The Bacillus subtilis open reading frame ysgA encodes the SPOUT methyltransferase RlmP forming 2′-O-methylguanosine at position 2553 in the A-loop of 23S rRNA

Localization of the position modified by YsgA. (A) Hybridization-protection studies to localize the site of methylation by YsgA. A total of 20 µg of methyl ³H-labeled 23S rRNA (see Materials and Methods) was hybridized with 100 pmol of oligonucleotide and digested by RNase T1. Samples were TCA-precipitated and radioactivity was measured using a scintillation counter; oligo Hyb-1 centered on 2553 (▴), oligo Hyb-2 centered on 1916 (♦), oligo Hyb-3 centered on 2251 (●), and oligo Hyb-4 centered on 2450 (▪). (B) YsgA is able to methylate the ribose of A but not C nor U when G2553 is mutated in 23S rRNA. Autoradiograms of 2D chromatograms of P1 hydrolysates of T7 transcripts of 23S rRNA bearing G2553, A2553, C2553, and U2553 methylated by YsgA in the presence of ¹⁴C-SAM. The methylated nucleotides were identified by comparison to reference maps (Grosjean et al. 2007).