title: Enhanced eIF1 binding to the 40S ribosome impedes conformational rearrangements of the preinitiation complex and elevates initiation accuracy.
abstract: In the current model of translation initiation by the scanning mechanism, eIF1 promotes an open conformation of the 40S subunit competent for rapidly loading the eIF2·GTP·Met-tRNAi ternary complex (TC) in a metastable conformation (POUT) capable of sampling triplets entering the P site while blocking accommodation of Met-tRNAi in the PIN state and preventing completion of GTP hydrolysis (Pi release) by the TC. All of these functions should be reversed by eIF1 dissociation from the preinitiation complex (PIC) on AUG recognition. We tested this model by selecting eIF1 Ssu(-) mutations that suppress the elevated UUG initiation and reduced rate of TC loading in vivo conferred by an eIF1 (Sui(-)) substitution that eliminates a direct contact of eIF1 with the 40S subunit. Importantly, several Ssu(-) substitutions increase eIF1 affinity for 40S subunits in vitro, and the strongest-binding variant (D61G), predicted to eliminate ionic repulsion with 18S rRNA, both reduces the rate of eIF1 dissociation and destabilizes the PIN state of TC binding in reconstituted PICs harboring Sui(-) variants of eIF5 or eIF2. These findings establish that eIF1 dissociation from the 40S subunit is required for the PIN mode of TC binding and AUG recognition and that increasing eIF1 affinity for the 40S subunit increases initiation accuracy in vivo. Our results further demonstrate that the GTPase-activating protein eIF5 and β-subunit of eIF2 promote accuracy by controlling eIF1 dissociation and the stability of TC binding to the PIC, beyond their roles in regulating GTP hydrolysis by eIF2.
authors: Pilar
authors: P
authors: Martin-Marcos
authors: Jagpreet S
authors: JS
authors: Nanda
authors: Rafael E
authors: RE
authors: Luna
authors: Fan
authors: F
authors: Zhang
authors: Adesh K
authors: AK
authors: Saini
authors: Vera A
authors: VA
authors: Cherkasova
authors: Gerhard
authors: G
authors: Wagner
authors: Jon R
authors: JR
authors: Lorsch
authors: Alan G
authors: AG
authors: Hinnebusch
mesh_terms: D000595:Amino Acid Sequence; D018387:Codon, Initiator; D015977:Eukaryotic Initiation Factor-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism; D040061:Eukaryotic Initiation Factor-5 / Q000737:chemistry / Q000378:metabolism; D055786:Gene Knockout Techniques; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011485:Protein Binding; D055550:Protein Stability; D054682:Ribosome Subunits, Small, Eukaryotic / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D061785:Transcription Initiation, Genetic
chemical_list: D018387:Codon, Initiator; D015977:Eukaryotic Initiation Factor-1; D015852:Eukaryotic Initiation Factor-2; D040061:Eukaryotic Initiation Factor-5; C072076:SUI1 protein, S cerevisiae; C584101:SUI3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D006160:Guanosine Triphosphate
keywords: AUG recognition; eIF1; eIF2; initiation; ribosome; translation