IF2 and unique features of initiator tRNA 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 and 30S•mRNA•tRNA complex formation. We found that complex stability varies over a large range with initiator tRNA, following the same trend as reported previously for initiation rate in vivo (AUG > GUG, UUG > CUG, AUC, AUA > ACG). With elongator tRNA, the codon dependence of binding differs qualitatively, with virtually no discrimination between GUG and CUG. A unique feature of initiator tRNA 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. Unexpectedly, for certain complexes containing fMet-tRNA, 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 help constrain mRNA positioning to set the correct reading frame during initiation.