Yeast eIF1 inhibits initiation at non-AUG triplets, but it was unknown whether it also discriminates against AUGs in suboptimal context. As in other eukaryotes, the yeast gene encoding eIF1 (SUI1) contains an AUG in poor context, which could underlie translational autoregulation. Previously, eIF1 mutations were identified that increase initiation at UUG codons (Sui(-) phenotype), and we obtained mutations with the opposite phenotype of suppressing UUG initiation (Ssu(-) phenotype). Remarkably, Sui(-) mutations in eukaryotic translation initiation factor 1 (eIF1), eIF1A, and eIF2? all increase SUI1 expression in a manner diminished by introducing the optimal context at the SUI1 AUG, whereas Ssu(-) mutations in eIF1 and eIF1A decrease SUI1 expression with the native, but not optimal, context present. Therefore, discrimination against weak context depends on specific residues in eIFs 1, 1A, and 2? that also impede selection of non-AUGs, suggesting that context nucleotides and AUG act coordinately to stabilize the preinitiation complex. Although eIF1 autoregulates by discriminating against poor context in yeast and mammals, this mechanism does not prevent eIF1 overproduction in yeast, accounting for the hyperaccuracy phenotype afforded by SUI1 overexpression.
Eukaryotic translation initiation factor (eIF) 1 is a central mediator of start codon recognition. Dissociation of eIF1 from the preinitiation complex (PIC) allows release of phosphate from the G-protein factor eIF2, triggering downstream events in initiation. Mutations that weaken binding of eIF1 to the PIC decrease the fidelity of start codon recognition (Sui(-) phenotype) by allowing increased eIF1 release at non-AUG codons. Consistent with this, overexpression of these mutant proteins suppresses their Sui(-) phenotypes. Here, we have examined mutations at the penultimate residue of eIF1, G107, that produce Sui(-) phenotypes without increasing the rate of eIF1 release. We provide evidence that, in addition to its role in gating phosphate release, dissociation of eIF1 triggers conversion from an open, scanning-competent state of the PIC to a stable, closed one. We also show that eIF5 antagonizes binding of eIF1 to the complex and that key interactions of eIF1 with its partners are modulated by the charge at and around G107. Our data indicate that eIF1 plays multiple roles in start codon recognition and suggest that prior to AUG recognition it prevents eIF5 from binding to a key site in the PIC required for triggering downstream events.
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