Fig. 1.
Rps3/uS3 plays a critical role in promoting mRNA binding at the 40S entry site and stabilizing the preinitiation complex at the start codon. (A) Model describing known conformational rearrangements of the PIC during scanning and start-codon recognition. (A, i) eIF1 and the scanning enhancers (SEs) in the C-terminal tail (CTT) of eIF1A stabilize an open conformation of the 40S subunit to which TC rapidly binds. Rps3 (uS3) is located on the solvent-exposed surface of the 40S subunit near the entry channel; the bulk of eIF3 binds on the solvent-exposed surface, with a prominent domain at the mRNA exit channel. (A, ii) The 43S PIC in the open conformation scans the mRNA for the start codon with Met-tRNAiMet bound in the POUT state. eIF2 can hydrolyze GTP to GDP•Pi, but release of Pi is blocked. (A, iii) On AUG recognition, Met-tRNAiMet moves from the POUT to the PIN state, clashing with eIF1 and the CTT of eIF1A, provoking displacement of the eIF1A CTT from the P site, dissociation of eIF1 from the 40S subunit, and Pi release from eIF2. The N-terminal tail (NTT) of eIF1A, harboring scanning inhibitor (SI) elements, adopts a defined conformation and interacts with the codon–anticodon helix. (Top) Arrows summarize that eIF1 and the eIF1A SE elements promote POUT and impede transition to the PIN state, whereas the SI element in the NTT of eIF1A stabilizes the PIN state. Results presented here indicate that uS3/Rps3 residues R116/R117, in contact with mRNA at the entry channel, stabilize the PIN state and also promote PIC interaction with mRNA at the entry channel, augmenting the role of eIF3 in PIC–mRNA interactions at the exit channel (adapted from ref. 1). (B) Alignment of a portion of uS3 sequences from diverse eukaryotes and Escherichia coli using Clustal Omega (www.ebi.ac.uk/Tools/msa/clustalo/). Boundaries of secondary structure on the top line refer to the Saccharomyces protein; symbolic summary of sequence conservation applies only to the upper seven eukaryotic sequences. Six conserved residues of Rps3 at the mRNA entry channel analyzed in this study are highlighted in black or pink. (C) Position of uS3/Rps3 in the yeast 48S PIC, and locations of conserved residues at the entry channel. The solvent-exposed surface of the partial yeast 48S PIC [Protein Data Bank (PDB) ID code 3J81] is depicted (Left) in cartoon format highlighting uS3 (green), mRNA (black), Met-tRNAiMet (pink), and rRNA residues of h18 or h34 that comprise the entry channel latch (blue). The boxed region is amplified (Right) where the six uS3/Rps3 residues analyzed here, which interact with mRNA or the rRNA latch, are highlighted in black or magenta (only for ease of visualization), shown in stick format, and labeled. Other ribosomal proteins and eIFs 1, 1A, and 2 are hidden for clarity.