Blocking IF3N delays bacterial translation initiation

Bacterial translation initiation factor IF3 is composed of two distinct domains—a well-characterized C-terminal domain (IF3C), which enhances the speed and fidelity of translation initiation, and a less understood N-terminal domain (IF3N). In this study, we developed an aptamer (Apt ³⁴³ ) that targets IF3N with the goal of elucidating its contribution to translation initiation. Rapid kinetics assays revealed that Apt ³⁴³ reduces the rate of IF3 association with the 30S ribosomal subunit by 13-fold, while inducing a pronounced rearrangement of both IF3 domains on the 30S. These changes compromise IF2-, mRNA-, and fMet-tRNA ^fMet -dependent movements of IF3, delaying 30S initiation complex (30S IC) formation by up to two orders of magnitude. Cryo-EM analysis suggests that Apt ³⁴³ may sterically clash with fMet-tRNA ^fMet , thereby perturbing the canonical pathway by which the initiator tRNA is accommodated after IF2-dependent recruitment and prior to start codon decoding. However, once the 30S IC is formed, blocking IF3N does not prevent 50S subunit joining or 70S IC assembly. Collectively, these findings support a role for IF3N in enhancing an efficient path for fMet-tRNA ^fMet accommodation towards the 30S IC and promoting IF3C displacement to unlock 50S recruitment. Moreover, this aptamer-based strategy offers a valuable tool for dissecting domain-specific activities of multidomain factors within complex environments such as the initiating ribosome.