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

Abstract

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 tRNAProUGG genes. Linkage analysis and suppression by SUF9G3: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 tRNAPro, we hypothesized that suppression results from mistranslation of Pro187 in Tti2L187P as Ala. A strain expressing Tti2L187A was not stress sensitive. In vitro, tRNAProUGG (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 SUF9G3:U70 induce a partial heat shock response but grow nearly identically to wild-type. Introducing the same G3:U70 mutation in SUF2 (tRNAProAGG) suppressed a second tti2 allele (tti2L50P). We have thus identified a strategy that allows mistranslation to suppress deleterious missense Pro mutations in Tti2.

Transfer RNA function and evolution

“…The authors visualized a rate of~3% mistranslation using a novel green fluorescent protein (D129P) reporter that fluoresces in response to mistranslation at proline codons. In contrast to previous studies in yeast [18], human cells in culture did not mount a detectable heat-shock response and tolerated the mistranslation without apparent impact on cell viability. Although a perfect proteome is not required for cellular viability, the cell contains several mechanisms to monitor and respond to errors in protein synthesis.…”

Global mistranslation increases cell survival under stress in Escherichia coli

“…Prior work also shows that Lon is required to alleviate the toxic effects of mistranslation [3]. However, thus far only two studies [49] [55] have linked the heat shock response with mistranslation. We now add to this work, clearly showing that the heat shock response is activated by general mistranslation and corroborating prior predictions that aberrant proteins activate heat shock chaperones [56].…”

Mistranslating tRNA identifies a deleterious S213P mutation in theSaccharomyces cerevisiae eco1-1allele

“…Loss of fidelity at either step can lead to mistranslation. Mistranslation is dramatically increased by tRNA variants that are inaccurately aminoacylated or contain mutations within their anticodon (Geslain et al 2010;Hoffman et al 2017;Lant et al 2017;Berg et al 2017;Zimmerman et al 2018). Nucleotides in the tRNA that are recognized by a specific aaRS are called identity elements and consist of single nucleotides, nucleotide pairs, and structural motifs (de Duve 1988;Hou and Schimmel 1988;Giegé et al 1998).…”