Single-turnover ligation kinetics of catalysis by T. fusca RtcB. (A) Urea–polyacrylamide gel depicting ligation product formation and a plot of product formation over time fitted to a single-exponential equation. 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 (5 μM), and RNA (1 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. (B) Plot of ligation product formation during catalysis by T. fusca RtcB in reaction mixtures that included the indicated concentration of either P. horikoshii or T. thermophilus Archease. 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 (5 μM), and RNA (1 μM). Reaction mixtures were incubated at 45°C for 4.5 min. Values are the mean ± SD for two separate experiments.
