Abstract Regulation of translation initiation is central to bacterial adaptation, but species-specific mechanisms remain poorly understood. We present high-resolution mapping of translation start sites in Staphylococcus aureus , revealing distinct features of initiation alongside numerous unannotated small ORFs. Our analysis, combined with cryo-EM of a native mRNA-ribosome complex, shows that S. aureus relies on extended, start codon proximal Shine-Dalgarno (SD) interactions, creating specificity against phylogenetically distant bacteria. Several natural S. aureus initiation sites are not correctly decoded by E. coli ribosomes. We identify new and conserved non-canonical start codons, whose regulatory initiation sites contain these characteristic extended SD sequence motifs. Finally, we characterize a novel example of uORF-mediated translational control in S. aureus , demonstrating that translation of a small leader peptide modulates expression of a key biofilm regulator. The described mechanism involves codon rarity, ribosome pausing, and arginine availability, linking nutrient sensing to biofilm formation in this major human pathogen.
Kohl et al. (Thu,) studied this question.