eIF1 Loop 2 interactions with Met-tRNA i control the accuracy of start codon selection by the scanning preinitiation complex

Significance In translation initiation, a preinitiation complex (PIC) of the 40S ribosomal subunit, eukaryotic initiation factors (eIFs), and initiator transfer RNA (tRNA i ) scans the mRNA for an AUG codon in favorable context. AUG recognition evokes a closed PIC conformation and triggers dissociation of eIF1. Structural data predict that a clash between eIF1 β-hairpin Loop 2 and tRNA i impedes selection of non-AUG start codons in the open, scanning conformation of the PIC. Supporting this model, we show that alanine substitutions of Loop 2 residues increase initiation at suboptimal start codons (UUGs and AUGs in poor context) in vivo and destabilize tRNA i binding at UUG codons in reconstituted PICs in vitro. These results establish Loop 2–RNA i interaction as a key determinant of initiation accuracy.