Evolving Mistranslating tRNAs Through a Phenotypically Ambivalent Intermediate in .

The genetic code converts information from nucleic acid into protein. The genetic code was thought to be immutable, yet many examples in nature indicate that variations to the code provide a selective advantage. We used a sensitive selection system involving suppression of a deleterious allele () in to detect mistranslation and identify mechanisms that allow genetic code evolution. Though tRNA containing a proline anticodon (UGG) is toxic, using our selection system we identified four tRNA variants, each with a single mutation, that mistranslate at a tolerable level. Mistranslating tRNA variants were also obtained, demonstrating the generality of the approach. We characterized two of the tRNA variants. One contained a G26A mutation, which reduced cell growth to 70% of the wild-type rate, induced a heat shock response, and was lost in the absence of selection. The reduced toxicity of tRNA-G26A is likely through increased turnover of the tRNA, as lack of methylation at G26 leads to degradation via the rapid tRNA decay pathway. The second tRNA variant, with a G9A mutation, had minimal effect on cell growth, was relatively stable in cells, and gave rise to less of a heat shock response. , the G9A mutation decreases aminoacylation and affects folding of the tRNA. Notably, the G26A and G9A mutations were phenotypically neutral in the context of an otherwise wild-type tRNA These experiments reveal a model for genetic code evolution in which tRNA anticodon mutations and mistranslation evolve through phenotypically ambivalent intermediates that reduce tRNA function.