Translation initiation factor 3 antagonizes authentic start codon selection on leaderless mRNAs

In this study, we have examined the influence of initiation factors on tran slation initiation of leaderless mRNAs whose 5'-terminal residues are the A of the AUG initiating codon. A 1:1 ratio of initiation factors to ribosome s abolished ternary complex formation at the authentic start codon of diffe rent leaderless mRNAs. Supporting this observation, in vitro translation as says using limiting ribosome concentrations with competing leaderless lambd a cl and Escherichia coil ompA mRNAs, the latter containing a canonical rib osome binding site, revealed reduced cl synthesis relative to OmpA in the p resence of added initiation factors. Using in vitro toeprinting and in vitr o translation assays, we show that this effect can be attributed to IF3. Mo reover, in vivo studies revealed that the translational efficiency of a lea derless reporter gene is decreased with increased IF3 levels. These studies are corroborated by the observed increased translational efficiency of a l eaderless reporter construct in an infC mutant strain unable to discriminat e against non-standard start codons. These results suggest that, in the abs ence of a leader or a Shine-Dalgarno sequence, the function(s) of IF3 limit s stable 30S ternary complex formation.