We study the molecular mechanisms involved in assembly and function of translation initiation complexes involved in protein synthesis, using yeast as a model system to exploit its powerful combination of genetics and biochemistry for dissecting complex cellular processes in vivo. The translation initiation pathway produces an 80S ribosome bound to mRNA with methionyl initiator tRNA (tRNAi) base-paired to the AUG start codon. The 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 reaction stimulated by eIFs 1, 1A, 3 and 5. The 43S PIC attaches to the 5' end of mRNA, 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 TC binding in a conformation suitable for scanning successive triplets entering the ribosomal P site (P-out) , and by eIF4F and other RNA helicases, such as Ded1, that remove secondary structure in the 5' UTR. AUG recognition leads to tighter binding of TC in the P-in state and evokes irreversible hydrolysis of the GTP bound to eIF2, dependent on the GTPase activating protein (GAP) eIF5, releasing eIF2-GDP from the PIC leaving tRNAi in the P site. After joining of the 60S subunit, producing the 80S initiation complex, the eIF2-GDP is recycled to eIF2-GTP by guanine nucleotide exchange factor (GEF) eIF2B for the next round of translation initiation. Structures of yeast preinitiation complexes reveal conformational changes from mRNA scanning to start-codon recognition. It is thought that the scanning PIC assumes an open conformation and that AUG recognition evokes a closed state that arrests scanning with more stable Met-tRNAi binding (P-in state), with attendant displacement of the eIF1A-CTT from the P site and dissociation of eIF1 from the 40S subunit. In collobaration with Venki Ramakrishnan's and Jon Lorsch's groups, cryo-EM reconstructions of yeast PICs have been obtained representing different stages of the initiation pathway, at 3.5-6.1 angstrom resolution. These include 40S-eIF1-eIF1A complexes and partial yeast 48S PICs in open and closed conformations (py48S-open and py48-closed). These structures provide a wealth of new information about conformational changes occurring in the transition from scanning to AUG selection. Comparing the 40S-eIF1-eIF1A complex to the free 40S reveals rotation of the 40S head that might promote TC binding to form the 43S PIC. The py48S-open, formed using mRNA with AUC start codon, reveals an upward shift of the 40S head that widens the mRNA entry channel and opens its latch--consistent with our eIF1A/Fe(II)-BABE cleavage data--which should facilitate mRNA insertion into the binding cleft to form the scanning PIC. Moreover, the P site is widened and lacks tRNAi contacts with the 40S body present in canonical 80S-tRNAi complexes. By contrast, py48S-closed, formed with mRNA(AUG), reveals downward head movement that closes the latch, clamps the mRNA into the binding cleft, and fully encloses tRNAi in the P site. The eIF1A NTT assumes a structured conformation and interacts with the AUG:anticodon duplex, consistent with its role in stabilizing P-in. eIF1 is repositioned on the 40S and deformed to prevent a clash with tRNAi, likely as a prelude to eIF1 dissociation from the 40S subunit. Both py48S-open and -closed complexes reveal eIF2beta and portions of the eIF3 complex. The eIF3 trimeric subcomplex eIF3b-CTD/eIF3i/eIF3g-NTD resides on the subunit-interface surface of the 40S, and appears to lock mRNA into the 40S binding cleft. eIF2beta interacts with tRNAi and segments of eIF1 and eIF1A exclusively in py48S-open, which could stabilize binding of TC and eIF1 to the scanning PIC prior to AUG recognition. A portion of eIF2alpha domain-I projects into the entry channel and contacts the beta-hairpin of uS7/Rps5 and nucleotides just upstream of the AUG codon, including the key -3 nucleotide of the Kozak consensus sequence for efficient AUG selection, consistent with a proposed role for eIF2alpha in start codon recognition. eIF4B and DEAD-box RNA helicases Ded1 and eIF4A preferentially stimulate translation of long mRNAs with structured 5UTRs and low closed-loop potential but weak dependence on eIF4G. RNA helicases eIF4A and Ded1 are believed to resolve mRNA secondary structures that impede ribosome attachment to the mRNA and scanning to the start codon, but whether they perform distinct functions in vivo was poorly understood. We compared the effects of mutations in Ded1 or eIF4A on translational efficiencies (TEs) by ribosome profiling. Despite similar reductions in bulk translation, inactivation of Ded1 substantially reduced the relative TEs of >600 mRNAs, whereas inactivation of eIF4A similarly affected <40 mRNAs. Ded1-dependent mRNAs show greater than average 5UTR length and propensity for secondary structure, implicating Ded1 in scanning though structured 5' UTRs. Thus, it appears that Ded1 is critically required are scanning through secondary structures in 5UTRs, while eIF4A promotes a step of initiation common to nearly all mRNAs, such as ribosome attachment. While eIF4B is regarded as a cofactor for eIF4A, it might also function independently of eIF4A. Ribosome profiling of a mutant lacking eIF4B showed that, while eliminating eIF4B preferentially impacts mRNAs with long structured 5UTRs, it reduces the relative TEs of many more genes than does inactivation of eIF4A. These findings support an eIF4A-independent role for eIF4B in addition to its function as eIF4A cofactor. eIF4B, eIF4A, and Ded1 mutations also preferentially impair translation of longer mRNAs in a manner mitigated by the ability to form closed-loop mRNPs via eIF4F-Pab1 association, suggesting cooperation between closed-loop assembly and eIF4B/helicase functions in stimulating initiation. Surprisingly, examining published datasets revealed that depleting eIF4G, the scaffold subunit of eIF4F, preferentially impacts short mRNAs with strong closed-loop potential and unstructured 5UTRs, exactly the opposite features associated with hyperdependence on eIF4B/helicases. Hence, short, highly efficient mRNAs depend primarily on the stimulatory effects of eIF4G-dependent closed-loop assembly versus helicase functions. Rli1/ABCE1 Recycles Terminating Ribosomes and Controls Translation Reinitiation in 3'UTRs In Vivo. Ribosome recycling is the final phase of protein synthesis, following release of the completed polypeptide chain from the ribosome at the stop codon. ABCE1 (yeast Rli1) has been shown to catalyze removal of the 60S subunit from post-termination complexes in vitro, but was not known to be crucial for recycling in vivo. Using ribosome profiling, in collaboration with Rachel Greens lab, we showed that depletion of Rli1 in yeast evokes accumulation of 80S ribosomes at stop codons and in the adjoining 3UTRs of most genes. We detected predicted 3UTR translation products by Western analysis and mass spectrometry of strains harboring epitope-tagged genes, and their small sizes indicate a non-canonical reinitiation mechanism in which, following polypeptide release at the canonical stop codon, unrecyled 80S ribosomes resume translation at a nearby sense codon. Thus, Rli1 is crucial for ribosome recycling in vivo and for overall gene expression as it modulates ribosome homeostasis and 3UTR translation.
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