrimination, has largely relied on the steady state kinetic pyrophosphate exchange and aminoacylation assays. Pre-steady state kinetic studies investigating a more limited set of aaRS systems have also been undertaken to assess the energetic contributions of individual enzyme-substrate interactions, particularly in the adenylation half reaction. More recently, a renewed interest in the use of rapid kinetics approaches for aaRSs has led to their application to several new aaRS systems, resulting in the identification of mechanistic differences that distinguish the two structurally distinct aaRS classes. Here, we review the techniques for thermodynamic and kinetic analysis of aaRS function. Following a brief survey of methods for the preparation of materials and for steady state kinetic analysis, this review will describe pre-steady state kinetic methods employing rapid quench and stopped-flow fluorescence for analysis of the activation and aminoacyl transfer reactions. Application of these methods to any aaRS system allows the investigator to derive detailed kinetic mechanisms for the activation and aminoacyl transfer reactions, permitting issues of substrate specificity, stereochemical mechanism, and inhibitor interaction to be addressed in a rigorous and quantitative fashion.

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“…Aminoacyl-tRNA synthetases (aaRSs) 1 catalyze the fundamental two-step reaction that provides aminoacyl-tRNA for protein synthesis [1][2][3]:…”

Section: Introductionmentioning

confidence: 99%

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

Francklyn

First

Perona

et al. 2008

Methods

106

120

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“…In bacteria, a single gene typically encodes these 20 enzymes, whereas in eukaryotes, separate genes usually code for cytoplasmic and mitochondrial forms. Because living systems are based on the genetic code, and because tRNA synthetases are key components of the code and only have single genes, a mutation that disrupts aminoacylation activity of any synthetase results in cell death (6,7). The existence of singlecopy genes is believed to reflect, in part, the result of selective pressure against any gene proliferations that could result in corruption of the code (8).…”

mentioning

confidence: 99%

Structure-specific tRNA Determinants for Editing a Mischarged Amino Acid

Beebe¹,

Merriman

Schimmel

2003

Journal of Biological Chemistry

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Alanyl-tRNA synthetase efficiently aminoacylates tRNA Ala and an RNA minihelix that comprises just one domain of the two-domain L-shaped tRNA structure. It also clears mischarged tRNA Ala using a specialized domain in its C-terminal half. In contrast to full-length tRNA Ala , minihelix Ala was robustly mischarged and could not be edited. Addition in trans of the missing anticodon-containing domain did not activate editing of mischarged minihelix Ala . To understand these differences between minihelix Ala and tRNA Ala , several chimeric full tRNAs were constructed. These had the acceptor stem of a non-cognate tRNA replaced with the stem of tRNA Ala . The chimeric tRNAs collectively introduced multiple sequence changes in all parts but the acceptor stem. However, although the acceptor stem in isolation (as the minihelix) lacked determinants for editing, alanyl-tRNA synthetase effectively cleared a mischarged amino acid from each chimeric tRNA. Thus, a covalently continuous two-domain structure per se, not sequence, is a major determinant for clearance of errors of aminoacylation by alanyl-tRNA synthetase. Because errors of aminoacylation are known to be deleterious to cell growth, structure-specific determinants constitute a powerful selective pressure to retain the format of the two-domain L-shaped tRNA.

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“…Eqns (2) and (3) together with a corresponding computer program were used to calculate the free ATP and PPi concentrations in the analysis of the ATP/PPi exchange reaction.…”

Section: Calculation Ofthe Concentrations Of Free Atp and Ppimentioning

confidence: 99%

On the roles of magnesium and spermidine in the isoleucyl‐tRNA synthetase reaction

Airas

1990

European Journal of Biochemistry

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The reaction of isoleucyl-tRNA synthetase from Escherichiu coli B was analysed by deriving total steady-state rate equations for the ATPjPP, exchange reaction and for the aminoacylation of tRNA, and by fitting these rate equations to series of experimental results. The analysis suggests that (a) a Mg2 + inhibits the aminoacylation of tRNA but not the activation of the amino acid. In the chosen mechanism, this enzyme-bound Mg2+ is required at the activation step. (b) Another Mg2+ is required at ATP, but the MgATP apparently can be replaced by the spermidine . ATP complex. Spermidine . ATP is a weaker substrate. The role of spermidine . ATP is especially suggested by the relative rates of the aminoacylation of tRNA when the spermidine and magnesium concentrations are varied. The aminoacylation measurements still suggest that (c) two (or more) Mg2+ are bound to the tRNA molecule and are required for enzyme activity at the transfer step, and that these Mg2+ can be replaced by spermidines.The general model for the aminoacyl-tRNA synthetase reactions contains the activation of the amino acid by ATP through formation of an enzyme-bound aminoacyl-adenylate intermediate, and thereafter transfer of the aminoacyl moiety to the tRNA [I -31. A Mg2+ can theoretically participate in the total reaction by binding to the enzyme [4, 51, ATP [6] or tRNA [7-91. It is obligatory in the activation reaction, but most of it can be replaced by spermidine or spermine in the transfer step or total reaction [4, 10, 111. Many of the characteristics of the Mg2+ dependence of the isoleucyl-tRNA synthetase reaction can be explained by a model where MgATP always is one of the substrates, and a second Mg2+ is required in the tRNA, and the second Mg2 + can be replaced by spermidine [8].In some recent papers we have developed total steady-state rate equations to describe various properties of the aminoacyltRNA synthetase reactions [12 -141. This approach inevitably has limitations due to the great complexity of the total reaction and due to the unknown values of most of the kinetic constants. However it has proved to be useful as a supplement to the earlier kinetic treatments. In the present paper, some features of the isoleucyl-tRNA synthetase reaction were analysed by expanding the total rate equation to the Mg2+ and spermidine effects on the aminoacylation of tRNA and the ATP/PPi exchange reaction. The results suggest a participation of two Mg2+ in the activation reaction. One of them can be replaced by a polyamine. In the best chosen mechanism the Mg2+ in ATP can be replaced by spermidine and the other, enzyme-bound Mg2 + is required only in the absence of tRNA.

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Aminoacyl-tRNA Synthetases: General Features and Recognition of Transfer RNAs

Cited by 620 publications

References 97 publications

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

Structure-specific tRNA Determinants for Editing a Mischarged Amino Acid

On the roles of magnesium and spermidine in the isoleucyl‐tRNA synthetase reaction

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