Cryo-EM studies of the interplay between uS2 ribosomal protein and leaderless mRNA during bacterial translation initiation.

E. coli ribosomes lacking ribosomal protein uS2 translate leaderless transcripts more efficiently than wild-type ribosomes 1 Without the Shine-Dalgarno sequence, translation initiation occurs on the mutant 70S complex rather than the 30S initiation complex. The ribosome uses the initiation codon (AUG) and the first bases of the transcript, including a downstream box (DB) sequence, as recognition signals. We used cryo-EM to solve the structures of the uS2-deficient 70S ribosome and the wild-type 70S complex bound to leaderless mRNA (lmRNA) in the presence of fmet-tRNA fMet . Importantly, the uS2-deficient 70S ribosome also lacks protein bS21. The anti-Shine-Dalgarno (aSD) region is supported by bS21, and the absence of the latter causes the aSD to divert from the normal mRNA exit pathway. We identified a π-stacking interaction between a monitor base (1493A) and the first base (A +4 ) of lmRNA following the AUG and potentially stabilizing it. Coulomb charge flow and swapping along with peristalsis-like dynamics within the mRNA entry channel are likely to facilitate the propagation of lmRNA through the ribosome.