Large-scale movement of eIF3 domains during translation initiation modulate start codon selection

Abstract

The eukaryotic initiation factor 3 (eIF3) complex is involved in every step of translation initiation, but there is limited understanding of its molecular functions. Here, we present a single particle electron cryomicroscopy (cryo-EM) reconstruction of yeast 48S ribosomal preinitiation complex (PIC) in an open conformation conducive to scanning, with core subunit eIF3b bound on the 40S interface near the decoding center in contact with the ternary complex eIF2·GTP·initiator tRNA. eIF3b is relocated together with eIF3i from their solvent interface locations observed in other PIC structures, with eIF3i lacking 40S contacts. Re-processing of micrographs of our previous 48S PIC in a closed state also suggests relocation of the entire eIF3b-3i-3g-3a-Cter module during the course of initiation. Genetic analysis indicates that high fidelity initiation depends on eIF3b interactions at the 40S subunit interface that promote the closed PIC conformation, or facilitate the relocation of eIF3b/eIF3i to the solvent interface, on start codon selection.

Conformational rearrangements upon start codon recognition in human 48S translation initiation complex

“…eIF1 binds close to the P-site codon, while the C-terminal domain (CTD) of eIF2β interacts with eIF1 and contacts the anticodon-stem loop of the tRNA, thereby stabilizing the tRNA P out conformation (left panels in Figure 2B and D ). eIF1A binds to the A site of the decoding center between h18 and h44 of 18S rRNA and proteins uS12 and eS30 of the 40S body domain (Figure 2E and F and Supplementary Figure S5B ), as in all reported 48S IC structures ( 21 , 34 , 44 , 46 , 49 ). An α-helical element (residues 265–278) in the N-terminal domain (NTD) of eIF3c, which is specific for human eIF3, interacts with eIF1 in a similar way as in the h48S·scan complex (Figure 2C and Supplementary Figure S5C ; ( 21 )).…”

“…h48S scan: human 48S IC with non-cognate codon CUC replacing the AUG codon (PDB: 6ZMW, ( 21 )). y48S·AUC: yeast 48S IC open state with a near-cognate AUC codon (PDB: 6GSM, ( 46 )). y48S·AUG: yeast 48S IC with cognate AUG and eIF1 bound in closed state (PDB: 3JAQ, ( 44 )).…”

“…This conformation is stabilized by contacts between the NTT of eIF1A with the tRNA-mRNA duplex ( 34 , 44–46 ). AUG recognition induces the release of eIF1 and eIF2β from the P site, Pi release from eIF2, and reorganization of eIF3 ( 33 , 34 , 44 , 46–48 ). The existing structures of mammalian 48S IC assembled on AUG are similar to the closed y48S AUG ( 49 , 50 ).…”

eIF3 and Its mRNA-Entry-Channel Arm Contribute to the Recruitment of mRNAs With Long 5′-Untranslated Regions

“… Disruption of the eIF3 complex or its mRNA-entry channel arm provoke strong and overlapping effects on the translation of mRNAs across the transcriptome. (A) Structural model of eIF3 bound to the PIC (PDB 6GSM) viewed looking down at top of the small (40S) ribosomal subunit head so as to visualize the path of mRNA as it enters and exits the PIC ( Llácer et al, 2021 ). The small (40S) ribosomal subunit is shown in grey, and the initiator tRNA and mRNA are shown in yellow and orange, respectively (with the path of the mRNA entering and exiting the PIC shown as a cartoon).…”

“…To that end, we constructed ribosome profiling and RNA-seq libraries from both the eIF3i DDKK and eIF3a/b Degron strains (and their corresponding isogenic WT strains) grown under restrictive conditions and calculated relative translational efficiency (TE rel ) values for coding sequences (CDS), ignoring reads obtained from the initial 15 codons and FIGURE 1 | Disruption of the eIF3 complex or its mRNA-entry channel arm provoke strong and overlapping effects on the translation of mRNAs across the transcriptome. (A) Structural model of eIF3 bound to the PIC (PDB 6GSM) viewed looking down at top of the small (40S) ribosomal subunit head so as to visualize the path of mRNA as it enters and exits the PIC (Llácer et al, 2021). The small (40S) ribosomal subunit is shown in grey, and the initiator tRNA and mRNA are shown in yellow and orange, respectively (with the path of the mRNA entering and exiting the PIC shown as a cartoon).…”