Ligation reactions under multiple-turnover conditions. (A) Urea-polyacrylamide gel depicting ligation product formation during catalysis by T. thermophilus RtcB under multiple-turnover conditions in the absence and presence of T. thermophilus Archease (2 μM). Reactions were performed in 50 mM Tris–HCl buffer (pH 7.4) containing NaCl (300 mM), MnCl2 (0.5 mM), GTP (0.1 mM), RtcB (0.5 μM), and RNA (2 μM). Reaction mixtures were incubated at 70°C, and aliquots were removed and quenched at the indicated times by adding an equal volume of RNA gel-loading buffer. (B) Urea–polyacrylamide gel depicting ligation product formation during catalysis by T. fusca RtcB under multiple-turnover conditions. Reactions were performed in 50 mM Tris–HCl buffer (pH 7.4) containing NaCl (300 mM), MnCl2 (0.5 mM), GTP (0.1 mM), RtcB (0.5 μM), and RNA (2 μM). Reaction mixtures were incubated at 45°C, and aliquots were removed and quenched at the indicated times by adding an equal volume of RNA gel-loading buffer. (C) Plots of ligation product formation during catalysis by T. thermophilus RtcB in the presence of T. thermophilus Archease (closed circles) and ligation product formation during catalysis by T. fusca RtcB (open diamonds) under multiple-turnover conditions. Plotted values were obtained from the above gels. Values are the mean ± SD for two separate experiments. (D) A plot of ligation product formation by T. fusca RtcB under multiple-turnover conditions in reactions that included either P. horikoshii or T. thermophilus Archease (8 μM). Reactions were performed in 50 mM Tris–HCl buffer (pH 7.4) containing NaCl (300 mM), MnCl2 (0.5 mM), GTP (0.1 mM), T. fusca RtcB (0.5 μM), and RNA (2 μM). Reaction mixtures were incubated at 45°C for 1 h. Values are the mean ± SD for two separate experiments.
