Partial purification and characterization of nuclear ribonuclease P from wheat.
Ribonuclease P (RNase P) from wheat nuclei has been purified over 1000-fold, using wheat germ extract as starting material and a combination of poly(ethylenglycol) precipitation and column chromatography. The enzyme was shown to be of nuclear origin by its characteristic ionic requirements; for optimum activity it requires 0.5-1.5 mM Mg2+, which can be partly replaced by Mn2+. With about 100 kDa, wheat nuclear RNase P has the lowest molecular mass reported so far for a eukaryotic RNase P. The enzyme has an isoelectric point of 5.0 and a buoyant density of 1.34 g/ml in CsCl, suggesting the presence of a nucleic acid component; it is, however, insensitive against treatment with micrococcal nuclease. Wheat germ RNase P requires an intact tertiary structure of the pre-tRNA substrate; its cleavage efficiency is also influenced by the presence of an intron, and by the nature of the 3' terminus of the substrate. The apparent Km and Vmax for an intronless plant pre-tRNA(Tyr) are 10.3 nM and 1.12 fmol/min, respectively.
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Institut für Biochemie, Bayerische Julius-Maximilians-Universität, Biozentrum, Würzburg, Germany.
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D001483:Base Sequence; D002467:Cell Nucleus / Q000201:enzymology; D004722:Endoribonucleases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007526:Isoelectric Point; D007700:Kinetics; D008836:Micrococcal Nuclease; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D016337:RNA, Catalytic / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D018749:RNA, Plant / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012365:RNA, Transfer, Tyr / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043262:Ribonuclease P; D013379:Substrate Specificity; D014908:Triticum / Q000201:enzymology* / Q000235:genetics
D009838:Oligodeoxyribonucleotides; D012322:RNA Precursors; D016337:RNA, Catalytic; D018749:RNA, Plant; D012365:RNA, Transfer, Tyr; D004722:Endoribonucleases; D043262:Ribonuclease P; D008836:Micrococcal Nuclease