A network of eIF2 interactions with eIF1 and Met-tRNAi promotes accurate start codon selection by the translation preinitiation complex

In translation initiation, a 43S preinitiation complex (PIC) containing eIF1 and a ternary complex (TC) of GTP-bound eIF2 and Met-RNA(i) scans the mRNA for the start codon. AUG recognition triggers eIF1 release and rearrangement from an open PIC conformation to a closed state with more tightly-bound Met-tRNA(i) (P-IN state). Cryo-EM models reveal eIF2 contacts with eIF1 and Met-tRNA(i) exclusive to the open complex that should destabilize the closed state. eIF2 or eIF1 substitutions disrupting these contacts increase initiation at UUG codons, and compound substitutions also derepress translation of GCN4, indicating slower TC recruitment. The latter substitutions slow TC loading while stabilizing TC binding at UUG codons in reconstituted PICs, indicating a destabilized open complex and shift to the closed/P-IN state. An eIF1 substitution that should strengthen the eIF2:eIF1 interface has the opposite genetic and biochemical phenotypes. eIF2 is also predicted to restrict Met-tRNA(i) movement into the closed/P-IN state, and substitutions that should diminish this clash increase UUG initiation in vivo and stabilize Met-tRNA(i) binding at UUG codons in vitro with little effect on TC loading. Thus, eIF2 anchors eIF1 and TC to the open complex, enhancing PIC assembly and scanning, while impeding rearrangement to the closed conformation at non-AUG codons.