S. aureus ribosome contains m7G2601. (A) Mass spectrometry analysis of the RNase T1 derived fragment of S. aureus HG001 23S rRNA region spanning from U2598 to G2603 (corresponding to E. coli U2571 to G2576). The fragment has a mass of 1948.2 Da (m/z 973.62) and carries the sequence UAC[mG]CG > p based on MSMS analysis. (B) 2D representation of the entire S. aureus 23S rRNA, zooming on the region containing helix H90 with G2601 (corresponding to E. coli G2574). Red and black numbers indicate S. aureus and E. coli numbering, respectively. The red bar highlights the fragment analyzed by LC/MSMS. (C) Structural analysis of G2601 from the deposited S. aureus 50S cryo-EM map at 2.3 Å (Halfon et al. 2019) (map: EMD-10077; PDB: 6s0z). The density around N7 indicates the presence of the methyl group, which is not modeled in the atomic coordinates. (D) PE analysis of HG001 and USA300 (LAC) S. aureus strains, including controls for the AlkAlanine treatment. AGCU lanes represent dideoxy sequencing, NT stands for the nontreated sample, and the red arrow points to the reverse transcriptase stop due to the m7G-modified nucleotide. (E) Details of the accommodation corridor for aminoacyl-tRNA CCA-end on the E. coli ribosome structure, with the A-site tRNA (gray surface) in the accommodated state (Watson et al. 2020). The E. coli 23S rRNA helices (H69–H71, H90–H91) are shown: G2524 (red sphere), corresponding to m7G2601 in S. aureus, is located in H90 (green), while m5C1962 (yellow sphere), corresponding to C1989 in S. aureus, is in H70/H71 (bright orange). The mRNA (blue transparent surface) in the 30S decoding channel (DC) interacts with the anticodon of the A-site tRNA. (Sau) S. aureus, (Eco) E. coli.
