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

YsgA is a methyltransferase that forms 2′O-methylguanosine in 23S rRNA in vitro. (A) 50 µg of total RNA extracted from B. subtilis wild-type (▪) or ΔysgA (●) cells or 50 µg of purified rRNA (○) or tRNA (□) from B. subtilis ΔysgA cells were incubated with 0.25 µM [methyl ³H] SAM and 100 ng of purified YsgA. After incubation, reaction mixtures were processed as described in Materials and Methods, and the radioactivity incorporated in the different RNA preparations was measured using a scintillation counter. (B) Total B. subtilis ΔysgA rRNA (20 µg) was incubated with 1 µg of purified YsgA and 1 µCi [methyl ³H] SAM at 37°C. After 30 min incubation, the reaction mixture was loaded in two slots of a 1% agarose gel. After migration, the bands corresponding to 23S and 16S rRNA were cut out of the gel and melted by heating. Scintillation cocktail was added, and radioactivity measured in a scintillation counter. The number of cpm present in 23S and 16S rRNAs is indicated. (C) Autoradiography of 2D chromatograms of P1 hydrolysates of ¹⁴C-methylated rRNA. Dotted circles show the migration of pA, pC, pG, and pU nucleotides used as ultraviolet markers. The methylated nucleotides were identified by comparison to reference maps (Grosjean et al. 2007).