Protein component of Bacillus subtilis RNase P specifically enhances the affinity for precursor-tRNAAsp.
Ribonuclease P (RNase P) is an endonuclease that cleaves precursor tRNA to form the 5'-end of mature tRNA and is composed of a catalytic RNA subunit and a small protein subunit. The function of the protein component of Bacillus subtilis RNase P in catalysis of B. subtilis precursor tRNAAsp cleavage has been elucidated using steady-state kinetics, transient kinetics, and ligand affinity measurements to compare the functional properties of RNase P holoenzyme to RNase P RNA in 10 mM MgCl2, 100 mM NH4Cl. The protein component modestly affects several steps including </=10-fold increases in the rate constant for tRNA dissociation, the affinity of tRNA, and the rate constant for phosphodiester bond cleavage. However, the protein principally affects substrate binding, increasing the affinity of RNase P for pre-tRNAAsp by a factor of 10(4) as determined from both the ratio of the pre-tRNAAsp dissociation and association rate constants measured in 10 mM MgCl2 and a binding isotherm measured in 10 mM CaCl2 using gel filtration to separate enzyme-bound and free pre-tRNAAsp. Therefore, the main role of the protein component in RNase P is to facilitate recognition of pre-tRNA by enhancing the interaction between the enzyme and the 5'-precursor segment of the substrate, rather than stabilizing the tertiary structure of the folded RNA as has been observed for protein-facilitated group I intron self-splicing. Furthermore, the protein component maximizes the efficiency of RNase P under physiological conditions and minimizes product inhibition.
Kurz
J C
JC
Department of Biochemistry, Box 3711, Duke University Medical Center, Durham, North Carolina 27710, USA.
Niranjanakumari
S
S
Fierke
C A
CA
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012349:RNA, Transfer, Asp / Q000235:genetics / Q000378:metabolism*; D043262:Ribonuclease P; D013816:Thermodynamics
D001426:Bacterial Proteins; D012322:RNA Precursors; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D012349:RNA, Transfer, Asp; D004722:Endoribonucleases; D043262:Ribonuclease P; D002118:Calcium