Purification and characterization of mitochondrial ribonuclease P from Aspergillus nidulans.
Mitochondrial ribonuclease (RNase) P from Aspergillus nidulans was purified to near homogeneity using whole-cell extract as the starting material. A 4400-fold purification with a yield of 5.2% was achieved by ammonium sulfate fractionation, heat treatment, and five types of column chromatography, including tRNA-affinity column chromatography. This enzyme, which has a molecular mass of 232 kDa determined by glycerol gradient sedimentation analysis, appears to be composed of seven polypeptides and an RNA moiety. These seven polypeptides consistently copurified with the RNase P activity through two ion-exchange chromatography columns and in a glycerol gradient. As judged by nuclease sensitivity, the enzyme requires an RNA component for its activity. The 3'-end-labeled RNAs that copurified with the enzyme displayed identical sequences but had variable lengths for the 5' end, indicating that they originated from a common RNA molecule, the putative RNA component of RNase P. The purified enzyme cleaved mitochondrial precursor tRNAHis, resulting in an 8-bp acceptor stem. This implies that the purified RNase P is a mitochondrial enzyme and that an additional guanylate residue (at position -1) of tRNAHis in A. nidulans mitochondria is generated by a mode that is analogous to the generation of their counterparts in prokaryotes and chloroplasts.
Lee
Y C
YC
Department of Microbiology, College of Natural Sciences, Seoul National University, Korea.
Lee
B J
BJ
Hwang
D S
DS
Kang
H S
HS
D001233:Aspergillus nidulans / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000235:genetics; D004722:Endoribonucleases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D016337:RNA, Catalytic / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012349:RNA, Transfer, Asp / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043262:Ribonuclease P
D017931:DNA Primers; D012322:RNA Precursors; D016337:RNA, Catalytic; D012331:RNA, Fungal; D012349:RNA, Transfer, Asp; D004722:Endoribonucleases; D043262:Ribonuclease P