The Bacillus subtilis ywbD gene encodes RlmQ, the 23S rRNA methyltransferase forming m⁷G2574 in the A-site of the peptidyl transferase center

YwbD is the 23S rRNA methyltransferase forming m⁷G2574. (A) Fifty micrograms of total RNA extracted from E. coli WT was incubated with 1 µCi [methyl-³H] SAM and 100 ng of purified YwbD, RsmG, or TrmB of B. subtilis. After incubation, the samples were processed as described in Materials and Methods. (B) Twenty micrograms of total RNA extracted from B. subtilis WT (▴) or ΔywbD (●) cells, or rRNA (▪) and tRNA (♦) extracted from B. subtilis ΔywbD cells were incubated with 1 µCi [methyl-³H] SAM and 100 ng of purified YwbD. 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. (C) B. subtilis ΔywbD rRNA (10 µg) was incubated with 1 µg of purified YwbD and 1 µCi [methyl-³H] SAM at 37°C. After 30 min incubation, the reaction mixture was loaded onto 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 was measured in a scintillation counter. The number of cpm present in 23S and 16S rRNAs is indicated. (D) Autoradiography of 2D chromatograms of P1 hydrolysates of rRNA extracted from B. subtilis ΔywbD, in vitro methylated using YwbD and [methyl-¹⁴C] SAM. Modified nucleotides were analyzed by two-dimensional thin-layer chromatography (2D-TLC) on cellulose plates (Merck). The first dimension was developed with solvent A and the second dimension was developed with solvent B (left panel) or with solvent C (right panel). The radioactive spots were visualized by autoradiography. The nucleotides were identified using reference maps (Grosjean et al. 2007). Dotted circles show the migration of pA, pC, pG, and pU nucleotides used as ultraviolet markers. The spot corresponding to pm⁷G is indicated. (E) Autoradiography of 2D chromatograms of P1 hydrolysates of methylated WT 23S rRNA transcript (left panel) or (G2574A) 23S rRNA transcript (right panel) by YwbD in the presence of [methyl-¹⁴C] SAM. The second chromatography dimension was developed with solvent C. (F) Purified 70S and 50S ribosomal particles are poor substrates of YwbD. A total of 0.5 A260 units of 70S or 50S ribosomal particles were incubated for 30 min with 1 µg of purified YwbD and 1 µCi [methyl-³H] SAM. Incorporated radioactivity was measured by scintillation counting. RNA fr70S or fr50S are for RNA freed from, respectively, 70S or 50S ribosomal particles.