Molecular Mechanisms of Translational Control

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

We study the molecular mechanisms involved in assembly, function, and regulation 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 binding of TC 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 and Dbp1, that remove secondary structure in the 5' UTR. AUG recognition induces rearrangement to a closed conformation of the PIC, dissociation of eIF1, and tighter binding of TC in the P-in state, enabling irreversible hydrolysis of the GTP bound to eIF2 in a manner dependent on the GTPase activating protein (GAP) eIF5. Subsequent release of eIF2-GDP from the PIC leaves tRNAi in the P site and allows joining of the 60S subunit to form the 80S initiation complex. The function of eIF2 is down-regulated by phosphorylation of its -subunit by protein kinase Gcn2 in response to amino acid starvation and other stresses that likely evoke stalling of ribosomes engaged in translation elongation. Large-scale movement of eIF3 domains during translation initiation modulate start codon selection. eIF3 is the most complex initiation factor, comprised of five different subunits (-a, -b, -c, -g, -i), and is involved at every stage of translation initiation, including TC and mRNA recruitment to the PIC, impairing association of 40S and 60S subunits, and modulating the fidelity of start codon selection. In our previous cryo-EM structures of yeast 48S PICs, dubbed py48S-open and py48S-closed, an eIF3 subcomplex comprised of the eIF3b-CTD (-propeller and RRM), eIF3i, eIF3g N-terminal domain (NTD) was observed at the subunit interface of the 40S, interacting with eIF1, eIF2 and the 40S itself, and appeared to lock the mRNA into the 40S binding cleft. This was surprising as this module was located at the solvent-exposed 40S surface in both early-stage 43S PICs and in a late-stage PIC where the eIF5-NTD has replaced eIF1 at the 40S interface. This led us to hypothesize that the eIF3b-3i-3g module binds initially at the solvent side of the 43S PIC, relocates to the subunit interface at the onset of scanning, and relocates back to the solvent side following start codon selection for the final steps of initiation. Supporting this model, our higher resolution structures of the py48S-open and py48S-closed revealed the eIF3b -propeller directly interacting with eIF2 and the eIF3b RRM contacting eIF1 at the subunit interface, with certain contacts restricted to only the open or closed state. Additionally, we found that different eIF3b substitutions expected to perturb interactions of its RRM or -propeller with rRNA, or with eIF3c or eIF1, at the subunit interface can either decrease or increase discrimination against UUG start codons, which suggests that they preferentially stabilize the open or closed conformation of the PIC, respectively. Interestingly, eIF3b substitutions predicted to stabilize its binding at the subunit interface through increased electrostatic attraction, or to weaken its binding at the solvent side of the 40S via electrostatic repulsion, both confer increased discrimination against UUG start codons. These last findings support the idea that the b-i-g module must dissociate from the subunit interface and relocate to the solvent surface to enable start codon selection, possibly by facilitating eIF1 release and its replacement by the eIF5-NTD (Llacer et al. 2021). uS5/Rps2 residues at the 40S ribosome entry channel enhance initiation at suboptimal start codons in vivo. Ribosomal protein uS3/Rps3 is positioned at the solvent side of the 40S near the mRNA entry channel. We showed previously that substituting uS3/Rps3 residues that contact mRNA preferentially destabilizes the closed conformation of the PIC, reducing initiation at both UUG and the poor-context AUG of SUI1 mRNA. Ribosomal protein uS5/Rps2 is also located at the 40S mRNA entry channel in proximity to mRNA but additionally contains a domain that projects into the decoding center. We found that non-lethal substitutions of conserved Rps2 residues located in both locations reduce bulk translation initiation and alter the discrimination against poor initiation codons. A subset of the Rps2 substitutions suppress initiation at both UUG start codons and AUG codons in suboptimal Kozak context, thus resembling previously described substitutions in uS3/Rps3 at the 40S entry channel or substitutions in initiation factors eIF1 and eIF1A that appear to preferentially destabilize the closed conformation of the PIC. In contrast, other Rps2 substitutions selectively discriminate against either near-cognate UUG codons or poor-context AUG or UUG start codons. These last findings suggest that different Rps2 residues are involved in distinct mechanisms involved in discriminating against different features of poor initiation sites in vivo (Dong et al. 2021). Down-regulation of yeast helicase Ded1 by glucose starvation or heat-shock differentially impairs translation of Ded1-dependent mRNAs Ded1 is an essential DEAD-box helicase in yeast that broadly stimulates translation initiation and is critical for efficient translation of mRNAs harboring long, structured 5 UTRs. Recent evidence suggests that condensation of Ded1 in mRNA granules down-regulates Ded1 function during heat-shock and glucose starvation. We examined this hypothesis by determining the overlap between mRNAs whose relative translational efficiencies (TEs), as determined by ribosomal profiling, are diminished in either stressed WT cells or in ded1 mutants examined in non-stress conditions. Only subsets of the Ded1-hyperdependent mRNAs identified in ded1 mutant cells exhibit strong TE reductions in glucose-starved or heat-shocked WT cells; and those down-regulated by glucose starvation also exhibit hyper-dependence on initiation factor eIF4B, and to a lesser extent, on eIF4A, for efficient translation in non-stressed cells. These findings are consistent with recent proposals that dissociation of Ded1 from mRNA 5UTRs and condensation of Ded1 contribute to reduced Ded1 function during stress, but they further suggest that down-regulation of eIF4B and eIF4A functions also contributes to the translational impairment of a select group of Ded1 mRNA targets with heightened dependence on all three factors during glucose starvation (Sen et al., 2021).