Rethinking the Primacy of the Shine-Dalgarno Sequence: Leaderless mRNA Translation as a Default Mechanism in Bacterial Ribosome Recruitment

Abstract

The Shine-Dalgarno (SD) sequence has long been considered the canonical determinant of ribosome recruitment in prokaryotic translation initiation. However, accumulating evidence from cryo-electron microscopy, ribosome profiling, and comparative genomics challenges this dogma. Here, we demonstrate through quantitative ribosome footprinting and in vitro reconstitution assays in Escherichia coli and Thermus thermophilus that leaderless mRNAs (lmRNAs) engage the 30S subunit via a SD-independent pathway involving direct codon–anticodon pairing at the peptidyl (P) site. Mutational dissection of 16S rRNA helices h44 and h45 further reveals that conformational dynamics of the decoding center, rather than anti-SD complementarity, govern initiation fidelity across stress conditions. These findings necessitate a fundamental revision of the prokaryotic translation initiation model and carry significant implications for the design of synthetic riboregulatory circuits.