FIGURE 5.

NTP donor specificity of the KlaTrl1 5′ kinase reaction. (A) Reaction mixtures (20 µL) containing 50 mM Tris-HCl, pH 8.0, 2 mM DTT, 10 mM MgCl₂, 20 nM 3′ ³²P-labeled 10-mer _HORNA>p substrate (depicted at the bottom of the panel with the ³²P-label denoted by •), 1 µM KlaTrl1-(385–813), and either no added NTP (lane –) or 0.1 µM of the indicated NTP were incubated at 22°C for 1 min, then quenched with an equal volume of 90% formamide, 30 mM EDTA. The products were analyzed by urea-PAGE and visualized by autoradiography. Enzyme was omitted from the control reaction in the leftmost lane. (B,C) Reaction mixtures (20 µL) containing 50 mM Tris-HCl (pH 8.0), 2 mM DTT, 10 mM MgCl₂, 20 nM _HOR10>p, 1 µM KlaTrl1-(385–813), and either 0.1, 1, 10, or 100 µM of the indicated NTP (either ATP, CTP, GTP, or UTP in panel B) or dNTP (dATP, dCTP, dGTP, dTTP, or dUTP in panel C) were incubated at 22°C for 1 min. The products were analyzed by urea-PAGE. The extents of 5′ phosphorylation were quantified by scanning the gel and are plotted in bar graph format as a function of NTP or dNTP concentration. Each datum is the average of three separate experiments ±SEM.