Fast kinetic study of yeast phenylalanyl-tRNA synthetase: an efficient discrimination between tyrosine and phenylalanine at the level of the aminoacyladenylate-enzyme complex

Lin, Sheng‐Xiang; Baltzinger, Mireille; Rémy, Pierre

doi:10.1021/bi00272a024

Cited by 48 publications

(50 citation statements)

References 16 publications

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“…Among class II ARSs, editing functions have similarly been demonstrated for phenylalanyl-(PheRS) (17)(18)(19), alanyl-(AlaRS) (20,21), threonyl-(ThrRS) (22), and prolyl-tRNA synthetases (ProRS) (23). Unlike class I enzymes, which share a conserved CP-1 editing domain, class II ARSs lack a common editing domain.…”

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confidence: 99%

Aminoacyl Transfer Rate Dictates Choice of Editing Pathway in Threonyl-tRNA Synthetase

Minajigi

Francklyn

2010

Journal of Biological Chemistry

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Aminoacyl-tRNA synthetases hydrolyze aminoacyl adenylates and aminoacyl-tRNAs formed from near-cognate amino acids, thereby increasing translational fidelity. The contributions of pre-and post-transfer editing pathways to the fidelity of Escherichia coli threonyl-tRNA synthetase (ThrRS) were investigated by rapid kinetics. In the pre-steady state, asymmetric activation of cognate threonine and noncognate serine was observed in the active sites of dimeric ThrRS, with similar rates of activation. In the absence of tRNA, seryl-adenylate was hydrolyzed 29-fold faster by the ThrRS catalytic domain than threonyl-adenylate. The rate of seryl transfer to cognate tRNA was only 2-fold slower than threonine. Experiments comparing the rate of ATP consumption to the rate of aminoacyl-tRNA AA formation demonstrated that pre-transfer hydrolysis contributes to proofreading only when the rate of transfer is slowed significantly. Thus, the relative contributions of pre-and posttransfer editing in ThrRS are subject to modulation by the rate of aminoacyl transfer.The accurate transfer of genetic information in living systems requires multiple mechanisms to enhance and preserve the fidelity of gene expression, particularly in protein synthesis. The 1-3 errors per 10,000 amino acids incorporated during protein synthesis largely originate from errors in decoding (1) but can arise from previous steps, including the aminoacylation reaction catalyzed by aminoacyl-tRNA synthetases (ARSs) 2 (2). In the first of two half-reactions, ARSs condense amino acid and ATP to form a noncovalently enzyme-bound adenylate, followed by a transfer reaction that produces aminoacyl-tRNA and AMP as products. This highly accurate reaction (on the order of 1 error in 10 5 ) reflects the ability of ARSs to carefully discriminate among chemically similar standard and nonstandard amino acids (3). Mutations in ARSs or their cognate tRNAs can nonetheless create errors associated with significant molecular pathologies, particularly in neural tissues (4).Amino acids that differ by a single methyl group pose a particular discrimination challenge for ARSs (3,5,6). An additional methyl group on an amino acid typically provides no more than ϳ1 kcal/mol of incremental binding energy, imposing an upper limit of discrimination on the order of 1 in 5 (7). To account for the enhanced discrimination seen in living systems, a "double sieve" model was proposed (8). Amino acids larger than cognate are excluded by the "coarse sieve" of the aminoacylation site, whereas smaller or isosteric amino acids are cleaved from the end of the tRNA by the "fine sieve" of the editing site. In principle, noncognate amino acids can be edited both by increased hydrolysis of the adenylate (pre-transfer editing) or by hydrolysis of the mis-acylated tRNA (post-transfer editing). Either or both reactions can formally occur in a dedicated editing site structurally distinct from the standard synthetic site. Because of differences between systems, and the absence of a comprehensive kinetic description ...

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confidence: 99%

Aminoacyl Transfer Rate Dictates Choice of Editing Pathway in Threonyl-tRNA Synthetase

Minajigi

Francklyn

2010

Journal of Biological Chemistry

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“…These findings are discussed in the context of other recent studies, which together with the data presented here, raise the possibility that mitochondrial protein synthesis may be less accurate than its cytosolic counterpart. (29). LB and M9 media were prepared as described previously (30).…”

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Loss of Editing Activity during the Evolution of Mitochondrial Phenylalanyl-tRNA Synthetase

Roy

Ling

Alfonzo

et al. 2005

Journal of Biological Chemistry

100

104

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Accurate selection of amino acids is essential for faithful translation of the genetic code. Errors during amino acid selection are usually corrected by the editing activity of aminoacyl-tRNA synthetases such as phenylalanyl-tRNA synthetases (PheRS), which edit misactivated tyrosine. Comparison of cytosolic and mitochondrial PheRS from the yeast Saccharomyces cerevisiae suggested that the organellar protein might lack the editing activity. Yeast cytosolic PheRS was found to contain an editing site, which upon disruption abolished both cis and trans editing of Tyr-tRNA Phe . Wild-type mitochondrial PheRS lacked cis and trans editing and could synthesize Tyr-tRNA Phe , an activity enhanced in active site variants with improved tyrosine recognition. Possible trans editing was investigated in isolated mitochondrial extracts, but no such activity was detected. These data indicate that the mitochondrial protein synthesis machinery lacks the tyrosine proofreading activity characteristic of cytosolic translation. This difference between the mitochondria and the cytosol suggests that either organellar protein synthesis quality control is focused on another step or that translation in this compartment is inherently less accurate than in the cytosol.The aminoacyl-tRNA synthetases (aaRS) 2 are a ubiquitous and essential protein family required for protein synthesis (1-3). Structurally and functionally the aaRSs are divided into two unrelated but biochemically analogous groups, class I and class II (4 -6). The aaRSs attach amino acids to the 3Ј-ends of tRNA containing the corresponding anticodon sequence, and the resulting aminoacyl-tRNAs (aa-tRNAs) are used as substrates for ribosomal translation of mRNA. The accuracy of aa-tRNA synthesis is generally assured by the existence of aaRSs specific for each particular amino acid-tRNA pair. Cognate tRNA recognition and discrimination of non-cognate RNAs are achieved by sequencespecific direct and indirect readout of the numerous combinations of bases present in tRNAs (7-10). The relative structural simplicity and inherent similarity between the amino acid substrates makes their accurate recognition and discrimination more challenging. Although some amino acids such as cysteine and tyrosine are different enough to allow their specific recognition by a particular aaRS (11, 12), others such as valine and isoleucine are less easily distinguished. For example the class I aaRS isoleucyl-tRNA synthetase (IleRS) is only able to poorly discriminate against valine, which has a misactivation rate of about 1:200 compared with the cognate substrate isoleucine. Despite this significant rate of misactivation and misaminoacylation, the accuracy of translation is not compromised because of the existence of an intrinsic proofreading and editing mechanism in IleRS that specifically hydrolyzes both misactivated Val-AMP and misaminoacylated Val-tRNA Ile (13,14). In addition to IleRS, many other class I and class II aaRSs also employ editing to prevent release of non-cognate aa-tRNA and subsequent l...

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“…A rapid loss of the non-cognate intermediates relative to the -phenylalanyl adenylate may explain an apparent corrective mechanism that could maintain the accuracy of aminoacylation. Only very dissimilar non-cognate complexes should dissociate rapidly to compete significantly with transfer [31][32][33]. The breakdown of the aminoacyl adenylate may take place by essentially two routes : first, dissociation of the complex into solution to give free aminoacyl adenylates with subsequent hydrolysis, and secondly, hydrolysis of the aminoacyl adenylate when bound to the enzyme.…”

Section: Discussionmentioning

confidence: 99%

Editing of non-cognate aminoacyl adenylates by peptide synthetases

Pavela-Vrančić

Dieckmann²,

Döhren³

et al. 1999

Biochemical Journal

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Non-ribosomally formed peptides display both highly conserved and variable amino acid positions, the variations leading to a wide range of peptide families. Activation of the amino acid substrate proceeds in analogy to the ribosomal biosynthetic mechanism generating aminoacyl adenylate and acyl intermediates. To approach the mechanism of fidelity of amino acid selection, the stability of the aminoacyl adenylates was studied by employing a continuous coupled spectrophotometric assay. The apo-form of tyrocidine synthetase 1 (apo-TY1) was used, generating an -phenylalanyl-adenylate intermediate stabilized by the interaction of two structural subdomains of the adenylation domain. Adenylates of substrate analogues have shown variable and reduced degrees of stability, thus leading to an enhanced generation of pyrophosphate due to hydrolysis and continuous adenylate formation. Discrimination of the non-

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Fast kinetic study of yeast phenylalanyl-tRNA synthetase: an efficient discrimination between tyrosine and phenylalanine at the level of the aminoacyladenylate-enzyme complex

Cited by 48 publications

References 16 publications

Aminoacyl Transfer Rate Dictates Choice of Editing Pathway in Threonyl-tRNA Synthetase

Aminoacyl Transfer Rate Dictates Choice of Editing Pathway in Threonyl-tRNA Synthetase

Loss of Editing Activity during the Evolution of Mitochondrial Phenylalanyl-tRNA Synthetase

Editing of non-cognate aminoacyl adenylates by peptide synthetases

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