title: Dissociation of eIF1 from the 40S ribosomal subunit is a key step in start codon selection in vivo.
abstract: Selection of the AUG start codon is a key step in translation initiation requiring hydrolysis of GTP in the eIF2*GTP*Met-tRNA(i)(Met) ternary complex (TC) and subsequent P(i) release from eIF2*GDP*P(i). It is thought that eIF1 prevents recognition of non-AUGs by promoting scanning and blocking P(i) release at non-AUG codons. We show that Sui(-) mutations in Saccharomyces cerevisiae eIF1, which increase initiation at UUG codons, reduce interaction of eIF1 with 40S subunits in vitro and in vivo, and both defects are diminished in cells by overexpressing the mutant proteins. Remarkably, Sui(-) mutation ISQLG(93-97)ASQAA (abbreviated 93-97) accelerates eIF1 dissociation and P(i) release from reconstituted preinitiation complexes (PICs), whereas a hyperaccuracy mutation in eIF1A (that suppresses Sui(-) mutations) decreases the eIF1 off-rate. These findings demonstrate that eIF1 dissociation is a critical step in start codon selection, which is modulated by eIF1A. We also describe Gcd(-) mutations in eIF1 that impair TC loading on 40S subunits or destabilize the multifactor complex containing eIF1, eIF3, eIF5, and TC, showing that eIF1 promotes PIC assembly in vivo beyond its important functions in AUG selection.
authors.affiliation: Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Yuen-Nei
authors.initials: YN
authors.lastname: Cheung
authors.forename: David
authors.initials: D
authors.lastname: Maag
authors.forename: Sarah F
authors.initials: SF
authors.lastname: Mitchell
authors.forename: Christie A
authors.initials: CA
authors.lastname: Fekete
authors.forename: Mikkel A
authors.initials: MA
authors.lastname: Algire
authors.forename: Julie E
authors.initials: JE
authors.lastname: Takacs
authors.forename: Nikolay
authors.initials: N
authors.lastname: Shirokikh
authors.forename: Tatyana
authors.initials: T
authors.lastname: Pestova
authors.forename: Jon R
authors.initials: JR
authors.lastname: Lorsch
authors.forename: Alan G
authors.initials: AG
authors.lastname: Hinnebusch
mesh_terms: D018387:Codon, Initiator / Q000235:genetics / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D015977:Eukaryotic Initiation Factor-1 / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D014176:Protein Biosynthesis / Q000502:physiology*; D054682:Ribosome Subunits, Small, Eukaryotic / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*
chemical_list: D018387:Codon, Initiator; D015977:Eukaryotic Initiation Factor-1