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 an AUG start codon in preferred sequence context. 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. eIF1A residues implicated in cancer stabilize translation preinitiation complexes and favor suboptimal initiation sites in yeast Our previous cryo-EM analysis of partial yeast PICs revealed distinct conformations relevant to different stages of initiation. A py48S-open complex exhibits upward movement of the 40S head from the body that widens both the mRNA binding cleft and the P site and eliminates interactions of the 40S subunit with Met-tRNAi and mRNA that are evident in the py48S-closed complex. The py48S-open conformation seems well-suited for scanning of successive triplets for complementarity to Met-tRNAi, with TC anchored in the unstable P-out conformation; whereas py48S-closed exhibits the more stable P-in conformation required for start codon selection. During the transition from py48S-open to py48S-closed, the unstructured N-terminal tail (NTT) of factor eIF1A assumes a defined structure and deploys five basic residues to interact extensively with the tRNAi anticodon or mRNA nucleotides surrounding the AUG codon or rRNA, thus suggesting that the eIF1A NTT directly stabilizes the P-in state. Interestingly, EIF1AX mutations altering the human eIF1A NTT are recurring mutations associated with uveal melanoma (UM). We found that substituting all five basic residues, and seven UM-associated substitutions, in yeast eIF1A suppresses initiation at near-cognate UUG codons and AUGs in poor sequence context. Ribosome profiling of the UM-associated NTT substitution R13P reveals heightened discrimination against unfavorable AUG context throughout the translatome. Both the R13P and K16D substitutions were shown to destabilize the closed complex at UUG codons in reconstituted PICs. Thus, we conclude that electrostatic interactions between eIF1A NTT basic residues and nucleotides in tRNAi, mRNA, or rRNA in the decoding center stabilize the closed conformation of the PIC and promote utilization of suboptimal start codons in vivo. We predict that UM-associated EIF1AX mutations alter the expression of human oncogenes or tumor suppressor genes by increasing discrimination against poor initiation codons. eIF1 Loop 2 interactions with Met-tRNAi control the accuracy of start codon selection by the scanning preinitiation complex As described above, AUG recognition evokes rearrangement from an open PIC conformation with TC in a P-out state to a closed conformation with TC more tightly bound in the P-in conformation. Factor eIF1 binds to the 40S subunit and exerts a dual role of enhancing TC binding to the open PIC conformation while antagonizing the P-in state, necessitating eIF1 dissociation for start codon selection to proceed. Our previous cryo-EM structures of partial yeast PICs revealed juxtaposition of eIF1 Loop 2 with the Met-tRNAi D loop in the P-in state and predict a distortion of Loop 2 from its conformation in the open complex to avoid a clash with Met-tRNAi. We showed that Ala substitutions in Loop 2 increase initiation at both near-cognate UUG codons and AUG codons in poor context in vivo. Consistently, the D71A-M74A double substitution stabilizes TC binding to 48S PICs reconstituted with mRNA harboring a UUG start codon, without affecting eIF1 affinity for 40S subunits. Similar but relatively stronger decreases in discrimination against poor start codons were conferred by arginine substitutions in Loop 2; and none of the Loop 2 substitutions perturbed the rate of TC loading on scanning 40S subunits in vivo. These findings indicate that electrostatic and steric clashing between the eIF1 Loop 2 and tRNAi D loop impede Met-tRNAi accommodation specifically in the P-in state of the closed complex without influencing the P-out mode of TC binding to the open complex; and Arg substitutions convert the Loop 2-tRNAi clash to an electrostatic attraction that stabilizes P-in and enhances selection of poor start codons in vivo. Thus, in contrast to the eIF1A NTT that specifically stabilizes the closed/P-in state of the PIC and enables recognition of poor start codons, eIF1 Loop 2 destabilizes the P-in state and helps to insure relatively greater initiation frequencies for optimal start codons in vivo. Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR) recycle post-termination 40S subunits in vivo Recycling of post-termination 40S subunits in vivo liberates free 40S subunits, deacylated tRNA, and mRNA for further rounds of translation. Rli1/ABCE1 catalyzes the first stage of recycling, splitting the 80S ribosome into a free 60S subunit and a tRNA/mRNA-bound 40S subunit. The second stage, dissociation of tRNA and mRNA from the 40S, has been reconstituted in vitro with ligatin/eIF2D, or the two interacting proteins MCT-1 and DENR, homologous to the N- and C-termini, respectively, of eIF2D. Initiation factors eIF1, eIF1A, eIF3 and eIF3j can also recycle 40S post-termination complexes, but it was unknown if either recycling mechanism operates in living cells. To address this, we performed ribosome profiling on yeast mutants lacking eIF2D (Tma64) together with MCT-1 (Tma20) or DENR (Tma22). Both double mutants revealed 80S ribosomes queued immediately upstream of stop codons, consistent with a genome-wide block in 40S recycling at stop codons. We also found evidence that unrecycled 40S complexes in the mutants could reinitiate translation at AUG codons located downstream in the 3UTR, as indicated by 80S peaks at 3'UTR AUG codons, and by detecting expression of 3UTR translation products that was diminished by eliminating the presumptive AUG start codons. In vitro translation experiments using reporter mRNAs containing upstream ORFs (uORFs) further established that reinitiation at coding sequences downstream of the uORFs increased in extracts devoid of these proteins. In some cases, 40S ribosomes appeared to rejoin with 60S subunits and undergo an alternative 80S reinitiation process in 3UTRs observed previously in cells depleted of Rli1 involving unrecycled 80S post-termination complexes. These results support a crucial role for eIF2D (Tma64), MCT-1 (Tma20), and DENR (Tma22) in recycling of 40S ribosomal subunits at stop codons and thereby diminishing translation reinitiation following termination at the uORFs and main coding sequences of many mRNAs.
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