Regulatory elements in eIF1A control the fidelity of start codon selection by modulating tRNA_i^Met binding to the ribosome

Abstract

eIF1A is the eukaryotic ortholog of bacterial translation initiation factor IF1, but contains a helical domain and long unstructured N-terminal tail (NTT) and C-terminal tail (CTT) absent in IF1. Here, we identify elements in these accessory regions of eIF1A with dual functions in binding methionyl initiator tRNA (Met-tRNA i Met ) to the ribosome and in selecting AUG codons. A pair of repeats in the eIF1A CTT, dubbed Scanning Enhancer 1 (SE 1 ) and SE 2 , was found to stimulate recruitment of Met-tRNA i Met in the ternary complex (TC) with eIF2ÁGTP and also to block initiation at UUG codons. In contrast, the NTT and segments of the helical domain are required for the elevated UUG initiation occurring in SE mutants, and both regions also impede TC recruitment. Remarkably, mutations in these latter elements, dubbed scanning inhibitors SI 1 and SI 2 , reverse the defects in TC loading and UUG initiation conferred by SE substitutions, showing that the dual functions of SE elements in TC binding and UUG suppression are mechanistically linked. It appears that SE elements enhance TC binding in a conformation conducive to scanning but incompatible with initiation, whereas SI elements destabilize this conformation to enable full accommodation of Met-tRNA i Met in the P site for AUG selection.[Keywords: Translation; initiation; eIF1A; eIF2; initiator; scanning]Supplemental material is available at http://www.genesdev.org.

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

“…eIF1 moderately autoregulates translation by exacerbating the effects of poor AUG context at SUI1. As noted above, overexpressing WT eIF1 confers an Ssu Ϫ phenotype (41,48). Consistent with this, hc SUI1 ϩ phenocopies the sui1 Ssu Ϫ mutations and reduces expression of the WT SUI1-lacZ fusion by a factor of 2 but diminishes expression of SUI1-opt-lacZ by only ϳ20% (Fig.…”

“…Similar to the effects of IF3 mutations in bacteria, hypomorphic mutations in yeast eIF1 (an essential protein) increase initiation from UUG codons in vivo (12,49), i.e., the Sui Ϫ phenotype, whereas overexpressing wild-type (WT) eIF1 suppresses UUG initiation in mutants with Sui Ϫ substitutions in other initiation factors, i.e., the Ssu Ϫ phenotype (41,48). Interestingly, Sui Ϫ mutations in eIF1 generally weaken its affinity for the 40S and enable inappropriate eIF1 dissociation and P i release from eIF2-GDP-P i at non-AUG codons.…”

“…The SE mutation, which impairs both scanning enhancer elements (SE 1 and SE 2 ) in the eIF1A C-terminal tail, greatly elevates the UUG/AUG ratio for the HIS4-lacZ fusion (41). We also examined the Ssu Ϫ mutation tif11-17-21, which introduces substitutions into the scanning inhibitory (SI) element in the N-terminal tail of eIF1A and lowers the UUG/AUG ratio in cells harboring SUI3-2 (14,41).…”

“…Ssu Ϫ mutations in eIF1A with these properties have been shown to shift the equilibrium toward the open conformation (41), with increased retention of eIF1 on the 40S subunit (8), suppressing both the defect in TC loading to the open complex and the inappropriate rearrangement to the closed complex at UUG provoked by Sui Ϫ mutations in the eIF1A SE elements (41). The fact that the 17-21 Ssu Ϫ mutation in eIF1A also diminishes the Sui Ϫ and TC loading defects conferred by the SUI3-2 mutation in eIF2␤ (14,41) suggests that SUI3-2 produces a Sui Ϫ phenotype, at least partly, by destabilizing TC binding to the open conformation (41). There is biochemical evidence that elevated GTP hydrolysis by the TC also plays a role (21), possibly shifting the equilibrium between eIF2-GTP and eIF2-GDP-P i to the right and driving P i release at non-AUG codons by mass action, or increasing the rate of P i release directly.…”

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

“…In contrast to the Sui 2 mutations, which relax the stringency for start codon selection, a second class of mutations enhances start codon selectivity. The Ssu 2 (suppressor of Sui 2 ) mutations block the ability of Sui 2 mutations to enhance initiation at a UUG codon in a mutant HIS4 allele (Asano et al 2001;Fekete et al 2007;Saini et al 2010). In general, Sui 2 mutations are thought to block scanning and promote conversion of the 40S subunit to its closed, scanning arrested conformation (P in ).…”