Pi Release from eIF2, Not GTP Hydrolysis, Is the Step Controlled by Start-Site Selection during Eukaryotic Translation Initiation

Abstract

Irreversible GTP hydrolysis by eIF2 is a critical step in translation initiation in eukaryotes because it is thought to commit the translational machinery to assembling the ribosomal complex at the selected point in the mRNA. Our quantitative analysis of the steps and interactions involved in activating GTP hydrolysis by eIF2 during translation initiation in vitro indicates that a structural rearrangement in the 43S preinitiation complex activates it to become fully competent to hydrolyze GTP. Contrary to the prevailing model, release of inorganic phosphate after GTP hydrolysis by eIF2, not hydrolysis itself, is controlled by recognition of the AUG codon. Release of P(i), which makes GTP hydrolysis irreversible, appears to be controlled by the AUG-dependent dissociation of eIF1 from the preinitiation complex.

Coupled Release of Eukaryotic Translation Initiation Factors 5B and 1A from 80S Ribosomes following Subunit Joining

“…Release of P i and eIF1 upon AUG start codon recognition triggers the release of eIF2 and fixes the reading frame for translation (2). Concurrent with these events, eIF1A binding to the 40S subunit is enhanced, indicating a conformational change in the complex (21,22).…”

“…Upon AUG codon recognition, GTP hydrolysis is completed, and P i is released, converting the 48S complex from an open to a closed complex with Met-tRNA i Met in the P site of the 40S subunit (29). The release of P i from the 48S complex appears to be coupled with displacement of eIF1 from its binding site near the P site (2,22). In addition, the majority of eIF2 either dissociates from or repositions on the 48S complex following AUG codon recognition (32).…”

“…The 43S complex then binds to an mRNA near the 5Ј cap, forming a 48S complex that scans along the mRNA in a 3Ј direction in search of an AUG start codon. In the 48S complex, some of the eIF2-GTP is hydrolyzed to eIF2-GDP ϩ P i (2). Upon AUG codon recognition, GTP hydrolysis is completed, and P i is released, converting the 48S complex from an open to a closed complex with Met-tRNA i Met in the P site of the 40S subunit (29).…”

Functional Elements in Initiation Factors 1, 1A, and 2β Discriminate against Poor AUG Context and Non-AUG Start Codons

“…eIF1 and IF3 occupy analogous locations on the platform of the small ribosomal subunit (11,27,38) and, similar to IF3, eIF1 blocks formation of stable 48S PICs at near-cognate start codons (20,36). eIF1/Sui1 and eIF1A (Tif11 in yeast) cooperate to promote an open conformation of the 40S subunit (34) thought to be conducive to scanning (35), and eIF1 also blocks the final step of GTP hydrolysis by the TC, the release of P i from eIF2-GDP-P i , until an AUG enters the P site (1). AUG recognition triggers dissociation of eIF1 from the 40S subunit (30), enabling P i release and stabilizing a closed conformation of the 40S subunit that is incompatible with scanning.…”

Regulation of protein synthesis and the role of eIF3 in cancer

“…AUGs surrounded by a poor consensus sequence may be passed over, a phenomenon called "leaky scanning". Following formation of a correct codon-anticodon interaction, the GTP bound to the eIF2 is hydrolyzed in a reaction promoted by eIF5, and eIF1 and inorganic phosphate are released (17). Then, eIF2-GDP (guanosine diphosphate) leaves to complete formation of the 40S initiation complex and is recycled by eIF2B to the active eIF2-GTP complex.…”