Figure 7.

Effect of Archease on the activity of active-site variants of RtcB in RNA ligation assays with GTP or ATP as a cofactor. (A) Reactions with GTP (0.10 mM) as a cofactor. (B) Reactions with ATP (0.10 mM) as a cofactor. Reaction mixtures included 100 nM Archease where indicated, and were incubated at 70°C for 30 min. (C) Graph of the ligation product obtained for each RtcB variant. Values are the mean ± SE for two separate experiments. (D) ATP-dependent K480A RtcB-catalyzed RNA ligation reactions titrated with increasing concentrations of Archease, as specified. ATP was included at 0.10 mM, and reaction mixtures were incubated at 70°C for 15 min. Values are the mean ± SE for three separate experiments. (E) Michaelis–Menten plot of reaction rate versus ATP cofactor concentration for K480A RtcB-catalyzed RNA ligation reactions under single-turnover conditions. Values are the mean ± SE for three separate experiments. (F) Single-turnover kinetics of ATP-dependent RNA ligation catalyzed by K480A RtcB with the inclusion of Archease (800 nM). Values are the mean ± SE for two separate experiments. Ligation reaction mixtures contained 50 mM Bis–Tris buffer (pH 7.0), NaCl (300 mM), MnCl₂ (0.25 mM), NTP as indicated, P. horikoshii RtcB (5 μM), 5′ RNA fragment (1.0 μM) and 3′ RNA fragment (1.0 μM).