Genetic selection for mistranslation rescues a defective co-chaperone in yeast

Despite the general requirement for translation fidelity, mistranslation can be an adaptive response. We selected spontaneous second site mutations that suppress the stress sensitivity caused by a Saccharomyces cerevisiae tti2 allele with a Leu to Pro mutation at residue 187, identifying a single nucleotide mutation at the same position (C70U) in four tRNA(Pro)(UGG) genes. Linkage analysis and suppression by SUF9(G3:U70) expressed from a centromeric plasmid confirmed the causative nature of the suppressor mutation. Since the mutation incorporates the G3:U70 identity element for alanyl-tRNA synthetase into tRNA(Pro), we hypothesized that suppression results from mistranslation of Pro187 in Tti2(L187P) as Ala. A strain expressing Tti2(L187A) was not stress sensitive. In vitro, tRNA(Pro)(UGG) (C70U) was mis-aminoacylated with alanine by alanyl–tRNA synthetase, but was not a substrate for prolyl–tRNA synthetase. Mass spectrometry from protein expressed in vivo and a novel GFP reporter for mistranslation confirmed substitution of alanine for proline at a rate of ∼6%. Mistranslating cells expressing SUF9(G3:U70) induce a partial heat shock response but grow nearly identically to wild-type. Introducing the same G3:U70 mutation in SUF2 (tRNA(Pro)(AGG)) suppressed a second tti2 allele (tti2(L50P)). We have thus identified a strategy that allows mistranslation to suppress deleterious missense Pro mutations in Tti2.