β-Hairpin Loop of Eukaryotic Initiation Factor 1 (eIF1) Mediates 40 S Ribosome Binding to Regulate Initiator tRNAMet Recruitment and Accuracy of AUG Selection in Vivo

Recognition of the translation initiation codon is thought to require dissociation of eIF1 from the 40 S ribosomal subunit, enabling irreversible GTP hydrolysis (P i release) by the eIF2·GTP·Met-tRNA i ternary complex (TC), rearrangement of the 40 S subunit to a closed conformation incompatible with scanning, and stable binding of Met-tRNA i to the P site. The crystal structure of a Tetrahymena 40 S·eIF1 complex revealed several basic amino acids in eIF1 contacting 18 S rRNA, and we tested the prediction that their counterparts in yeast eIF1 are required to prevent premature eIF1 dissociation from scanning ribosomes at non-AUG triplets. Supporting this idea, substituting Lys-60 in helix α1, or either Lys-37 or Arg-33 in β-hairpin loop-1, impairs binding of yeast eIF1 to 40 S·eIF1A complexes in vitro , and it confers increased initiation at UUG codons (Sui − phenotype) or lethality, in a manner suppressed by overexpressing the mutant proteins or by an eIF1A mutation ( 17–21 ) known to impede eIF1 dissociation in vitro . The eIF1 Sui − mutations also derepress translation of GCN4 mRNA, indicating impaired ternary complex loading, and this Gcd − phenotype is likewise suppressed by eIF1 overexpression or the 17–21 mutation. These findings indicate that direct contacts of eIF1 with 18 S rRNA seen in the Tetrahymena 40 S·eIF1 complex are crucial in yeast to stabilize the open conformation of the 40 S subunit and are required for rapid TC loading and ribosomal scanning and to impede rearrangement to the closed complex at non-AUG codons. Finally, we implicate the unstructured N-terminal tail of eIF1 in blocking rearrangement to the closed conformation in the scanning preinitiation complex. Background: Start codon selection requires eIF1 dissociation from its 40 S-binding site. Results: eIF1 residues in β-hairpin loop-1 and helix α1 make functionally critical contacts with the 40 S subunit. Conclusion: Direct 40 S contacts of eIF1 regulate the rate of Met-tRNA i recruitment and block non-AUG recognition. Significance: eIF1's direct contacts with the 40 S subunit are crucial for AUG recognition in vivo .