Abstract
Non-protein amino acids, particularly isomers of the proteinogenic amino acids, present a threat to proteome integrity if they are mistakenly inserted into proteins. Quality control during aminoacyl-tRNA synthesis reduces non-protein amino acid incorporation by both substrate discrimination and proofreading. For example phenylalanyl-tRNA synthetase (PheRS) proofreads the non-protein hydroxylated phenylalanine derivative m-Tyr after its attachment to tRNA(Phe) We now show in Saccharomyces cerevisiae that PheRS misacylation of tRNA(Phe) with the more abundant Phe oxidation product o-Tyr is limited by kinetic discrimination against o-Tyr-AMP in the transfer step followed by o-Tyr-AMP release from the synthetic active site. This selective rejection of a non-protein aminoacyl-adenylate is in addition to known kinetic discrimination against certain non-cognates in the activation step as well as catalytic hydrolysis of mispaired aminoacyl-tRNA(Phe) species. We also report an unexpected resistance to cytotoxicity by a S. cerevisiae mutant with ablated post-transfer editing activity when supplemented with o-Tyr, cognate Phe, or Ala, the latter of which is not a substrate for activation by this enzyme. Our phenotypic, metabolomic, and kinetic analyses indicate at least three modes of discrimination against non-protein amino acids by S. cerevisiae PheRS and support a non-canonical role for SccytoPheRS post-transfer editing in response to amino acid stress.
Highlights
The units of genetic information contained in mRNA codons need to be precisely translated at the ribosome into corresponding amino acid sequences in proteins
SccytoPheRS Post-transfer Editing Limits the Cytotoxicity of Oxidative Stress—After our observations that phenylalanyl-tRNA synthetase (PheRS) posttransfer editing activity is required to protect E. coli from m-Tyr, we examined the role that post-transfer editing activity plays in a eukaryotic species, S. cerevisiae
We found that SccytoPheRS post-transfer editing activity limited the toxicity of paraquat (Fig. 2), suggesting a similar protective role of PheRS post-transfer editing in both bacteria and eukarya under oxidative stress
Summary
Either by generating non-protein amino acids, which must be proofread [10], or by direct inactivation of such proofreading (10 –12). The post-transfer editing activity of the wild-type enzyme can hydrolyze m-Tyr-tRNAPhe species that are synthesized as m-Tyr accumulates under conditions of oxidative stress. In contrast to the E. coli enzyme, Saccharomyces cerevisiae cytoplasmic PheRS (SccytoPheRS) exhibits poor discrimination in the amino acid activation step against p-Tyr (ϳ20-fold lower than EcPheRS), but p-Tyr-tRNAPhe production is limited by post-transfer editing [10]. As with EcPheRS, SccytoPheRS post-transfer editing of m-Tyr-tRNAPhe limits cytotoxicity of m-Tyr. Here, we show unexpected phenotypic effects of direct media supplementation of S. cerevisiae with cognate Phe, proteinogenic non-cognate Ala, and non-protein oxidized Phe derivatives in a SccytoPheRS post-transfer quality control mutant. We highlight striking differences in the promiscuity of the yeast and bacterial enzymes for non-protein amino acids and discuss multiple strategies employed by SccytoPheRS to maintain product specificity in the face of conditional threats to translational quality control in a biological context
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