title: The β-hairpin of 40S exit channel protein Rps5/uS7 promotes efficient and accurate translation initiation in vivo.
abstract: The eukaryotic 43S pre-initiation complex bearing tRNAi(Met) scans the mRNA leader for an AUG start codon in favorable context. Structural analyses revealed that the β-hairpin of 40S protein Rps5/uS7 protrudes into the 40S mRNA exit-channel, contacting the eIF2∙GTP∙Met-tRNAi ternary complex (TC) and mRNA context nucleotides; but its importance in AUG selection was unknown. We identified substitutions in β-strand-1 and C-terminal residues of yeast Rps5 that reduced bulk initiation, conferred 'leaky-scanning' of AUGs; and lowered initiation fidelity by exacerbating the effect of poor context of the eIF1 AUG codon to reduce eIF1 abundance. Consistently, the β-strand-1 substitution greatly destabilized the 'PIN' conformation of TC binding to reconstituted 43S·mRNA complexes in vitro. Other substitutions in β-hairpin loop residues increased initiation fidelity and destabilized PIN at UUG, but not AUG start codons. We conclude that the Rps5 β-hairpin is as crucial as soluble initiation factors for efficient and accurate start codon recognition.
authors.affiliation: Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States.
authors.forename: Jyothsna
authors.initials: J
authors.lastname: Visweswaraiah
authors.affiliation: Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States.
authors.forename: Yvette
authors.initials: Y
authors.lastname: Pittman
authors.affiliation: Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Dever
authors.affiliation: Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States.
authors.forename: Alan G
authors.initials: AG
authors.lastname: Hinnebusch
mesh_terms: D019943:Amino Acid Substitution; D018387:Codon, Initiator / Q000378:metabolism; D004252:DNA Mutational Analysis; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010442:Peptide Chain Initiation, Translational; D014176:Protein Biosynthesis; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*; D054682:Ribosome Subunits, Small, Eukaryotic / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
chemical_list: D018387:Codon, Initiator; D050505:Mutant Proteins; D012269:Ribosomal Proteins; C030831:ribosomal protein S5
keywords: Rps5/uS7; S. cerevisiae; chromosomes; genes; initiation; regulation; ribosome; translation; yeast