Paper ID: 348
1 National Institutes of Health (USA)
2 CSIC/Universidad de Salamanca (Spain)
3 University Duisburg-Essen (Germany)
In eukaryotic translation initiation, a preinitiation complex (PIC) comprised of the 40S ribosomal subunit, initiation factors 1, 1A and 3, and a ternary complex (TC) of eIF2-GTP and initiator tRNAi attaches to mRNA and scans the leader for an AUG codon in optimal context. eIF1 stimulates recruitment of TC with tRNAi bound in a state (POUT) suitable for scanning, and opposes transition to a more stable PIN state at the start codon, necessitating eIF1 release on AUG recognition. Structures of reconstituted PICs reveal an open 40S conformation at a near-cognate AUC codon, and a more closed configuration at AUG in which eIF1 shifts on the 40S and its β-hairpin loops are distorted to avoid clashing with tRNAi. Substitutions in eIF1 loop-2 that remove ionic repulsion or create attraction with tRNAi increase initiation at UUG codons (Sui- phenotype) and AUGs in poor context in vivo, and stabilize TC binding at UUG start codons in reconstituted PICs. Thus, the loop-2/tRNAi clash destabilizes the PIN state to disfavor suboptimal start codons. eIF2β contacts eIF1 and tRNAi exclusively in the open complex, and substitutions at the eIF2β/eIF1 interface confer Sui- phenotypes, indicating eIF1:eIF2β interactions also impede rearrangement to PIN at suboptimal start sites. In contrast, the unstructured N-terminal tail (NTT) of eIF1A contacts the codon:anticodon helix only in the closed/PIN state, and substituting its basic residues suppresses initiation at UUG codons (Ssu- phenotype) and destabilizes TC binding at UUG codons in vitro. Ribosome profiling of the eIF1A-R13P mutant reveals decreased initiation at AUGs in poor context genome-wide, implicating the NTT in selecting suboptimal start codons. Similar genetic/biochemical findings identify roles for ribosomal proteins at the mRNA exit (uS7) and entry (uS3) channels in stabilizing the closed/PIN conformation via contacts with eIF2α (uS7) or mRNA (uS3), to enable recognition of suboptimal start sites. eIF3 is a central player in PIC recruitment to mRNA, and we used model mRNAs lacking contacts with the 40S entry or exit channels to identify a critical role for eIF3a in stabilizing PIC:mRNA interactions at the exit channel, and an ancillary role at the entry channel that is functionally redundant with uS3 residues that contact mRNA. Thus, multiple interactions within the PIC serve to stabilize the open or closed states and set the proper stringency level for initiation at non-optimum start codons in vivo.