title: 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.
authors.affiliation: Department of Biochemistry, The University of Western Ontario, London, ON N6A 5C1, Canada.
authors.forename: Kyle S
authors.initials: KS
authors.lastname: Hoffman
authors.affiliation: Department of Biochemistry, The University of Western Ontario, London, ON N6A 5C1, Canada.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Berg
authors.affiliation: Department of Biochemistry, The University of Western Ontario, London, ON N6A 5C1, Canada.
authors.forename: Brian H
authors.initials: BH
authors.lastname: Shilton
authors.affiliation: Department of Biochemistry, The University of Western Ontario, London, ON N6A 5C1, Canada.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Brandl
authors.affiliation: Department of Biochemistry, The University of Western Ontario, London, ON N6A 5C1, Canada.|Department of Chemistry, The University of Western Ontario, London, ON N6A 5B7, Canada.
authors.forename: Patrick
authors.initials: P
authors.lastname: O'Donoghue
mesh_terms: D000483:Alleles; D019943:Amino Acid Substitution; D007438:Introns; D018832:Molecular Chaperones / Q000096:biosynthesis / Q000235:genetics*; D014176:Protein Biosynthesis; D012361:RNA, Transfer, Pro / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics*; D012641:Selection, Genetic; D013489:Suppression, Genetic
chemical_list: D018832:Molecular Chaperones; D012361:RNA, Transfer, Pro; D029701:Saccharomyces cerevisiae Proteins; C576144:Tti2 protein, S cerevisiae