Abstract
SummaryIn bacterial translational initiation, three initiation factors (IFs 1–3) enable the selection of initiator tRNA and the start codon in the P site of the 30S ribosomal subunit. Here, we report 11 single-particle cryo-electron microscopy (cryoEM) reconstructions of the complex of bacterial 30S subunit with initiator tRNA, mRNA, and IFs 1–3, representing different steps along the initiation pathway. IF1 provides key anchoring points for IF2 and IF3, thereby enhancing their activities. IF2 positions a domain in an extended conformation appropriate for capturing the formylmethionyl moiety charged on tRNA. IF3 and tRNA undergo large conformational changes to facilitate the accommodation of the formylmethionyl-tRNA (fMet-tRNAfMet) into the P site for start codon recognition.
Highlights
Translational initiation is highly regulated and involves initiation factor (IF)-mediated positioning of an initiator tRNA over an mRNA start codon at the P site of the small ribosomal subunit
Maps derived from sample 1 are named from pre-initiation complex (PIC) 1–4, with the numbering intended to reflect a plausible order of events in the initiation process
We suggest that PIC-1C is a good candidate for a state just prior to the binding of the fMet-tRNAfMet, as it has an open conformation of 30S with a widened P site, and G1338 and A1339 of 16S rRNA are correctly positioned for interactions with the three highly conserved G:C base pairs in the anticodon stem loop (ASL) (Figure S5D)
Summary
Translational initiation is highly regulated and involves initiation factor (IF)-mediated positioning of an initiator tRNA over an mRNA start codon at the P site of the small ribosomal subunit. Translational initiation is controlled by just three IFs, which bind cooperatively to the small ribosomal subunit (30S). IF1 is a small protein that binds in the 30S A site. IF2 is a large GTPase that helps recruit formylmethionyl-tRNA (fMettRNAfMet), with a C-terminal (C2) domain that recognizes the N-formylmethionine moiety. IF3 is a two-domain protein that plays a important role in the fidelity of selection of tRNAfMet and correct start codon. Eukaryotic initiation is far more complex, each of these three IFs has a homolog in eukaryotes and in archaea
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