Purification and characterization of the nuclear ribonuclease P of Aspergillus nidulans.
In Aspergillus nidulans, the nuclear ribonuclease P was separated from its mitochondrial counterpart by Q-Sepharose chromatography, and a precursor-tRNA(His) processing assay system was used to discriminate nuclear ribonuclease P activity from the mitochondrial counterpart. The nuclear ribonuclease P was purified to near homogeneity from whole-cell extracts. A 2150-fold purification with a yield of 2.3% was achieved by five types of chromatography including tRNA affinity chromatography and glycerol gradient velocity sedimentation. This enzyme, which had a molecular mass of 580 kDa determined by both glycerol-gradient sedimentation analysis and gel-permeation chromatography, appeared to be composed of seven polypeptides and an RNA molecule. Seven polypeptides, with masses of 125, 85, 45, 33, 30, 21, 19 kDa, were consistently copurified with nuclear ribonuclease P activity through MonoS and tRNA affinity chromatography and in a glycerol gradient. As judged by a micrococcal-nuclease-sensitivity assay, nuclear ribonuclease P required an RNA component for its activity, as do other ribonuclease Ps. Analysis of the radiolabeled 5' end of RNAs copurified with nuclear ribonuclease P implied that RNA molecules in the purified nuclear ribonuclease P originated from a common RNA molecule, the putative RNA molecule of nuclear ribonuclease P. Comparison of the two ribonuclease Ps in A. nidulans showed that the protein and RNA components of the nuclear ribonuclease P were different from those of the mitochondrial counterpart.
Han
S J
SJ
Department of Microbiology, College of Natural Sciences, Korea.
Lee
B J
BJ
Kang
H S
HS
D001233:Aspergillus nidulans / Q000201:enzymology*; D002853:Chromatography, Liquid / Q000379:methods; D004722:Endoribonucleases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology; D009687:Nuclear Proteins / Q000302:isolation & purification; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000737:chemistry; D016337:RNA, Catalytic / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012354:RNA, Transfer, His / Q000737:chemistry; D043262:Ribonuclease P; D012261:Ribonucleoproteins / Q000302:isolation & purification; D014158:Transcription, Genetic
D009687:Nuclear Proteins; D012322:RNA Precursors; D016337:RNA, Catalytic; D012354:RNA, Transfer, His; D012261:Ribonucleoproteins; D004722:Endoribonucleases; D043262:Ribonuclease P