We study molecular mechanisms involved in translation initiation using budding yeast as a model system, owing to the powerful combination of genetics and biochemistry available to dissect complex pathways in yeast cells. The translation initiation pathway produces an 80S ribosome bound to mRNA with methionyl initiator tRNA (Met-tRNAi) base-paired to the AUG start codon. Met-tRNAi is recruited to the 40S subunit in a ternary complex (TC) with GTP-bound eIF2, to produce the 43S preinitiation complex (PIC), in a manner stimulated by eIFs -1, -1A, and -3. The 43S PIC attaches to the 5'end of mRNA, in a manner facilitated by cap-binding complex eIF4F (comprised of eIF4E, eIF4G, and RNA helicase eIF4A) and PABP bound to the poly(A) tail, and scans the 5'untranslated region (UTR) for the AUG start codon. Scanning is promoted by eIFs 1 and 1A, which induce an open conformation of the 40S, and by eIF4F and other RNA helicases that remove secondary structure in the 5'UTR. AUG recognition by Met-tRNAi evokes irreversible hydrolysis of the GTP bound to eIF2, dependent on GTPase activating protein (GAP) eIF5, releasing eIF2-GDP from the PIC and Met-tRNAi into the ribosomal P site. After joining of the 60S subunit, producing the completed 80S initiation complex, the eIF2-GDP is recycled to eIF2-GTP by guanine nucleotide exchange factor (GEF) eIF2B to prepare for the next round of translation initiation. β-hairpin loop of eIF1 mediates 40S ribosome binding to regulate Met-tRNAi recruitment and accuracy of AUG selection in vivo. Recognition of the start codon is thought to require dissociation of eIF1 from the 40S subunit, enabling Pi release from eIF2-GDP (following GTP hydrolysis by the TC), rearrangement of the 40S subunit to a closed conformation incompatible with scanning, and stable binding of Met-tRNAi to the P site. Supporting this model, we showed that substituting basic amino acids in yeast eIF1 helix α1 or β-hairpin loop-1, which contact 18S rRNA in the Tetrahymena 40S∙eIF1 crystal structure, impairs eIF1 binding to 40S∙eIF1A complexes in vitro, and increases initiation at UUG codons (Sui- phenotype) in a manner suppressed by overexpressing the mutant proteins or an eIF1A mutation (17-21) known to impede eIF1 dissociation in vitro. The eIF1 Sui- mutations also derepress translation of GCN4 mRNA, indicating impaired TC loading, and this Gcd- phenotype is likewise suppressed by eIF1 overexpression or the 17-21 mutation. Thus, direct contacts of eIF1 with 18S rRNA seen in the Tetrahymena 40S∙eIF1 complex are crucial in yeast to stabilize the open conformation of the 40S subunit, required for rapid TC loading and ribosomal scanning, and impede rearrangement to the closed complex at non-AUG codons. Coordinated movements of eukaryotic translation initiation factors eIF1, eIF1A, and eIF5 trigger phosphate release from eIF2 in response to start codon recognition by the ribosomal preinitiation complex. Using fluorescence resonance energy transfer (FRET) between fluorophores on the C-terminus of eIF1A and on eIF5 in reconstituted yeast PICs, we found that the CTT of eIF1A moves closer to the NTD of eIF5 in response to start codon recognition in a manner controlled by the rate of eIF1 dissociation from the PIC and dependent on the SE elements we identified previously in the eIF1A CTT. Remarkably, mutations in the SE elements uncouple eIF1 release from Pi release from eIF2-GDP, dramatically impairing both Pi release and movement of the eIF1A CTT while minimally affecting eIF1 release. These findings demonstrate that eIF1 dissociation is not sufficient for Pi release and that movement of the eIF1A-CTT towards the eIF5-NTD is additionally required for this key step in start codon recognition. We propose that eIF1 release determines the timing of these events by setting the rate of accommodation of Met-tRNAi into the P site, which in turn triggers movement of the eIF1A-CTT towards the eIF5-NTD. Exome sequencing identifies recurrent somatic mutations in the eIF1A structural gene (EIF1AX) in uveal melanoma with disomy 3. Uveal melanoma (UM) is the most frequent malignant tumor of the eye, and exome sequencing identified somatic mutations in EIF1AX as prevalent mutations found in UM associated with chromosome 3 disomy. The EIF1AX mutations produce amino acid substitutions or short deletions in the conserved unstructured N-terminal tail (NTT) of eIF1A. As we previously showed that NTT substitutions in yeast eIF1A reduce PIC assembly, the EIF1AX mutations could diminish general translation, and might also induce transcription factors (eg. ATF4) whose mRNA translation is inversely coupled to TC concentration by the same mechanism governing translation of yeast GCN4. We also implicated the yeast eIF1A NTT in initiation accuracy, as NTT substitutions suppress initiation at near-cognate triplets (Ssu- phenotype), and cause the PIC to bypass the first AUG encountered while scanning from the 5 end of the mRNA. Hence, the EIF1AX mutations might suppress recognition of near-cognate initiation siteswhich appear to be more prevalent than previously suspectedor of 5proximal AUG codons to alter the relative utilization of different start codons for tumor-promoting or tumor-suppressing genes. Yeast eIF4B binds to the head of the 40S ribosomal subunit and promotes mRNA recruitment through its N-terminal and internal repeat domains. eIF4B stimulates recruitment of mRNA to the 43S PIC. While the mammalian factor stimulates eIF4A helicase activity, this function is lacking in yeast. Yeast (y)eIF4B consists of an N-terminal domain (NTD) predicted to be unstructured in solution;an RNA-recognition motif (RRM);and an unusual domain comprised of seven imperfect repeats of 26 amino acids;and previous studies implicated the RRM and its RNA binding activity in promoting translation initiation. By analyzing the effects of deletions and mutations of yeIF4Bs domains on PIC attachment to mRNA in vitro and translation initiation in vivo, we found that the 7-repeats domain is critical for productive interaction with the PIC and other components of the initiation machinery, particularly eIF4A, in promoting PIC attachment to mRNA. The NTD also plays a role in accelerating mRNA binding to the PIC but, surprisingly, the RRM and its associated ssRNA binding activity are dispensable in vitro and in vivo. We also found that yeIF4B binds to ribosomal protein Rps20 in the head of the 40S subunit, and induces structural changes in the ribosome mRNA entry channel, suggesting that yeIF4B modulates the 40S head conformation to promote a receptive state of the mRNA entry channel. Yeast eukaryotic initiation factor 4B (eIF4B) enhances complex assembly between eIF4A and eIF4G in vivo. Association of eIF4A with eIF4G in eIF4F activates eIF4A helicase activity, as the eIF4G HEAT domains juxtapose the eIF4A RecA-like domains in a conformation poised for catalysis. We identified Ts- mutations in the HEAT domains of yeast eIF4G1 and eIF4G2 that are suppressed by overexpressing either yeIF4B or eIF4A, whereas others are suppressed only by eIF4A overexpression. Importantly, suppression of HEAT domain substitutions by yeIF4B overexpression was correlated with the restoration of native eIF4A∙eIF4G complexes in vivo, and the rescue of specific mutant eIF4A∙eIF4G complexes by yeIF4B was reconstituted in vitro. Association of eIF4A with WT eIF4G in vivo also was enhanced by yeIF4B overexpression, and was impaired in cells lacking yeIF4B. Furthermore, we detected native complexes containing eIF4G and yeIF4B but lacking eIF4A. These and other findings lead us to propose that yeIF4B acts in vivo to promote eIF4F assembly by enhancing a conformation of the HEAT domain of yeast eIF4G conducive for stable binding to eIF4A.
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