Extended Shine-Dalgarno motifs govern translation initiation inStaphylococcus aureus

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 inS. aureus, revealing distinct features of initiation alongside numerous unannotated small ORFs. Our analysis, combined with cryo-EM of a native mRNA-ribosome complex, shows thatS. aureusrelies on extended, start codon proximal Shine-Dalgarno (SD) interactions, creating specificity against phylogenetically distant bacteria. Several naturalS. aureusinitiation sites are not correctly decoded byE. coliribosomes. We identify new and conserved non-canonical start codons, whose regulatory initiation sites contain these characteristic extended SD sequence motifs. Finally, we characterize a striking example of uORF-mediated translational control inS. aureus, demonstrating that translation of a novel 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.