Archease titration of T. thermophilus RtcB and single-turnover ligation kinetics. (A) T. thermophilus RtcB-catalyzed RNA ligation reactions titrated with increasing concentrations of T. thermophilus Archease, as specified. 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). (RNA substrate is shown at top and has an internal 6-carboxyfluorescein label.) Reaction mixtures were incubated at 70°C for 1 min, and quenched with an equal volume of RNA gel-loading buffer (5× TBE containing 7 M urea, 20% v/v glycerol, and 15 mg/mL blue dextran). Reaction products were resolved by electrophoresis through an 18% w/v urea–polyacrylamide gel and visualized by fluorescence scanning of the 6-carboxyfluorescein label. (B) Single-turnover kinetics of catalysis by T. thermophilus RtcB in the absence of 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), RtcB (5 μM), and RNA (1 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. Ligation product formation over time is plotted and fitted to a single-exponential equation. (C) Single-turnover kinetics of catalysis by T. thermophilus RtcB in the presence of T. thermophilus Archease (2 μM). Ligation product formation over time is plotted and fitted to a single-exponential equation. Values are the mean ± SD for two separate experiments.
