Extended Shine-Dalgarno motifs govern translation initiation in Staphylococcus 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 in S. 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 striking example of uORF-mediated translational control in S. 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/660503v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1ea5094org.highwire.dtl.DTLVardef@505be5org.highwire.dtl.DTLVardef@1c14ad6org.highwire.dtl.DTLVardef@118549c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG