Kinetics of single-turnover ligation by MciRNL–AMP. (A) Ligation of 2′-OH RNA. Reaction mixtures (80 µL) containing 50 mM Tris-acetate, pH 6.5, 5 mM MgCl2, 0.1 µM 5′ 32P-labeled 10-mer RNA with a 3′-OH,2′-OH end, and 1 µM MciRNL were incubated at 37°C. The reactions were initiated by adding MciRNL to a prewarmed reaction mixture. Aliquots (10 µL) were withdrawn before adding enzyme (time 0) or at the times specified after enzyme addition, and quenched immediately with an equal volume of 95% formamide/50 mM EDTA. The products were analyzed by urea-PAGE. The distribution of radiolabeled RNAs is plotted as a function of reaction time. Each datum in the graph is an average of three independent experiments ± SEM, except for the 10-min time point, which is an average of two independent experiments. (B) Ligation of 2′-PO4 RNA. Individual replicate reaction mixtures (10 µL) containing 50 mM Tris-acetate, pH 6.5, 5 mM MgCl2, 0.1 µM 5′ 32P-labeled 10-mer RNA with a 3′-OH,2′-PO4 end, and 1 µM MciRNL were incubated at 37°C. The reactions were initiated by adding MciRNL to a prewarmed reaction mixture. The reactions were quenched after 5 or 15 sec with an equal volume of 95% formamide/50 mM EDTA. The products were analyzed by urea-PAGE. The distribution of radiolabeled RNAs is plotted as a function of reaction time. Each datum in the graph is an average of nine replicate reactions ± SEM. The data in panels A and B were fit by nonlinear regression in Prism to a unidirectional two-step kinetic mechanism.
