SummaryDuring eukaryotic translation initiation, initiator tRNA does not insert fully into the P decoding site on the 40S ribosomal subunit. This conformation (POUT) is compatible with scanning mRNA for the AUG start codon. Base pairing with AUG is thought to promote isomerization to a more stable conformation (PIN) that arrests scanning and promotes dissociation of eIF1 from the 40S subunit. Here, we present a cryoEM reconstruction of a yeast preinitiation complex at 4.0 Å resolution with initiator tRNA in the PIN state, prior to eIF1 release. The structure reveals stabilization of the codon-anticodon duplex by the N-terminal tail of eIF1A, changes in the structure of eIF1 likely instrumental in its subsequent release, and changes in the conformation of eIF2. The mRNA traverses the entire mRNA cleft and makes connections to the regulatory domain of eIF2α, eIF1A, and ribosomal elements that allow recognition of context nucleotides surrounding the AUG codon.

SUMMARY Recognition of the proper start codon on mRNAs is essential for protein synthesis, which requires scanning and involves eukaryotic initiation factors (eIFs) eIF1, eIF1A, eIF2 and eIF5. The carboxyl-terminal domain (CTD) of eIF5 stimulates 43S preinitiation complex (PIC) assembly; however, its precise role in scanning and start codon selection has remained unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we identified the binding sites of eIF1 and eIF2β on eIF5-CTD and find that they are partially overlapped. Mutating select eIF5 residues in the common interface specifically disrupts interaction with both factors. By abrogating eIF5-CTD binding to eIF2β, genetic and biochemical evidence indicate that these eIF5-CTD mutations impair start codon recognition and impede eIF1 release from the PIC. This study provides mechanistic insight into the novel role of eIF5-CTD’s dynamic interplay with eIF1 and eIF2β in switching PICs from an open to closed state at start codons.

Eukaryotic translation initiation factor (eIF) 1 is a central mediator of start codon recognition. Dissociation of eIF1 from the pre-initiation complex allows release of phosphate from the G-protein factor eIF2, triggering downstream events in initiation. Mutations that weaken binding of eIF1 to the pre-initiation complex decrease the fidelity of start codon recognition (Sui− phenotype) by allowing increased eIF1 release at non-AUG codons. Consistent with this, over-expression of these mutant proteins suppresses their Sui− phenotypes. Here, we have examined mutations at the penultimate residue of eIF1, G107, that produce Sui− phenotypes without increasing the rate of eIF1 release. We provide evidence that, in addition to its role in gating phosphate release, dissociation of eIF1 triggers conversion from an open, scanning-competent state of the pre-initiation complex to a stable, closed one. We also show that eIF5 antagonizes binding of eIF1 to the complex and that key interactions of eIF1 with its partners are modulated by the charge at and around G107. Our data indicate that eIF1 plays multiple roles in start codon recognition and suggest that prior to AUG recognition it prevents eIF5 from binding to a key site in the pre-initiation complex required for triggering downstream events.

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 ...

Summary During eukaryotic translation initiation, eIF3 binds the solvent-accessible side of the 40S ribosome and recruits the gate-keeper protein eIF1 and eIF5 to the decoding center. This is largely mediated by the N-terminal domain (NTD) of eIF3c, which can be divided into three parts: 3c0, 3c1 and 3c2. The N-terminal part, 3c0, binds eIF5 strongly, but only weakly to the ribosome-binding surface of eIF1, whereas 3c1 and 3c2 form a stoichiometric complex with eIF1. 3c1 contacts eIF1 through Arg-53 and Leu-96, while 3c2 faces 40S protein uS15/S13, to anchor eIF1 to the scanning pre-initiation complex (PIC). We propose that the 3c0:eIF1 interaction diminishes eIF1 binding to the 40S, whereas 3c0:eIF5 interaction stabilizes the scanning PIC by precluding this inhibitory interaction. Upon start codon recognition, interactions involving eIF5, and ultimately 3c0:eIF1 association facilitate eIF1 release. Our results reveal intricate molecular interactions within the PIC, programmed for rapid scanning-arrest at the start codon.

We identified a mutation in the 60S ribosomal protein L33A (rpl33a-G76R) that elicits derepression of GCN4 translation (Gcd ؊ phenotype) by allowing scanning preinitiation complexes to bypass inhibitory upstream open reading frame 4 (uORF4) independently of prior uORF1 translation and reinitiation. At 37°C, rpl33a-G76R confers defects in 60S biogenesis comparable to those produced by the deletion of RPL33A (⌬A). At 28°C, however, the 60S biogenesis defect is less severe in rpl33a-G76R than in ⌬A cells, yet rpl33a-G76R confers greater derepression of GCN4 and a larger reduction in general translation. Hence, it appears that rpl33a-G76R has a stronger effect on ribosomal-subunit joining than does a comparable reduction of wild-type 60S levels conferred by ⌬A. We suggest that rpl33a-G76R alters the 60S subunit in a way that impedes ribosomalsubunit joining and thereby allows 48S rRNA complexes to abort initiation at uORF4, resume scanning, and initiate downstream at GCN4. Because overexpressing tRNA i Met suppresses the Gcd ؊ phenotype of rpl33a-G76R cells, dissociation of tRNA iMet from the 40S subunit may be responsible for abortive initiation at uORF4 in this mutant. We further demonstrate that rpl33a-G76R impairs the efficient processing of 35S and 27S pre-rRNAs and reduces the accumulation of all four mature rRNAs, indicating an important role for L33 in the biogenesis of both ribosomal subunits.Cell growth and division are highly interconnected processes that require protein synthesis, which in turns requires the biogenesis of ribosomes, soluble translation factors, and charged tRNA species. In Saccharomyces cerevisiae, ribosome biogenesis consumes a great amount of energy and is tightly regulated according to nutrient availability and to signals depending on other macromolecular pathways (77). The production of 60S and 40S ribosomal subunits is a highly dynamic process that begins with the transcription of rRNA by RNA polymerase I (35S rRNA precursor to 25S, 18S, and 5.8S rRNAs) and RNA polymerase III (5S rRNA) in the nucleolus and ends with export of preribosomal 60S and 40S subunits to the cytoplasm, where final steps of maturation occur. The maturation pathway of the 35S pre-rRNA to yield 25S, 18S, and 5.8S rRNAs (see Fig. 4A) is closely coordinated with the assembly of 79 ribosomal proteins (r-proteins) and more than 150 trans-acting factors involved in the synthesis, maturation, and transport of the ribosomal subunits (reviewed in references 11, 41, 53, and 73). The association of 35S pre-rRNA with the U3-snoRNP complex, transiently associated trans-acting factors, and several r-proteins, mostly belonging to the 40S subunit, leads to the formation of the 90S nucleolar complexes, where cleavage at the A0, A1, and A2 processing sites occurs (16, 28; reviewed in reference 29). The pre-40S particle, which contains some newly associated maturation factors and some already present in the 90S particle, is released after A0-A1-A2 cleavage and transported to the cytoplasm, where cleavage of 20S pre-rRNA to matur...

Background: Start codon selection requires eIF1 dissociation from its 40 S-binding site. Results: eIF1 residues in ␤-hairpin loop-1 and helix ␣1 make functionally critical contacts with the 40 S subunit. Conclusion: Direct 40 S contacts of eIF1 regulate the rate of Met-tRNA i recruitment and block non-AUG recognition. Significance: eIF1's direct contacts with the 40 S subunit are crucial for AUG recognition in vivo.

The 90S pre-ribosome, also known as the small subunit (SSU) processome, is a large multisubunit particle required for the production of the 18S rRNA from a pre-rRNA precursor. Recently, it has been shown that the formation of this particle entails the initial association of the tUTP subunit with the nascent pre-RNA and, subsequently, the binding of Rrp5/UTP-C and U3 snoRNP/UTP-B subunits in two independent assembly branches. However, the mode of assembly of other 90S pre-ribosome components remains obscure as yet. In this study, we have investigated the assembly of three proteins (Utp20, Imp4 and Bms1) previously regarded as potential nucleating factors of the 90S particle. Here, we demonstrate that the loading of those three proteins onto the pre-rRNA takes place independently of Rrp5/UTP-C and, instead, occurs downstream of the tUTP and U3/UTP-B subcomplexes. We also demonstrate that Bms1 and Utp20 are required for the recruitment of a subset of proteins to nascent pre-ribosomes. Finally, we show that proteins associated through secondary steps condition the stability of the two assembly branches in partially assembled pre-ribosomes. These results provide new information about the functional relationships among 90S particle components and the events that are required for their stepwise incorporation onto the primary pre-rRNA.