title: β-Hairpin loop of eukaryotic initiation factor 1 (eIF1) mediates 40 S ribosome binding to regulate initiator tRNA(Met) recruitment and accuracy of AUG selection in vivo.
abstract: Recognition of the translation initiation codon is thought to require dissociation of eIF1 from the 40 S ribosomal subunit, enabling irreversible GTP hydrolysis (Pi release) by the eIF2·GTP·Met-tRNAi ternary complex (TC), rearrangement of the 40 S subunit to a closed conformation incompatible with scanning, and stable binding of Met-tRNAi to the P site. The crystal structure of a Tetrahymena 40 S·eIF1 complex revealed several basic amino acids in eIF1 contacting 18 S rRNA, and we tested the prediction that their counterparts in yeast eIF1 are required to prevent premature eIF1 dissociation from scanning ribosomes at non-AUG triplets. Supporting this idea, substituting Lys-60 in helix α1, or either Lys-37 or Arg-33 in β-hairpin loop-1, impairs binding of yeast eIF1 to 40 S·eIF1A complexes in vitro, and it confers increased initiation at UUG codons (Sui(-) phenotype) or lethality, in a manner suppressed by overexpressing the mutant proteins or by an eIF1A mutation (17-21) known to impede eIF1 dissociation in vitro. The eIF1 Sui(-) mutations also derepress translation of GCN4 mRNA, indicating impaired ternary complex loading, and this Gcd(-) phenotype is likewise suppressed by eIF1 overexpression or the 17-21 mutation. These findings indicate that direct contacts of eIF1 with 18 S rRNA seen in the Tetrahymena 40 S·eIF1 complex are crucial in yeast to stabilize the open conformation of the 40 S subunit and are required for rapid TC loading and ribosomal scanning and to impede rearrangement to the closed complex at non-AUG codons. Finally, we implicate the unstructured N-terminal tail of eIF1 in blocking rearrangement to the closed conformation in the scanning preinitiation complex.
authors.affiliation: Laboratory of Gene Regulation and Development, Eunice K. Shriver NICHD, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Pilar
authors.initials: P
authors.lastname: Martin-Marcos
authors.affiliation: Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Jagpreet
authors.initials: J
authors.lastname: Nanda
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Rafael E
authors.initials: RE
authors.lastname: Luna
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Wagner
authors.affiliation: Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205. Electronic address: jlorsch@jhmi.edu.
authors.forename: Jon R
authors.initials: JR
authors.lastname: Lorsch
authors.affiliation: Laboratory of Gene Regulation and Development, Eunice K. Shriver NICHD, National Institutes of Health, Bethesda, Maryland 20892. Electronic address: ahinnebusch@nih.gov.
authors.forename: Alan G
authors.initials: AG
authors.lastname: Hinnebusch
mesh_terms: D050976:Basic-Leucine Zipper Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D018387:Codon, Initiator / Q000235:genetics / Q000378:metabolism*; D015977:Eukaryotic Initiation Factor-1 / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010442:Peptide Chain Initiation, Translational / Q000502:physiology*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000235:genetics / Q000378:metabolism*; D054682:Ribosome Subunits, Small, Eukaryotic / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D016808:Tetrahymena thermophila / Q000235:genetics / Q000378:metabolism
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D018387:Codon, Initiator; D015977:Eukaryotic Initiation Factor-1; C501391:GCN4 protein, S cerevisiae; D015800:Protozoan Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D012358:RNA, Transfer, Met; C072076:SUI1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins
keywords: AUG Recognition; Ribosome Function; Ribosomes; Transfer RNA (tRNA); Translation; Translation Initiation Factors; eIF1; eIF2
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