Enhanced eIF1 binding to the 40S ribosome impedes conformational rearrangements of the preinitiation complex and elevates initiation accuracy

Abstract

In the current model of translation initiation by the scanning mechanism, eIF1 promotes an open conformation of the 40S subunit competent for rapidly loading the eIF2·GTP·Met-tRNA i ternary complex (TC) in a metastable conformation (P OUT ) capable of sampling triplets entering the P site while blocking accommodation of Met-tRNA i in the P IN state and preventing completion of GTP hydrolysis (P i release) by the TC. All of these functions should be reversed by eIF1 dissociation from the preinitiation complex (PIC) on AUG recognition. We tested this model by selecting eIF1 Ssu − mutations that suppress the elevated UUG initiation and reduced rate of TC loading in vivo conferred by an eIF1 (Sui − ) substitution that eliminates a direct contact of eIF1 with the 40S subunit. Importantly, several Ssu − substitutions increase eIF1 affinity for 40S subunits in vitro, and the strongest-binding variant (D61G), predicted to eliminate ionic repulsion with 18S rRNA, both reduces the rate of eIF1 dissociation and destabilizes the P IN state of TC binding in reconstituted PICs harboring Sui − variants of eIF5 or eIF2. These findings establish that eIF1 dissociation from the 40S subunit is required for the P IN mode of TC binding and AUG recognition and that increasing eIF1 affinity for the 40S subunit increases initiation accuracy in vivo. Our results further demonstrate that the GTPase-activating protein eIF5 and β-subunit of eIF2 promote accuracy by controlling eIF1 dissociation and the stability of TC binding to the PIC, beyond their roles in regulating GTP hydrolysis by eIF2.

Rps3/uS3 promotes mRNA binding at the 40S ribosome entry channel and stabilizes preinitiation complexes at start codons

“…Met were chased with excess unlabeled TC, incubated for increasing time periods, and then resolved via native gel electrophoresis to separate 40S-bound and unbound fractions of TC. In agreement with previous findings (11,29,30), little to no TC dissociation occurred from WT PICs formed with the AUG-containing mRNA [mRNA(AUG)] over the time course Fig. 2A with the indicated RPS3 alleles were cultured in SD+His+Trp+Ura medium to an A 600 of ∼1.0 and WCEs were subjected to Western analysis using antibodies against eIF1 or Hcr1 (as loading control).…”

Conserved residues in yeast initiator tRNA calibrate initiation accuracy by regulating preinitiation complex stability at the start codon

“…One explanation might be that the predicted reduction in k on for 43SÁmRNA(UUG) complexes conferred by slower TC loading to the open/P OUT state is offset by an increase of nearly equal magnitude in the rate of P OUT -to-P IN isomerization at UUG but not AUG codons. To explain why G70A would selectively accelerate the P OUT -to-P IN transition at UUG, it could be proposed that G70A perturbs interaction of Met-tRNA i with eIF2 in a way that alters the orientation of TC in the P site to favor base-pairing with UUG, which entails a U:U mismatch at the first position of the codon:anticodon duplex but not for the perfect codon:anticodon duplex at AUG. We came to a similar conclusion recently regarding the SUI5 mutation in eIF5 that stabilizes P IN at UUG while destabilizing it at AUG (Martin-Marcos et al 2014).…”

Human Eukaryotic Initiation Factor 2 (eIF2)-GTP-Met-tRNAi Ternary Complex and eIF3 Stabilize the 43 S Preinitiation Complex

“…It is also clear that eIF5 plays an important role in initiation codon selection, most likely through changes in direct and indirect interactions with eIF1, eIF1A, and eIF2 on the surface of the 40 S subunit (11,15,48). Although we have not determined whether there is a contribution of eIF5 on the overall stability of the 43 S PIC, we do not observe any influence of eIF5 on the stability of eIF1, eIF1A, or eIF3j individually with the 40 S subunit (Table 1).…”