Dissociation of eIF1 from the 40S ribosomal subunit is a key step in start codon selection in vivo

Abstract

Selection of the AUG start codon is a key step in translation initiation requiring hydrolysis of GTP in the eIF2•GTP•Met-tRNA iMet ternary complex (TC) and subsequent P i release from eIF2•GDP•P i . It is thought that eIF1 prevents recognition of non-AUGs by promoting scanning and blocking P i release at non-AUG codons. We show that Sui − mutations in Saccharomyces cerevisiae eIF1, which increase initiation at UUG codons, reduce interaction of eIF1 with 40S subunits in vitro and in vivo, and both defects are diminished in cells by overexpressing the mutant proteins. Remarkably, Sui − mutation ISQLG 93-97 ASQAA (abbreviated 93-97) accelerates eIF1 dissociation and P i release from reconstituted preinitiation complexes (PICs), whereas a hyperaccuracy mutation in eIF1A (that suppresses Sui − mutations) decreases the eIF1 off-rate. These findings demonstrate that eIF1 dissociation is a critical step in start codon selection, which is modulated by eIF1A. We also describe Gcd − mutations in eIF1 that impair TC loading on 40S subunits or destabilize the multifactor complex containing eIF1, eIF3, eIF5, and TC, showing that eIF1 promotes PIC assembly in vivo beyond its important functions in AUG selection.[Keywords: AUG selection; Saccharomyces cerevisiae; translation initiation; eIF1] 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

“…It is thought that increasing the cellular concentration of eIF1 proteins harboring such Sui Ϫ substitutions overcomes their 40S binding defects by mass action, reversing premature eIF1 release at non-AUG codons. This is the same mechanism evoked to explain how overexpressing WT eIF1 suppresses the Sui Ϫ phenotypes of mutations in other eIFs (8,48).…”

“…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. Furthermore, a mutation in the N-terminal tail (NTT) of eIF1A (17)(18)(19)(20)(21) that suppresses UUG initiation in vivo retards eIF1 dissociation on start codon recognition (8). Thus, the rate of eIF1 dissociation and P i release are critical determinants of AUG recognition.…”

“…Because the Sui Ϫ phenotype generally results from a reduction in eIF1 function (8,48,49), it was important to demonstrate that the Sui Ϫ substitutions reduce eIF1 activity rather than its expression. Western analysis of WCEs revealed that the sui1 mutants actually contain ϳ5-to ϳ12-fold higher than WT levels of eIF1 (normalized to eIF2Bε subunit Gcd6), with the largest increase observed for the strongest Sui Ϫ mutant (L96P) (Fig.…”

“…Although the reduced 40S occupancy of a Sui Ϫ eIF1 mutant decreases the rate of TC loading, since this reaction occurs in the open 40S conformation, once TC is bound it can isomerize more readily to the fully accommodated mode of P-site binding in the absence of a perfect codon-anticodon match, e.g., at UUG codons, thus accounting for the Sui Ϫ phenotype (8,33). We presume that the novel Sui Ϫ mutations in eIF1 described here similarly reduce its affinity for the 40S because they exhibit the hallmark of this class of eIF1 mutations, that their Sui Ϫ phenotypes are suppressed by overexpressing the mutant proteins (unpublished observations).…”

uORFs with unusual translational start codons autoregulate expression of eukaryotic ornithine decarboxylase homologs

“…However, eukaryotic initiation factor 1 (eIF1) is pertinent. Dissociation of eIF1 from the ternary initiation complex (eIF2, GTP, and Met-tRNA i Met ) is a key step in initiation codon selection (27)(28)(29). Specifically, mutants in eIF1 that reduce its affinity to the ternary complex increase initiation at non-AUG codons.…”