title: A yeast tRNA mutant that causes pseudohyphal growth exhibits reduced rates of CAG codon translation.
abstract: In Saccharomyces cerevisiae, the SUP70 gene encodes the CAG-decoding tRNA(Gln)(CUG). A mutant allele, sup70-65, induces pseudohyphal growth on rich medium, an inappropriate nitrogen starvation response. This mutant tRNA is also a UAG nonsense suppressor via first base wobble. To investigate the basis of the pseudohyphal phenotype, 10 novel sup70 UAG suppressor alleles were identified, defining positions in the tRNA(Gln)(CUG) anticodon stem that restrict first base wobble. However, none conferred pseudohyphal growth, showing altered CUG anticodon presentation cannot itself induce pseudohyphal growth. Northern blot analysis revealed the sup70-65 tRNA(Gln)(CUG) is unstable, inefficiently charged, and 80% reduced in its effective concentration. A stochastic model simulation of translation predicted compromised expression of CAG-rich ORFs in the tRNA(Gln)(CUG)-depleted sup70-65 mutant. This prediction was validated by demonstrating that luciferase expression in the mutant was 60% reduced by introducing multiple tandem CAG (but not CAA) codons into this ORF. In addition, the sup70-65 pseudohyphal phenotype was partly complemented by overexpressing CAA-decoding tRNA(Gln)(UUG), an inefficient wobble-decoder of CAG. We thus show that introducing codons decoded by a rare tRNA near the 5' end of an ORF can reduce eukaryote translational expression, and that the mutant tRNA(CUG)(Gln) constitutive pseudohyphal differentiation phenotype correlates strongly with reduced CAG decoding efficiency.
authors.affiliation: Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK.
authors.forename: Alain J
authors.initials: AJ
authors.lastname: Kemp
authors.forename: Russell
authors.initials: R
authors.lastname: Betney
authors.forename: Luca
authors.initials: L
authors.lastname: Ciandrini
authors.forename: Alexandra C M
authors.initials: AC
authors.lastname: Schwenger
authors.forename: M Carmen
authors.initials: MC
authors.lastname: Romano
authors.forename: Ian
authors.initials: I
authors.lastname: Stansfield
mesh_terms: D003062:Codon; D003470:Culture Media / Q000737:chemistry; D017930:Genes, Reporter; D025301:Hyphae / Q000254:growth & development*; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D009154:Mutation; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development*
chemical_list: D003062:Codon; D003470:Culture Media; D012343:RNA, Transfer; D008156:Luciferases