IF2 and unique features of initiator tRNA^fMet help establish the translational reading frame.

Translation begins at AUG, GUG, or UUG codons in bacteria. Start codon recognition occurs in the P site, which may help explain this first-position degeneracy. However, the molecular basis of start codon specificity remains unclear. In this study, we measured the codon dependence of 30S•mRNA•tRNA^fMet and 30S•mRNA•tRNA^Met complex formation. We found that complex stability varies over a large range with initiator tRNA^fMet, following the same trend as reported previously for initiation rate in vivo (AUG > GUG, UUG > CUG, AUC, AUA > ACG). With elongator tRNA^Met, the codon dependence of binding differs qualitatively, with virtually no discrimination between GUG and CUG. A unique feature of initiator tRNA^fMet is a series of three G-C basepairs in the anticodon stem, which are known to be important for efficient initiation in vivo. A mutation targeting the central of these G-C basepairs causes the mRNA binding specificity pattern to change in a way reminiscent of elongator tRNA^Met. Unexpectedly, for certain complexes containing fMet-tRNA^fMet, we observed mispositioning of mRNA, such that codon 2 is no longer programmed in the A site. This mRNA mispositioning is exacerbated by the anticodon stem mutation and suppressed by IF2. These findings suggest that both IF2 and the unique anticodon stem of fMet-tRNA^fMet help constrain mRNA positioning to set the correct reading frame during initiation.

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