Abstract
The eukaryotic 43S preinitiation complex (PIC), bearing initiator methionyl transfer RNA (Met-tRNA(i)) in a ternary complex (TC) with eukaryotic initiation factor 2 (eIF2)-GTP, scans the mRNA leader for an AUG codon in favorable context. AUG recognition evokes rearrangement from an open PIC conformation with TC in a "P(OUT)" state to a closed conformation with TC more tightly bound in a "P(IN)" state. eIF1 binds to the 40S subunit and exerts a dual role of enhancing TC binding to the open PIC conformation while antagonizing the P(IN) state, necessitating eIF1 dissociation for start codon selection. Structures of reconstituted PICs reveal juxtaposition of eIF1 Loop 2 with the Met-tRNA(i) D loop in the P(IN) state and predict a distortion of Loop 2 from its conformation in the open complex to avoid a clash with Met-tRNA(i) We show that Ala substitutions in Loop 2 increase initiation at both near-cognate UUG codons and AUG codons in poor context. Consistently, the D71A-M74A double substitution stabilizes TC binding to 48S PICs reconstituted with mRNA harboring a UUG start codon, without affecting eIF1 affinity for 40S subunits. Relatively stronger effects were conferred by arginine substitutions; and no Loop 2 substitutions perturbed the rate of TC loading on scanning 40S subunits in vivo. Thus, Loop 2-D loop interactions specifically impede Met-tRNA(i) accommodation in the P(IN) state without influencing the P(OUT) mode of TC binding; and Arg substitutions convert the Loop 2-tRNA(i) clash to an electrostatic attraction that stabilizes P(IN) and enhances selection of poor start codons in vivo.