Aminoacyl-tRNA synthetases use a variety of mechanisms to ensure fidelity of the genetic code and ultimately select the correct amino acids to be used in protein synthesis. The physiological necessity of these quality control mechanisms in different environments remains unclear, as the cost vs benefit of accurate protein synthesis is difficult to predict. We show that in Escherichia coli, a non-coded amino acid produced through oxidative damage is a significant threat to the accuracy of protein synthesis and must be cleared by phenylalanine-tRNA synthetase in order to prevent cellular toxicity caused by mis-synthesized proteins. These findings demonstrate how stress can lead to the accumulation of non-canonical amino acids that must be excluded from the proteome in order to maintain cellular viability.DOI: http://dx.doi.org/10.7554/eLife.02501.001

During mRNA decoding at the ribosome, deviations from stringent codon identity, or “mistranslation,” are generally deleterious and infrequent. Observations of organisms that decode some codons ambiguously, and the discovery of a compensatory increase in mistranslation frequency to combat environmental stress have changed the way we view “errors” in decoding. Modern tools for the study of the frequency and phenotypic effects of mistranslation can provide quantitative and sensitive measurements of decoding errors that were previously inaccessible. Mistranslation with non-protein amino acids, in particular, is an enticing prospect for new drug therapies and the study of molecular evolution.

Background: Translation of Tyr codons is highly prone to Phe misincorporation during amino acid limitation in CHO cells. Results: CHO TyrRS is error-prone and readily aminoacylates tRNA Tyr with Phe. Conclusion: Mammalian TyrRS has evolved to be significantly less accurate than its bacterial counterpart. Significance: Different evolutionary constraints determine the accuracy of translation quality control in eukaryotes and bacteria.

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 noncognates 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.

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