Accurate translation of the genetic code is maintained in part by aminoacyl-tRNA synthetases (aaRS) proofreading mechanisms that ensure correct attachment of a cognate amino acid to a transfer RNA (tRNA). During environmental stress, such as oxidative stress, demands on aaRS proofreading are altered by changes in the availability of cytoplasmic amino acids. For example, oxidative stress increases levels of cytotoxic tyrosine isomers, noncognate amino acids normally excluded from translation by the proofreading activity of phenylalanyl-tRNA synthetase (PheRS). Here we show that oxidation of PheRS induces a conformational change, generating a partially unstructured protein. This conformational change does not affect Phe or Tyr activation or the aminoacylation activity of PheRS. However, in vitro and ex vivo analyses reveal that proofreading activity to hydrolyze Tyr-tRNA Phe is increased during oxidative stress, while the cognate Phe-tRNA Phe aminoacylation activity is unchanged. In HPX − , Escherichia coli that lack reactive oxygen-scavenging enzymes and accumulate intracellular H 2 O 2 , we found that PheRS proofreading is increased by 11%, thereby providing potential protection against hazardous cytoplasmic m-Tyr accumulation. These findings show that in response to oxidative stress, PheRS proofreading is positively regulated without negative effects on the enzyme's housekeeping activity in translation. Our findings also illustrate that while the loss of quality control and mistranslation may be beneficial under some conditions, increased proofreading provides a mechanism for the cell to appropriately respond to environmental changes during oxidative stress.aminoacyl-tRNA | oxidative stress | proofreading | quality control

The aminoacylation status of the cellular tRNA pool regulates both general amino acid control (GAAC) and target of rapamycin (TOR) stress response pathways in yeast. Consequently, fidelity of translation at the level of aminoacyl-tRNA synthesis plays a central role in determining accuracy and sensitivity of stress responses. To investigate effects of translational quality control (QC) on cell physiology under stress conditions, phenotypic microarray analyses were used to identify changes in QC deficient cells. Nitrogen source growth assays showed QC deficient yeast grew differently compared to WT. The QC deficient strain was more tolerant to caffeine treatment than wild type through altered interactions with the TOR and GAAC pathways. Increased caffeine tolerance of the QC deficient strain was consistent with the observation that the activity of Gln3p, a transcription factor controlled by the TOR pathway, is decreased in the QC deficient strain compared to WT. GCN4 translation, which is typically repressed in the absence of nutritional stress, was enhanced in the QC deficient strain through TOR inhibition. QC did not impact cell cycle regulation; however, the chronological lifespan of QC deficient yeast strains decreased compared to wild type, likely due to translational errors and alteration of the TOR-associated regulon. These findings support the idea that changes in translational fidelity provide a mechanism of cellular adaptation by modulating TOR activity. This, in turn, supports a central role for aminoacyl-tRNA synthesis QC in the integrated stress response by maintaining the proper aa-tRNA pools necessary to coordinate the GAAC and TOR.