Characterization of the CthHen1 RNA methyltransferase activity. (A) Methyl transfer to RNA. Reaction mixtures (10 μL) containing 25 mM Tris-HCl (pH 8.0), 5 mM DTT, 0.5 mM MnCl2, 20 μM [3H-CH3]AdoMet, 10 μM 24-mer RNA, and increasing concentrations of CthHen1 (0.06, 0.6, 1.25, 2.5, 5, and 9.8 μM, proceeding from left to right) were incubated for 60 min at 45°C and then quenched with EDTA. Aliquots of the mixtures were analyzed by PEI-cellulose TLC in 0.2 M ammonium sulfate. The 3H-labeled material was visualized by autoradiography. (B) Time course. Reaction mixtures containing 25 mM Tris-HCl (pH 8.5), 0.5 mM MnCl2, 20 μM [3H-CH3]AdoMet, 10 μM 24-mer RNA, and 4 or 8 μM CthHen1 were incubated at 45°C. Aliquots (10 μL) were withdrawn at the times specified and quenched immediately with EDTA. The extents of 3H-methyl transfer to RNA are plotted as a function of reaction time. (C) pH dependence. Reaction mixtures (10 μL) containing 25 mM Tris buffer (either Tris-acetate at pH 4.5–6.5 or Tris-HCl at pH 7.0–9.5), 0.5 mM MnCl2, 20 μM [3H-CH3]AdoMet, and 4 μM CthHen1 as specified were incubated for 30 min at 45°C. The extents of 3H-methyl transfer to RNA are plotted as a function of pH. (The pH values represent that of a 1M solution of Tris buffer at 22°C.) (D) Divalent cation dependence. Reaction mixtures (10 μL) containing 25 mM Tris-HCl (pH 8.0), either 0, 0.2, 0.5, 1, 2, 5, or 10 mM MnCl2 or MgCl2 as specified, 20 μM [3H-CH3]AdoMet, 10 μM 24-mer RNA, and 5 μM CthHen1 were incubated for 30 min at 45°C. The extents of 3H-methyl transfer to RNA are plotted as a function of divalent cation concentration. (E) Methyl acceptor specificity. Reaction mixtures (10 μL) containing 25 mM Tris-HCl (pH 8.5), 0.5 mM MnCl2, 20 μM [3H-CH3]AdoMet, 8 μM CthHen1, and either 10 μM 24-mer RNA, 10 μM 24-mer DNA, or no added nucleic acid (none) were incubated for 30 min at 45°C. The extents of 3H-methyl transfer to the TLC origin are plotted.
