GTP hydrolysis controls stringent selection of the AUG start codon during translation initiation inSaccharomyces cerevisiae

Abstract

We have isolated and characterized two suppressor genes, SUI4 and SUI5, that can initiate translation in the absence of an AUG start codon at the HIS4 locus in Saccharomyces cerevisiae. Both suppressor genes are dominant in diploid cells and lethal in haploid cells. The SUI4 suppressor gene is identical to the GCD11 gene, which encodes the ␥ subunit of the eIF-2 complex and contains a mutation in the G 2 motif, one of the four signature motifs that characterizes this subunit to be a G-protein. The SUI5 suppressor gene is identical to the TIF5 gene that encodes eIF-5, a translation initiation factor known to stimulate the hydrolysis of GTP bound to eIF-2 as part of the 43S preinitiation complex. Purified mutant eIF-5 is more active in stimulating GTP hydrolysis in vitro than wild-type eIF-5, suggesting that an alteration of the hydrolysis rate of GTP bound to the 43S preinitiation complex during ribosomal scanning allows translation initiation at a non-AUG codon. Purified mutant eIF-2␥ complex is defective in ternary complex formation and this defect correlates with a higher rate of dissociation from charged initiator-tRNA in the absence of GTP hydrolysis. Biochemical characterization of SUI3 suppressor alleles that encode mutant forms of the ␤ subunit of eIF-2 revealed that these mutant eIF-2 complexes have a higher intrinsic rate of GTP hydrolysis, which is eIF-5 independent. All of these biochemical defects result in initiation at a UUG codon at the his4 gene in yeast. These studies in light of other analyses indicate that GTP hydrolysis that leads to dissociation of eIF-2 ⅐ GDP from the initiator-tRNA in the 43S preinitiation complex serves as a checkpoint for a 3-bp codon/anticodon interaction between the AUG start codon and the initiator-tRNA during the ribosomal scanning process.

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

“…To confirm these last findings, we examined whether the sui1 Ssu Ϫ mutations also suppress the Sui Ϫ phenotype of the dominant eIF2␤ Sui Ϫ allele SUI3-2 (21). Indeed, all of the sui1 alleles resemble hc SUI1 ϩ in suppressing the His ϩ phenotype of SUI3-2 ( Fig.…”

“…8A). Importantly, these differences in expression are nearly eliminated by Sui Ϫ mutations in eIF1 (K60E), eIF2␤ (SUI3-2), and eIF1A (SE), while the difference in expression between SUI1-UUU -lacZ and SUI1-opt-lacZ is exacerbated by the Ssu Ϫ mutations affecting eIF1 (E48V) and eIF1A (17)(18)(19)(20)(21) (Fig. 8).…”

“…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.…”

“…Finally, we examined the effect of the eIF5 Sui Ϫ substitution (G31R) encoded by the SUI5 allele of TIF5. As noted above, SUI5 is lethal as the only source of eIF5 but produces a dominant Sui Ϫ phenotype and a large increase in the UUG/AUG ratio in cells coexpressing WT eIF5 (21). Hence, we first examined the effect of SUI5 on eIF1 and SUI1-lacZ expression in a strain containing chromosomal P GAL -TIF5, and cultured cells on galactose to allow coexpression of WT eIF5 and attendant cell growth.…”

Non-AUG Initiation ofAGAMOUSmRNA Translation inArabidopsis thaliana

“…Mammal and insect non-AUG codon recognition is largely dependent on the alternative codon being in an optimal sequence context, which by itself is an important component of the scanning model. In contrast, yeast appears to be unable to efficiently use codons other than AUG as translation initiators (10,12,19), perhaps because sequence context in yeast has a very minor role in AUG recognition (9), which would leave the 3-bp codon-anticodon interaction as the only requirement for protein synthesis initiation (19).…”