FIGURE 3.
Kinetics and NTP specificity of the 5′ kinase reaction. (A) Reaction mixtures containing 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 10 mM MgCl2, 0.1 mM ATP, 2 mM DTT, 20 nM HORNA>p substrate, and 1 µM AtRNL-T1001A were incubated at 22°C. Aliquots (40 µL) were removed at the times specified and quenched immediately with an equal volume of 90% formamide, 30 mM EDTA. The products were resolved by urea-PAGE. An autoradiogram of the gel is shown in the top panel. Product formation, (pRNA>p/[pRNA>p + HORNA>p]) normalized to input RNA concentration is plotted as a function of reaction time. Each datum is the average of four separate experiments ±SEM. A nonlinear regression curve fit of the data to a single exponential is shown. (B) NTP dependence. Kinase reaction mixtures containing 20 nM HORNA>p substrate, 1 µM AtRNL-T1001A, and ATP or GTP as specified were incubated for 1 min at 22°C. The products were resolved by urea-PAGE. (C) NTP specificity. Reaction mixtures containing 20 nM HORNA>p substrate, 1 µM AtRNL-T1001A, and 0.1 mM ATP, CTP, GTP, UTP, or dATP as specified were incubated for 1 min at 22°C. The extents of product formation are shown; each datum in the graph is the average of three separate experiments ±SEM.