Residues in a Class I tRNA Synthetase Which Determine Selectivity of Amino Acid Recognition in the Context of tRNA

Schmidt, Eric; Schimmel, Paul

doi:10.1021/bi00035a028

Cited by 84 publications

(81 citation statements)

References 53 publications

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“…Class I enzymes are characterized by a Rossmann nucleotide binding fold (48) that forms the catalytic site for amino acid activation (3, 42, 45, 49, 50 -52). For the closely related class I isoleucyl-, leucyl-, and valyl-tRNA synthetases, an insertion known as CP1 splits the active site (42,(52)(53)(54)(55). This insertion harbors the center for editing that, in the threedimensional structures, is about 30 Å from the site for amino acid activation (15, 52, 54, 56 -58).…”

Section: Discussionmentioning

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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Section: Discussionmentioning

confidence: 99%

Structure-specific tRNA Determinants for Editing a Mischarged Amino Acid

Beebe¹,

Merriman

Schimmel

2003

Journal of Biological Chemistry

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“…The hs mt LeuRS appears to lack editing activity and achieves aminoacylation fidelity solely using its precise synthetic active site, which is not characteristic of other bacterial and eukaryotic LeuRSs studied to date (6)(7)(8)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)38). The hs mt LeuRS must possess special features within its active site that provide the uniquely high levels of amino acid specificity.…”

Section: Roles Of a Flexible Domain Containing A Critical Residue In mentioning

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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“…Through a series of mutational analyses, the centers for editing and the aminoacylation were clearly resolved even before a three-dimensional structure was available (11-13). Strikingly, the two sites are functionally independent in that mutations in one do not affect the other (12,21,29,37,38). However, the translocation step has been most difficult to resolve.…”

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confidence: 99%

“…These ancient proteins catalyze aminoacylation reactions that are the basis of the genetic code, that is, the matching of amino acids with specific nucleotide triplets imbedded in transfer RNAs (4)(5)(6)(7)(8). Inherent limitations to the capacity of active sites to discriminate between closely similar amino acids (9) was compensated by the introduction of a second active center where misactivated amino acids are cleared (10)(11)(12)(13)(14)(15). Without this active site for editing, ambiguity is introduced into the code as demonstrated by the extensive misincorporation of amino acids into cellular proteins when editing is disrupted (1).…”

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confidence: 99%

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Interstice mutations that block site-to-site translocation of a misactivated amino acid bound to a class I tRNA synthetase

Bishop

Beebe

Schimmel

2003

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

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Class I aminoacyl-tRNA synthetases catalyze editing reactions that prevent ambiguity from entering the genetic code. Misactivated amino acids are translocated in cis from the active site for aminoacylation to the center for editing, located Ϸ30 Å away. Mutational analysis has functionally separated the two sites by creating mutations that disrupt the catalytic center for editing but not for aminoacylation and vice versa. What is not known is whether translocation per se can be disrupted without an effect on either catalytic center. Here we describe mutations in a presumptive ''hinge region'' of isoleucyl-tRNA synthetase that is situated between the two sites. Interstice mutations had little or no effect on either catalytic center. In contrast, the same specific mutations disrupted translocation. Thus, with these mutations all three functions, translocation, catalysis of aminoacylation, and editing, have been mutationally separated. The results are consistent with translocation involving a hinge-region conformational shift that does not perturb the two catalytic centers.C onsiderable evidence supports the concept that the universal tree of life could not be generated without the editing activities of aminoacyl-tRNA synthetases (1-3). These ancient proteins catalyze aminoacylation reactions that are the basis of the genetic code, that is, the matching of amino acids with specific nucleotide triplets imbedded in transfer RNAs (4-8). Inherent limitations to the capacity of active sites to discriminate between closely similar amino acids (9) was compensated by the introduction of a second active center where misactivated amino acids are cleared (10-15). Without this active site for editing, ambiguity is introduced into the code as demonstrated by the extensive misincorporation of amino acids into cellular proteins when editing is disrupted (1).Class I aminoacyl-tRNA synthetases such as isoleucyl-, valyl-, and leucyl-tRNA synthetase are characterized by a catalytic center based on a Rossmann nucleotide-binding fold of alternating ␤-strands and ␣-helices (6, 16-21). The fold is split by an insertion known as connective polypeptide 1 (CP1) that joins one half of the catalytic domain to the other (12,(21)(22)(23). This insertion contains the active site for editing, located Ϸ30 Å from the catalytic center for aminoacylation (11-13, 15, 21, 24). The editing reactions require specific tRNAs as cofactors (25,26). The role of the tRNA is to trigger translocation of the misactivated residue from the catalytic center to the active site for editing (27)(28)(29).Amino acids are condensed with ATP to form aminoacyl adenylates bound to their cognate tRNA synthetases in the first step of protein synthesis. Occasional errors lead to activation of the wrong amino acid such as activation of valine by isoleucyltRNA synthetase (IleRS) or activation of threonine by valyltRNA synthetase (10, 30). Editing proceeds through two distinct pathways (Fig. 1). One is designated as the pretransfer step (10, 31). Here the misactivated amino acid (i...

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Residues in a Class I tRNA Synthetase Which Determine Selectivity of Amino Acid Recognition in the Context of tRNA

Cited by 84 publications

References 53 publications

Structure-specific tRNA Determinants for Editing a Mischarged Amino Acid

Structure-specific tRNA Determinants for Editing a Mischarged Amino Acid

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

Interstice mutations that block site-to-site translocation of a misactivated amino acid bound to a class I tRNA synthetase

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