title: Conformational changes in the P site and mRNA entry channel evoked by AUG recognition in yeast translation preinitiation complexes.
abstract: The translation preinitiation complex (PIC) is thought to assume an open conformation when scanning the mRNA leader, with AUG recognition evoking a closed conformation and more stable P site interaction of Met-tRNAi; however, physical evidence is lacking that AUG recognition constrains interaction of mRNA with the 40S binding cleft. We compared patterns of hydroxyl radical cleavage of rRNA by Fe(II)-BABE tethered to unique sites in eIF1A in yeast PICs reconstituted with mRNA harboring an AUG or near-cognate (AUC) start codon. rRNA residues in the P site display reduced cleavage in AUG versus AUC PICs; and enhanced cleavage in the AUC complexes was diminished by mutations of scanning enhancer elements of eIF1A that increase near-cognate recognition in vivo. This suggests that accessibility of these rRNA residues is reduced by accommodation of Met-tRNAi in the P site (PIN state) and by their interactions with the anticodon stem of Met-tRNAi. Our cleavage data also provide evidence that AUG recognition evokes dissociation of eIF1 from its 40S binding site, ejection of the eIF1A-CTT from the P-site and rearrangement to a closed conformation of the entry channel with reduced mobility of mRNA.
authors: Laboratory of Gene Regulation and Development, NICHD, NIH, Bethesda, MD 20892, USA.
authors: Fan
authors: F
authors: Zhang
authors: Laboratory of Gene Regulation and Development, NICHD, NIH, Bethesda, MD 20892, USA Department of Biotechnology, Shoolini University of Biotechnology and Management Sciences, Solan, Himachal Pradesh-173212, India.
authors: Adesh K
authors: AK
authors: Saini
authors: Laboratory of Gene Regulation and Development, NICHD, NIH, Bethesda, MD 20892, USA.
authors: Byung-Sik
authors: BS
authors: Shin
authors: Laboratory on the Mechanism and Regulation of Protein Synthesis, NICHD, NIH, Bethesda, MD 20892, USA.
authors: Jagpreet
authors: J
authors: Nanda
authors: Laboratory of Gene Regulation and Development, NICHD, NIH, Bethesda, MD 20892, USA ahinnebusch@nih.gov.
authors: Alan G
authors: AG
authors: Hinnebusch
mesh_terms: D019943:Amino Acid Substitution; D018387:Codon, Initiator; D003545:Cysteine / Q000235:genetics; D004492:Edetic Acid / Q000031:analogs & derivatives; D015977:Eukaryotic Initiation Factor-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010442:Peptide Chain Initiation, Translational; D012333:RNA, Messenger / Q000378:metabolism; D012337:RNA, Ribosomal, 18S / Q000737:chemistry / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000378:metabolism; D054682:Ribosome Subunits, Small, Eukaryotic / Q000737:chemistry* / Q000378:metabolism; D015003:Yeasts / Q000235:genetics
chemical_list: D018387:Codon, Initiator; D015977:Eukaryotic Initiation Factor-1; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S; D012358:RNA, Transfer, Met; C026022:eukaryotic peptide initiation factor-1A; C042007:1-(4-bromoacetamidobenzyl)EDTA; D004492:Edetic Acid; D003545:Cysteine