FIGURE 4.

MimiTgs catalyzes a single methyl addition at cap guanine-N2. Reaction mixtures containing 50 mM Tris-HCl (pH 8.0), 5 mM DTT, 100 μM [³H-CH₃]AdoMet, 50 μM m⁷GDP, and either 1.8 μM purified recombinant human Tgs1(576–853) (hTgs1) or 0.8 μM MimiTgs were incubated for 30 min at 37°C. The MimiTgs reaction mixture was then split in half and supplemented with 1 mM “cold” AdoMet and either 7 μM hTgs1 or 7 μM MimiTgs, and incubation was continued for another 30 min. Aliquots were removed after the 30-min pulse-labeling phase and again after the cold AdoMet chase phase. The products were analyzed by PEI cellulose TLC in 0.1 M ammonium sulfate. The chromatogram was treated with Enhance and the ³H-labeled material was visualized by autoradiography.