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

(A) Mutations in the RNR motif of RNase P decrease the affinity of pre-tRNA^Asp. RNase P-bound and free pre-tRNA^Asp were separated using a gel filtration centrifuge column. RNase P was reconstituted with wild-type (triangles, solid line), R60A (squares, solid line), or R68A (circles, dotted line) P protein. The bound ³²P-labeled pre-tRNA^Asp (≤0.2 nM) was measured as a function of RNase P concentration in 50 mM MES, 50 mM Tris, 380 mM KCl, and 10 mM CaCl₂ (pH 6.0, 37°C). The values of K_D,pre-tRNA, determined from a fit of a binding isotherm to these data, are listed in Table 1. (B) Measurement of the effects of mutations on the stability of the initial RNase P•pre-tRNA (ES) complex. The affinity of wild-type (triangles, solid line), R60A (squares, solid line), and R62A (circles, dotted line) RNase P for pre-tRNA was measured as described in A except that the buffer contained 50 mM MES, 50 mM Tris, 200 mM KCl, 2 mM Co(NH₃)₆(III), and 10 mM DTT (pH 6.0, 37°C). Under these conditions, the ES conformer predominates. The values of K₁, determined from a fit of a binding isotherm to these data, are listed in Table 1.