The RNR motif of B. subtilis RNase P protein interacts with both PRNA and pre-tRNA to stabilize an active conformer

The effect of mutations in P protein on ES* formation and RNase P-catalyzed pre-tRNA cleavage in calcium. (A) Stopped-flow experiments measuring changes in the fluorescence of Fl-pre-tRNA^Asp (30 nM) with a 5-nt-long 5′ leader upon mixing with wild-type (0.25 μM) or R62A RNase P (0.215 μM) in 20 mM CaCl₂ (380 mM KCl, 50 mM MES, and 50 mM Tris at 37°C, pH 6). As previously described (Hsieh and Fierke 2009), the first and second phases correspond to the formation of the ES complex and ES* conformer, respectively. Equation 6 was fit to the data. (B) Time courses for cleavage of ³²P-labeled pre-tRNA (≤1 nM) catalyzed by RNase P (400 nM) reconstituted with WT (diamonds, solid line) or R62A (circles, dashed line) P protein under single turnover conditions in 10 mM CaCl₂ (pH 8) in buffer (50 mM MES, 50 mM Tris, 200 mM KCl at 37°C). (C) pH dependence of single turnover rate constants catalyzed by WT (circles, solid line), R60A (squares), and R62A (diamonds) RNase P in 10 mM CaCl₂ (pH 6–8) and buffer.