A kinetic mechanism for cleavage of precursor tRNA(Asp) catalyzed by the RNA component of Bacillus subtilis ribonuclease P.
A kinetic mechanism is presented for the cleavage of Bacillus subtilis precursor tRNA(Asp) catalyzed by the RNA component of B. subtilis ribonuclease P (RNase P) under optimal conditions (50 mM Tris Cl (pH 8.0), 100 mM MgCl2, and 800 mM NH4Cl, 37 degrees 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-tRNA(Asp) to RNase P RNA rapidly (6.3 x 10(6) M-1 s-1), but slower than the diffusion-controlled limit; (2) cleavage of the phosphodiester bond with a rate constant of 6 s-1; (3) dissociation of products in a kinetically preferred pathway, with the 5' RNA fragment dissociating first (> or = 0.2 s-1) followed by rate-limiting tRNA dissociation (0.02 s-1); and (4) formation of a second conformer of RNase P RNA during the catalytic cycle that is less stable and binds pre-tRNA(Asp) significantly more slowly (7 x 10(4) M-1 s-1). 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.
Beebe
J A
JA
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Fierke
C A
CA
D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites; D004722:Endoribonucleases / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D012349:RNA, Transfer, Asp / Q000737:chemistry / Q000378:metabolism*; D043262:Ribonuclease P; D013816:Thermodynamics
D012329:RNA, Bacterial; D016337:RNA, Catalytic; D012349:RNA, Transfer, Asp; D004722:Endoribonucleases; D043262:Ribonuclease P