Figure 4.

Characterization of TbCom1 RNA 2′-O methyltransferase activity. (A) Protein titration. Reaction mixtures (10 μl) containing 50 mM Tris–HCl (pH 7.5), 5 mM DTT, 50 μM AdoMet, 1 pmol of either cap 1-, cap 0- or unmethylated cap RNA-IV, and TbCom1 as specified were incubated for 30 min at 27°C. Products were subjected to nuclease P1 digestion and analyzed by TLC. The extent of 2′-O methylation at position +2 is plotted as a function of input enzyme. (B) AdoMet dependence. Reaction mixtures (10 μl) contained 50 mM Tris–HCl (pH 7.5), 5 mM DTT, 0.1 μM of cap 0 RNA-IV, 4 pmol TbCom1, and AdoMet as indicated. Formation of pGm is plotted as a function of AdoMet concentration. (C) Inhibition by AdoHcy. Reaction mixtures (10 μl) contained 50 mM Tris–HCl (pH 7.5), 5 mM DTT, 0.1 μM m⁷GpppRNA, 50 μM AdoMet, 4 pmol TbCom1, with the indicated amounts of AdoHcy. The extent of methylation is plotted as a function of AdoHcy concentration. (D) pH dependency. Reaction mixtures (10 μl) containing 50 mM Tris buffer [either Tris–acetate (pH 5.0), 5.5, 6.5, 7.0 or Tris–HCl (pH 6.5, 7.0, 7.5, 8.0, 8.5, 9.0)], 5 mM DTT, 0.1 μM of cap 0 RNA-IV and 4 pmol of TbCom1. The extent of 2′-O methylation is plotted as function of pH.