Activation of T. thermophilus RtcB by P. horikoshii Archease. (A) T. thermophilus RtcB-catalyzed RNA ligation reactions titrated with increasing concentrations of P. horikoshii 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). Reaction mixtures were incubated at 70°C for 10 min, and quenched with an equal volume of RNA gel-loading buffer. (B) Single-turnover kinetics of catalysis by T. thermophilus RtcB in the presence of P. horikoshii 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), 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. Values are the mean ± SD for two separate experiments.
