ESTABLISHING THE FIDELITY OF START CODON RECOGNITION: ROLE OF EUKARYOTIC INITIATION FACTOR 2

In eukaryotes, start codon selection is governed by base pairing between the start codon and the anticodon of the initiator tRNA (Met-tRNAi) and is achieved via a complex machinery involving at least twelve initiation factors. eIF2 is a heterotrimeric G-protein that, in its active GTP-bound state, binds and delivers the Met-tRNAi to the P-site of the small (40S) subunit of the ribosome, in a process facilitated by multiple other initiation factors forming a pre-initiation complex (PIC). The process of initiation is composed of two phases with distinct conformations of the PIC. First, the PIC in an open conformation scans the mRNA 5’ UTR in a manner allowing for the Met-tRNAi to sample the nucleotides for complementarity. Upon a cognate codon:anticodon interaction, the PIC then adopts a closed conformation accompanied by the irreversible hydrolysis of eIF2-bound GTP and Pi release. The molecular mechanisms by which cognate codon:anticodon base pairing is linked to the hydrolysis of eIF2-bound GTP and Pi release, formation of the closed conformation, and start codon selection are not well understood. In this study, we provide genetic and biochemical evidence that suggests a new function for domain-III of eIF2γ in coordinating these processes and maintaining the equilibrium between the two conformations of the PIC. In order to identify the structural elements in eIF2 essential for start codon selection, we isolated novel mutations in the yeast γ subunit that alter the accuracy of this process. We identified two classes of mutations with opposing effects: mutations that reduce the stringency of start codon recognition and those that, conversely, restore initiation fidelity. The isolated mutations localize to distinct regions on the surface of domain-III in close proximity of the proposed binding interface between eIF2 and the 40S subunit. We propose a model in which eIF2(γ) maintains a dynamic interaction with the 40S subunit during the scanning of the mRNA 5’ UTR. Upon cognate codon:anticodon base pairing, however, new contact points between eIF2γ domain-III and the 40S subunit are created that stabilize the closed conformation, hence stabilizing the accommodation of the Met-tRNAi in the P-site and allowing for the progress of translation initiation