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

Abstract

Yeast eIF1 inhibits initiation at non-AUG triplets, but it was unknown whether it also discriminates against AUGs in suboptimal context. As in other eukaryotes, the yeast gene encoding eIF1 (SUI1) contains an AUG in poor context, which could underlie translational autoregulation. Previously, eIF1 mutations were identified that increase initiation at UUG codons (Sui ؊ phenotype), and we obtained mutations with the opposite phenotype of suppressing UUG initiation (Ssu ؊ phenotype). Remarkably, Sui ؊ mutations in eukaryotic translation initiation factor 1 (eIF1), eIF1A, and eIF2␤ all increase SUI1 expression in a manner diminished by introducing the optimal context at the SUI1 AUG, whereas Ssu ؊ mutations in eIF1 and eIF1A decrease SUI1 expression with the native, but not optimal, context present. Therefore, discrimination against weak context depends on specific residues in eIFs 1, 1A, and 2␤ that also impede selection of non-AUGs, suggesting that context nucleotides and AUG act coordinately to stabilize the preinitiation complex. Although eIF1 autoregulates by discriminating against poor context in yeast and mammals, this mechanism does not prevent eIF1 overproduction in yeast, accounting for the hyperaccuracy phenotype afforded by SUI1 overexpression.Bacterial translation initiation factor 3 (IF3) promotes the fidelity of initiation at AUG codons by discriminating against non-AUG triplets as start sites (17,26,40,45). This discriminatory function forms the basis for IF3's ability to negatively autoregulate translation of its mRNA, which initiates with an AUU start codon (5, 6). IF3 also destabilizes initiation complexes formed on AUG codons at the 5Ј ends of leaderless mRNAs (47), which lack the Shine-Dalgarno sequence that stabilizes mRNA association with the small (30S) ribosomal subunit at the AUG codon.In eukaryotes, the 43S preinitiation complex (PIC), harboring the eIF2-GTP-Met-tRNA i Met ternary complex (TC) and various other eIFs, attaches to the capped 5Ј end of the mRNA and identifies the AUG codon by scanning the mRNA leader base-by-base for complementarity with the anticodon of MettRNA i Met. Efficient initiation is influenced by the sequence immediately upstream from the AUG, but it is unclear how this sequence context is recognized or regulates AUG selection (20,36). The functional counterpart of IF3 in eukaryotes appears to be eIF1 (Sui1 in yeast). 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 ...

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

“…Moreover, overexpression of eIF1 suppresses Sui 2 mutations in other translation factors, indicating that eIF1 dissociation from the 48S complex is a key commitment step in start codon selection Cheung et al 2007;Martin-Marcos et al 2011;Martin-Marcos et al 2013;Martin-Marcos et al 2014). Cryo-EM structures of 48S PICs in open and closed states revealed a clash between eIF1 and Met-tRNA i Met in the P in state (Llacer et al 2015).…”

“…At odds with these studies, flanking nucleotides have been shown to be important in selection of the non-AUG start codon on the GRS1 mRNA (Chen et al 2008) as well as in the selection of alternate AUG start codons on the MOD5 and CCA1 mRNAs (Werner et al 1987;Slusher et al 1991;Wolfe et al 1994). In addition, the poor start codon context (C 23 GUAUG) of the SUI1 gene encoding translation factor eIF1 in yeast impairs expression and enables autoregulation due to the role of eIF1 in start codon selection (Martin-Marcos et al 2011).…”

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

“…The eIF1 mutations also mitigated the elevated UUG initiation provoked by SUI5 and by the dominant Sui − substitution in eIF2β (S264Y) conferred by the SUI3-2 mutation (summarized in Fig. 2C; Martin-Marcos et al 2011). There is currently no biochemical evidence that either SUI5 or SUI3-2 confers Sui − phenotypes by reducing eIF1 affinity for the 40S subunit.…”

“…(C) eIF1 Ssu − substitutions reduce the HIS4 UUG:AUG initiation ratio in SUI5 and SUI3-2 cells. These data were published previously (Martin-Marcos et al 2011) and are presented here in a different format for ease of comparison. For the SUI5 results (black bars), wholecell extracts (WCEs) prepared from derivatives of sui1Δ his4-301-myc SUI5 and sui1Δ HIS4-myc SUI5 yeast strains harboring the indicated SUI1 alleles were subjected to Western analysis with antibodies against myc epitope or Gcd6 (examined as a loading control).…”

Regulation of translation by upstream translation initiation codons of surfactant protein A1 splice variants

“…Although there are exceptions to this rule, the context of the putative start codon appears to be of high importance as far as the association of ribosomes to the mRNA is concerned (26,45). Recent work mechanistically proved the importance of nucleotides in positions Ϫ3 and ϩ4 by associating these with the recruitment of certain initiation factors (53,66). The bioinformatic analysis of the SP-A1 uAUG context performed herein with several different algorithms is consistent with the prevalence of the leaky scanning mechanism in the translation of SP-A1.…”