A distinct mechanism governs translation initiation and its regulation in Staphylococcus aureus [Ribo-Seq]

Retapamulin-assisted ribosome profiling (Ribo-Ret) reveals translation initiation sites at a translatome-wide scale. Here we employed Ribo-Ret in S. aureus, utilizing either MNase or RNAse 1 for ribosomal protected footprint (RPF) generation, significanlty improving the obtainable resolution at translation start sites with the latter. The improved resolution helped to identify precise start codon selection, revealing distinct features of the S. aureus translation initiation mechanism as well as multiple unrecognized small open reading frames (sORFs). Furthermore, we identified novel regulators of the translation initiation step in this major human pathogen. Overall design: For the MNase as well as the RNase 1 Ribo-Ret experiments, two biological replicates were analyzed for Retapamulin-treated and untreated samples. These samples were processed and analyzed by our method for ribosome profiling in S. aureus.