title: Eukaryotic translation initiation factor eIF5 promotes the accuracy of start codon recognition by regulating Pi release and conformational transitions of the preinitiation complex.
abstract: eIF5 is the GTPase activating protein (GAP) for the eIF2 · GTP · Met-tRNAi (Met) ternary complex with a critical role in initiation codon selection. Previous work suggested that the eIF5 mutation G31R/SUI5 elevates initiation at UUG codons by increasing GAP function. Subsequent work implicated eIF5 in rearrangement of the preinitiation complex (PIC) from an open, scanning conformation to a closed state at AUG codons, from which Pi is released from eIF2 · GDP · Pi. To identify eIF5 functions crucial for accurate initiation, we investigated the consequences of G31R on GTP hydrolysis and Pi release, and the effects of intragenic G31R suppressors on these reactions, and on the partitioning of PICs between open and closed states. eIF5-G31R altered regulation of Pi release, accelerating it at UUG while decreasing it at AUG codons, consistent with its ability to stabilize the closed complex at UUG. Suppressor G62S mitigates both defects of G31R, accounting for its efficient suppression of UUG initiation in G31R,G62S cells; however suppressor M18V impairs GTP hydrolysis with little effect on PIC conformation. The strong defect in GTP hydrolysis conferred by M18V likely explains its broad suppression of Sui(-) mutations in numerous factors. We conclude that both of eIF5's functions, regulating Pi release and stabilizing the closed PIC conformation, contribute to stringent AUG selection in vivo.
authors.affiliation: Laboratory of Gene Regulation and Development, Eunice K. Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA Laboratory on the Mechanism and Regulation of Protein Synthesis, Eunice K. Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA sainiade@gmail.com.
authors.forename: Adesh K
authors.initials: AK
authors.lastname: Saini
authors.affiliation: Shoolini University of Biotechnology and Management Sciences, Department of Biotechnology, Bajhol, Solan, Himachal Pradesh 173229, India.
authors.forename: Jagpreet S
authors.initials: JS
authors.lastname: Nanda
authors.affiliation: Laboratory of Gene Regulation and Development, Eunice K. Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Pilar
authors.initials: P
authors.lastname: Martin-Marcos
authors.affiliation: Laboratory of Gene Regulation and Development, Eunice K. Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Jinsheng
authors.initials: J
authors.lastname: Dong
authors.affiliation: Laboratory of Gene Regulation and Development, Eunice K. Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Fan
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Laboratory on the Mechanism and Regulation of Protein Synthesis, Eunice K. Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Monika
authors.initials: M
authors.lastname: Bhardwaj
authors.affiliation: Shoolini University of Biotechnology and Management Sciences, Department of Biotechnology, Bajhol, Solan, Himachal Pradesh 173229, India jon.lorsch@nih.gov.
authors.forename: Jon R
authors.initials: JR
authors.lastname: Lorsch
authors.affiliation: Laboratory of Gene Regulation and Development, Eunice K. Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA ahinnebusch@nih.gov.
authors.forename: Alan G
authors.initials: AG
authors.lastname: Hinnebusch
mesh_terms: D018387:Codon, Initiator; D015977:Eukaryotic Initiation Factor-1 / Q000235:genetics; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics; D040061:Eukaryotic Initiation Factor-5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D009154:Mutation; D010442:Peptide Chain Initiation, Translational; D010710:Phosphates / Q000378:metabolism; D013489:Suppression, Genetic
chemical_list: D018387:Codon, Initiator; D015977:Eukaryotic Initiation Factor-1; D015852:Eukaryotic Initiation Factor-2; D040061:Eukaryotic Initiation Factor-5; D010710:Phosphates; C026022:eukaryotic peptide initiation factor-1A; D006160:Guanosine Triphosphate
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