title: Chloroplast ribonuclease P does not utilize the ribozyme-type pre-tRNA cleavage mechanism.
abstract: The transfer RNA 5' maturation enzyme RNase P has been characterized in Bacteria, Archaea, and Eukarya. The purified enzyme from all three kingdoms is a ribonucleoprotein containing an essential RNA subunit; indeed, the RNA subunit of bacterial RNase P RNA is the sole catalytic component. In contrast, the RNase P activity isolated from spinach chloroplasts lacks an RNA component and appears to function as a catalytic protein. Nonetheless, the chloroplast enzyme recognizes a pre-tRNA substrate for E. coli RNase P and cleaves it as efficiently and precisely as does the bacterial enzyme. To ascertain whether there are differences in catalytic mechanism between an all-RNA and an all-protein RNase P, we took advantage of the fact that phosphodiester bond selection and hydrolysis by the E. coli RNase P ribozyme is directed by a Mg2+ ion coordinated to the nonbridging pro-Rp oxygen of the scissile bond, and is blocked by sulfur replacement of this oxygen. We therefore tested the ability of the chloroplast enzyme to process a precursor tRNA containing this sulfur substitution. Partially purified RNase P from spinach chloroplasts can accurately and efficiently process phosphorothioate-substituted pre-tRNAs; cleavage occurs exclusively at the thio-containing scissile bond. The enzymatic throughput is fivefold slower, consistent with a general chemical effect of the phosphorothioate substitution rather than with a metal coordination deficiency. The chloroplast RNase P reaction mechanism therefore does not involve a catalytic Mg2+ bonded to the pro-Rp phosphate oxygen, and hence is distinct from the mechanism of the bacterial ribozyme RNase P.
authors.affiliation: Department of Molecular Biosciences, University of Kansas, Lawrence 66045-2106, USA.
authors.forename: B C
authors.initials: BC
authors.lastname: Thomas
authors.forename: X
authors.initials: X
authors.lastname: Li
authors.forename: P
authors.initials: P
authors.lastname: Gegenheimer
mesh_terms: D001483:Base Sequence; D002384:Catalysis; D002736:Chloroplasts / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009946:Organothiophosphorus Compounds / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012360:RNA, Transfer, Phe / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012260:Ribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D018724:Spinacia oleracea / Q000166:cytology / Q000201:enzymology*; D013379:Substrate Specificity; D013455:Sulfur / Q000378:metabolism; D013816:Thermodynamics; D015003:Yeasts / Q000235:genetics; D003313:Zea mays / Q000235:genetics
chemical_list: D009946:Organothiophosphorus Compounds; D012322:RNA Precursors; D012360:RNA, Transfer, Phe; C407303:RNase P, spinach; D013455:Sulfur; D012260:Ribonucleases; D008274:Magnesium; D010100:Oxygen