Partial purification of RNase P from Schizosaccharomyces pombe.
Ribonuclease P from the fission yeast Schizosaccharomyces pombe was partially purified using DEAE-cellulose and phosphocellulose column chromatography. The yeast RNase P enzyme cleaves Escherichia coli tRNATyr precursor to give tRNATyr containing its mature 5' end. The enzyme activity is inhibited after treatment with nucleases; this indicates the requirement of a nucleic acid component for activity. The enzyme purification was greatly facilitated by using a synthetically prepared radioactive ApApApCOH ligated to the 5'-terminal phosphate of E. coli tRNAfMet (ApApApCp-tRNA) substrate. (p denotes a [32P]phosphate group.) This substrate was cleaved by yeast RNase P to the mature tRNA and a tetranucleoside triphosphate ApApApCOH. The synthetic substrate allowed the utilization of a simple assay procedure measuring the trichloroacetic acid solubility of the ApApApC product, thus avoiding the more cumbersome gel electrophoric separation of reaction products.
D001203:Ascomycota / Q000201:enzymology*; D001483:Base Sequence; D004720:Endonucleases / Q000302:isolation & purification* / Q000378:metabolism; D029968:Escherichia coli Proteins; D007700:Kinetics; D008836:Micrococcal Nuclease; D012343:RNA, Transfer; D043262:Ribonuclease P; D012260:Ribonucleases / Q000302:isolation & purification* / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology*; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D012343:RNA, Transfer; D004720:Endonucleases; D012260:Ribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli; D008836:Micrococcal Nuclease