Mistranslation is typically deleterious for cells, although specific mistranslated proteins can confer a short-term benefit in a particular environment. However, given its large overall cost, the prevalence of high global mistranslation rates remains puzzling. Altering basal mistranslation levels of Escherichia coli in several ways, we show that generalized mistranslation enhances early survival under DNA damage, by rapidly activating the SOS response. Mistranslating cells maintain larger populations after exposure to DNA damage, and thus have a higher probability of sampling critical beneficial mutations. Both basal and artificially increased mistranslation increase the number of cells that are phenotypically tolerant and genetically resistant under DNA damage; they also enhance survival at high temperature. In contrast, decreasing the normal basal mistranslation rate reduces cell survival. This wideranging stress resistance relies on Lon protease, which is revealed as a key effector that induces the SOS response in addition to alleviating proteotoxic stress. The new links between error-prone protein synthesis, DNA damage, and generalised stress resistance indicate surprising coordination between intracellular stress responses and suggest a novel hypothesis to explain high global mistranslation rates. Author summaryCells make mistakes all the time. Protein synthesis is exceptionally error-prone, which is puzzling because such mistakes are usually harmful. Prior work shows that specific erroneous proteins can be beneficial under specific stresses. However, this cannot explain a high background error rate, because most mistakes will not be useful. We offer a solution to this conundrum: cells that make more mistakes accumulate a key quality-control enzyme, tripping the switch for a broad stress response (the "SOS" response, that repairs damaged DNA). In turn, this increases cell survival. The unexpected link between protein synthesis and DNA damage suggests surprising cross talk across important quality-control processes, and motivates a new hypothesis to explain how and why cells tolerate so many mistakes.

In all domains of life, initiator tRNA functions exclusively at the first step of protein synthesis while elongator tRNAs extend the polypeptide chain. Unique features of initiator tRNA enable it to preferentially bind the ribosomal P site and initiate translation. Recently, we showed that the abundance of initiator tRNA also contributes to its specialized role. This motivates the question, can a cell also use elongator tRNA to initiate translation under certain conditions? To address this, we introduced non-AUG initiation codons CCC (Pro), GAG (Glu), GGU (Gly), UCU (Ser), UGU (Cys), ACG (Thr), AAU (Asn), and AGA (Arg) into the uracil DNA glycosylase gene (ung) used as a reporter gene. Enzyme assays from log-phase cells revealed initiation from non-AUG codons when intracellular initiator tRNA levels were reduced. The activity increased significantly in stationary phase. Further increases in initiation from non-AUG codons occurred in both growth phases upon introduction of plasmid-borne genes of cognate elongator tRNAs. Since purine-rich Shine-Dalgarno sequences occur frequently on mRNAs (in places other than the canonical AUG codon initiation contexts), initiation with elongator tRNAs from the alternate contexts may generate proteome diversity under stress without compromising genomic integrity. Thus, by changing the relative amounts of initiator and elongator tRNAs within the cell, we have blurred the distinction between the two classes of tRNAs thought to be frozen through years of evolution. Most organisms possess two kinds of tRNAs, the initiator tRNA, which decodes the initiation codon, and the elongator tRNAs, which decode the subsequent codons within the open reading frame (ORF). In Escherichia coli, there are four copies of the initiator tRNA genes; three of these, metZ, metW, and metV, are present at 63.5 min, whereas the fourth one, metY, is found at 71.5 min in the genome (1). It is also known that either (but not both) of these loci may be deleted from E. coli. The strain deleted for metZWV becomes cold sensitive. However, at temperatures of 30°C or higher the strain grows normally (2, 3).Initiator and elongator tRNAs are believed to perform their respective decoding functions without functional interference from one another. Although they share the same cloverleaf-like backbone structure, years of evolution have led to their separate and distinct functions, now held in place by an intricate support system comprising translation factors, quality control mechanisms, and the ribosome itself. Elongator tRNAs are excluded from participation at the start of protein synthesis by sequence features specific to the initiator tRNA that are recognized exclusively at the P site of the ribosome (4-6). Various other details, such as the fact that the initiation and elongation factors bind specifically to their cognate species of tRNAs, further aid the specificity of their functions.However, earlier studies have enabled initiation by elongator tRNAs, by transplanting the critical features of initiator tRNA onto the...

Of all tRNAs, initiator tRNA is unique in its ability to start protein synthesis by directly binding the ribosomal P-site. This ability is believed to derive from the almost universal presence of three consecutive G-C base (3G-C) pairs in the anticodon stem of initiator tRNA. Consistent with the hypothesis, a plasmid-borne initiator tRNA with one, two, or all 3G-C pairs mutated displays negligible initiation activity when tested in a WT Escherichia coli cell. Given this, the occurrence of unconventional initiator tRNAs lacking the 3G-C pairs, as in some species of Mycoplasma and Rhizobium , is puzzling. We resolve the puzzle by showing that the poor activity of unconventional initiator tRNAs in E. coli is because of competition from a large pool of the endogenous WT initiator tRNA (possessing the 3G-C pairs). We show that E. coli can be sustained on an initiator tRNA lacking the first and third G-C pairs; thereby reducing the 3G-C rule to a mere middle G-C requirement. Two general inferences following from our findings, that the activity of a mutant gene product may depend on its abundance in the cell relative to that of the WT, and that promiscuous initiation with elongator tRNAs has the potential to enhance phenotypic diversity without affecting genomic integrity, have been discussed.

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