The molecular mechanisms that govern translation initiation to ensure accuracy remain unclear. Here, we provide evidence that the subunit-joining step of initiation is controlled in part by a conformational change in the 1408 region of helix h44. First, chemical probing of 30S initiation complexes formed with either a cognate (AUG) or near-cognate (AUC) start codon shows that an IF1-dependent enhancement at A1408 is reduced in the presence of AUG. This change in reactivity is due to a conformational change rather than loss of IF1, because other portions of the IF1 footprint are unchanged and high concentrations of IF1 fail to diminish the reactivity difference seen at A1408. Second, mutations in h44 such as A1413C stimulate 50S docking and cause reduced reactivity at A1408. Third, streptomycin, which has been shown by Rodnina and coworkers to stimulate 50S docking by reversing the inhibitory effects of IF1, also causes reduced reactivity at A1408. Collectively, these data support a model in which IF1 alters the A1408 region of h44 in a way that makes 50S docking unfavorable, and canonical codon-anticodon pairing in the P site restores h44 to a docking-favorable conformation. We also find that, in the absence of factors, the cognate 30S•AUG•fMet-tRNA ternary complex is >1000-fold more stable than the near-cognate 30S•AUC•fMet-tRNA complex. Hence, the selectivity of ternary complex formation is inherently high, exceeding that of initiation in vivo by more than 10-fold.