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ABSTRACT Mechanisms have evolved to prevent errors in replication, transcription, and translation of genetic material, with translational errors occurring most frequently. Errors in protein synthesis can occur at two steps, during tRNA aminoacylation and ribosome decoding. Recent advances in protein mass spectrometry have indicated that previous reports of translational errors have potentially underestimated the frequency of these events, but also that the majority of translational errors occur during ribosomal decoding, suggesting that aminoacylation errors are evolutionarily less tolerated. Despite that interpretation, there is evidence that some aminoacylation errors may be regulated, and thus provide a benefit to the cell, while others are clearly detrimental. Here, we show that while it has been suggested that regulated Thr-to-Ser substitutions may be beneficial, there is a threshold beyond which these errors are detrimental. In contrast, we show that errors mediated by alanyl-tRNA synthetase (AlaRS) are not well tolerated and induce a global stress response that leads to gross perturbation of the Escherichia coli proteome, with potentially catastrophic effects on fitness and viability. Tolerance for Ala mistranslation appears to be much lower than with other translational errors, consistent with previous reports of multiple proofreading mechanisms targeting mischarged tRNA Ala . These results demonstrate the essential role of aminoacyl-tRNA proofreading in optimizing cellular fitness and suggest that any potentially beneficial effects of mistranslation may be confined to specific amino acid substitutions. IMPORTANCE Errors in protein synthesis have historically been assumed to be detrimental to the cell. While there are many reports that translational errors are consequential, there is a growing body of evidence that some mistranslation events may be tolerated or even beneficial. Using two models of mistranslation, we compare the direct phenotypic effects of these events in Escherichia coli . This work provides insight into the threshold for tolerance of specific mistranslation events that were previously predicted to be broadly neutral to proteome integrity. Furthermore, these data reveal the effects of mistranslation beyond the general unfolded stress response, leading to global translational reprogramming.