A kinetic mechanism is presented for the cleavage of Bacillus subtilis precursor tRNAAsp catalyzed by the RNA component of B. subtilis ribonuclease P (RNase P) under optimal conditions (50 mM Tris C1 (pH 8.0), 100 mM MgC12, and 800 mM NH&l, 37 "C). This kinetic mechanism was derived from measuring pre-steady-state, steady-state, single-turnover, and binding kinetics using a combination of quench-flow, gel filtration, and gel shift techniques. A minimal kinetic description involves the following: (1) binding of pre-tRNAASp to RNase P RNA rapidly (6.3 X lo6 M-l s-l), but slower than the diffusion- controlled limit; (2) cleavage of the phosphodiester bond with a rate constant of 6 s-l; (3) dissociation of products in a kinetically preferred pathway, with the 5' RNA fragment dissociating first (10.2 s-l) followed by rate-limiting tRNA dissociation (0.02 s-l); and (4) formation of a second conformer of RNase P RNA during the catalytic cycle that is less stable and binds pre-tRNAAsP significantly more slowly (7 X lo4 M-l s-l). This scheme involves the isolation of individual steps in the reaction sequence, is consistent with steady-state data, and pinpoints the rate-determining step under a variety of conditions. This kinetic mechanism will facilitate a more accurate definition of the role of metals, pH, and the protein component in each step of the reaction and provide an essential background for understanding the influence of structural changes on the catalytic activity. Ribonuclease P (RNase Pl), a ribonucleoprotein complex, catalyzes the essential 5' maturation of precursor tRNA (pre- tRNA) (see reviews: Altman (1989) and Pace and Smith (1990)l. In diverse organisms, this enzyme is composed of two subunits: an RNA moiety (about 400 nucleotides) and a protein (about 120 amino acids) moiety. While both are essential for in vivo activity, the RNA component from bacterial RNase P is sufficient to catalyze the specific cleavage of pre-tRNA in vitro in the presence of high salt, demonstrating that the RNA component is catalytic. Other RNAs that can specifically cleave nucleic acids are known (see review: Pyle (1993)l; however, RNase P is the only RNA-containing nuclease that truly behaves as an enzyme in vivo since it catalyzes multiple turnovers and is unchanged during catalysis. Moreover, this enzyme plays an essential physiological role in the formation of functional tRNA molecules (Abelson, 1979; Robertson et al., 1972). Investigation of the chemical mechanism and structure-function properties of RNase P should lead to a greater understanding of the fundamentals of ribozyme function and aid in the design of more efficient ribozymes directed toward gene inactivation. RNase P catalyzes the cleavage of P-3'0 bonds to produce 5'-phosphate and 3'-hydroxyl end groups at a specific site on