RlmQ is a m7G methyltransferase responsible for the enzymatic modification of m7G2601. (A) AlkAniline PE analysis of S. aureus 23S rRNA from USA300 JE2 WT and seven selected strains from the Nebraska collection with disrupted genes for methyltransferases of unknown function. AGCU dideoxy sequencing lanes. (Left) Lane 1, USA300 WT; lane 2, NE553; lane 3, NE1671; lane 4, NE1442; lane 5, NE189; lane 6, NE807; lane 7, NE1546 (corresponding to rlmQ-disruption mutant); lane 8, NE1337. (Right) Analysis of m7G restoration in the complemented mutant strain. Lane 1, S. aureus USA300 Lac WT-pCN38:wP:TT; lane 2, NE1546-pCN38:wP:TT; lane 3, NEB1546-pCN38:wP:rlmQ:TT. Red arrows indicate m7G modification. (B) Sequence alignment of S. aureus RlmQ and E. coli RlmI. Distinct domains are highlighted with colored bars as follows, starting from the amino terminus: PUA domain (blue bar over the sequence), EEHEE domain (magenta bar), β-hairpin (green bar), and MTase domain (red bar). Colored boxes spotlight the most significative signatures discriminating RlmQ and RlmI enzymes, as determined by BIS2 analysis (Dib and Carbone 2012). Red boxes represent cluster 1, with a P-value cutoff at 4.3 × 10−6, revealing the substitution of the conserved catalytic cysteine, typically found in m5C methyltransferases, by asparagine in RlmQ. Within this cluster, another substitution (His/Phe) correlates with the former, contributing to the distinctive motifs for the two classes of methyltransferases. Blue boxes denote cluster 2, with a P-value cutoff at 2.6 × 10−5, encompassing Lys/Met, Pro/Gly, and Arg/Trp covariations in RlmI/RlmQ, respectively. The brown box indicates cluster 3, with a P-value cutoff at 3.7 × 10−5, associating the fragment sequences DRSDVAVRKK/EKVRFK (RlmI/RlmQ) with the covariations of cluster 1. (C) Structure analysis illustrating the specific covariation clusters characterizing RlmQ (on the right) and RlmI (on the left). The structures of RlmQ and RlmI are sourced from the pdb 3VSE (Kita et al. 2013) and pdb 3C0K, respectively. The domains and motifs in the figure follow the same color code as in B. At the bottom, a comparison of the MTase active sites is presented for S. aureus RlmQ (orange background structure) and E. coli RlmI (cyan background structure). In the motif FSCSG, E. coli RlmI contains a cysteine (Cys), whereas S. aureus RlmQ has an asparagine (Asn) in the motif CTNAS. To facilitate comparison, the substrate S-adenosyl-l-homocysteine (SAH), present only in the RlmQ crystal structure, has also been modeled in the RlmI structure.
