N- and C-terminal residues of eIF1A have opposing effects on the fidelity of start codon selection

Abstract

Translation initiation factor eIF1A stimulates preinitiation complex (PIC) assembly and scanning, but the molecular mechanisms of its functions are not understood. We show that the F131A,F133A mutation in the C-terminal tail (CTT) of eIF1A impairs recruitment of the eIF2-GTP-MettRNA iMet ternary complex to 40S subunits, eliminating functional coupling with eIF1. Mutating residues 17-21 in the N-terminal tail (NTT) of eIF1A also reduces PIC assembly, but in a manner rescued by eIF1. Interestingly, the 131,133 CTT mutation enhances initiation at UUG codons (Sui À phenotype) and decreases leaky scanning at AUG, while the NTT mutation 17-21 suppresses the Sui À phenotypes of eIF5 and eIF2b mutations and increases leaky scanning. These findings and the opposite effects of the mutations on eIF1A binding to reconstituted PICs suggest that the NTT mutations promote an open, scanning-conducive conformation of the PIC, whereas the CTT mutations 131,133 have the reverse effect. We conclude that tight binding of eIF1A to the PIC is an important determinant of AUG selection and is modulated in opposite directions by residues in the NTT and CTT of eIF1A.

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

“…This latter defect of SUI3-2 could also be mitigated by the ability of the 17-21 mutation to stabilize the open conformation and retard eIF1 dissociation. Suppression of the eIF5 Sui Ϫ mutation SUI5 by 17-21 (14) can be explained similarly, since this eIF5 substitution both destabilizes the open conformation of the PIC (29) and accelerates GTP hydrolysis at UUG codons (21). Hence, we propose that the novel Ssu Ϫ substitutions in eIF1 described here likewise impede the open-to-closed transition of the PIC, possibly by retarding eIF1 dissociation, as a means of suppressing UUG initiation in cells harboring the SUI5 or SUI3-2 Sui Ϫ substitutions in eIF5 or eIF2␤, respectively.…”

Should I Stay or Should I Go? Eukaryotic Translation Initiation Factors 1 and 1A Control Start Codon Recognition

“…Mutations in both eIF1A and eIF5 that increase initiation at non-AUG codons in vivo strengthen this shift in the presence of a UUG codon and weaken it in the presence of an AUG codon. In addition, mutants of eIF1A that have a leaky scanning phenotype strongly shift the equilibrium toward the state in which eIF1A makes fewer stabilizing interactions (25). When eIF1A is bound more tightly, a reduction in the conformational freedom of its C terminus is also observed, indicating that a structural change has taken place in which this region has become more confined (24,25).…”

“…In addition, mutants of eIF1A that have a leaky scanning phenotype strongly shift the equilibrium toward the state in which eIF1A makes fewer stabilizing interactions (25). When eIF1A is bound more tightly, a reduction in the conformational freedom of its C terminus is also observed, indicating that a structural change has taken place in which this region has become more confined (24,25). These data are consistent with a model in which the more tightly bound state is the closed one and the less tightly bound state is the open, scanning-competent complex.…”