Magnesium Ions Are Required by Bacillus subtilis Ribonuclease P RNA for both Binding and Cleaving Precursor tRNAAsp

Abstract

The multiple roles Mg²⁺ plays in ribozyme-catalyzed reactions in stabilizing RNA structure, enhancing the affinity of bound substrates, and increasing catalysis are delineated for the RNA component of ribonuclease P (RNase P RNA) by a combination of steady-state kinetics, transient kinetics, and equilibrium binding measurements. Divalent metal ions cooperatively increase the affinity of Bacillus subtilis RNase P RNA for B. subtilis tRNA^Asp more than 10³-fold, consistent with at least two additional magnesium ions binding to the RNase P RNA·tRNA complex. Monovalent cations also decrease K_D^tRNA and reduce, but do not eliminate, the dependence on magnesium ions, demonstrating that nonspecific electrostatic shielding is not sufficient to explain the requirement for high salt. Both di- and monovalent cations promote the high affinity of tRNA by forming contacts in the binary complex that reduce the dissociation rate constant for tRNA. Additionally, the hyperbolic dependence of the hydrolytic rate constant on the concentration of magnesium with a K_1/2 ≈ 36 mM suggests that a third low-affinity divalent metal ion stabilizes the transition state for pre-tRNA cleavage. Furthermore, many (about 100) magnesium ions bind independently to RNase P RNA with higher affinity than the K_1/2 of any of the functionally characterized magnesium binding sites. Therefore, the magnesium binding sites that have differential affinity in either the “folded” species or binary complex are a small subset of the total number of associated magnesium ions. In summary, the importance of magnesium bound to RNase P RNA can be separated functionally into three crucial roles: at least three sites stabilize the folded RNA tertiary structure [Pan, T. (1995) Biochemistry 34, 902−909], at least two sites enhance the formation of complexes of RNase P RNA with pre-tRNA or tRNA, and at least one site stabilizes the transition state for pre-tRNA cleavage.