RlmQ/RlmI evolution changes 23S methylation maps in S. aureus and E. coli. (A) Detection of nonmodified C1989 in S. aureus (upper panel) and m5C1962 in E. coli (lower panel) using mass spectrometry. The RNase H generated fragment including H70 and H71 of the 23S rRNAs has been further digested with RNase T1 before analysis. S. aureus MSMS spectrum (upper panel) contains three fragments including the sequence of interest ACCCGp showing no methylation. E. coli MSMS spectrum (lower panel) contains only the methylated fragment of interest AC[m5C]UGp. (B) 2D representation of the entire S. aureus 23S rRNA, zooming on the region containing H70 and H71, with C1962 (E. coli numbering, corresponding to S. aureus C1989). Red and black numbers indicate S. aureus and E. coli numbering, respectively. The red bar highlights the fragment analyzed by LC/MSMS. (C) Structure analysis of C1962 from the deposited S. aureus 50S cryo-EM map at 2.3 Å (Halfon et al. 2019) (map: EMD-10077; PDB: 6s0z) and E. coli deposited 70S cryo-EM map at 2 Å (Watson et al. 2020) (map: EMD-22586; PDB: 7k00). While a clear density for methylation of position 5 on C1962 is observed in E. coli 23S allowing building this modification in the fitted model, no extra density is present on S. aureus 23S.
