Mapping the active site of ribonuclease P RNA using a substrate containing a photoaffinity agent.
Ribonuclease P RNA is the catalytic moiety of the ribonucleoprotein enzyme that removes precursor sequences from 5'-ends of pre-tRNAs. A photoaffinity cross-linking agent was coupled to the substrate phosphate on which RNase P acts and used to map nucleotides in the vicinity of the catalytic site of this ribozyme. Mature tRNA(Phe) containing a 5'-thiophosphate was synthesized by transcription in vitro using phage T7 RNA polymerase in the presence of guanosine 5'-phosphorothioate. The photoagent (azidophenacyl) was coupled uniquely to the 5'-thiophosphate of the tRNA, the site of action by RNase P. The photoagent-containing tRNA binds to RNase P RNA and is cross-linked by UV irradiation to it at high efficiency (10-30%). Cross-linked conjugates are enzymatically inactive, consistent with the occupancy of the active site of the RNase P RNA by the tRNA. Reversal of the cross-link by phenylmercuric acetate restores activity. The sites of cross-linking in RNase P RNA were determined by primer extension. In order to identify generalities and detect idiosyncrasies, analyses were carried out using RNase P RNAs from three phylogenetically diverse organisms: Bacillus subtilis, Chromatium vinosum and Escherichia coli. In the context of a phylogenetic structure model, two regions of cross-linking are observed in all three RNAs. Two of the RNAs cross-link to a lesser extent at a third structural region and one of the RNAs is cross-linked to a small extent to a fourth region. All the sites of cross-linking between the substrate phosphate in tRNA and the RNase P RNAs are in the conserved core of the structure model, consistent with the importance of the cross-linked residues to the action of this RNA enzyme.
Burgin
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Department of Biology, Indiana University, Bloomington 47405.
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D000345:Affinity Labels; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002844:Chromatium / Q000201:enzymology / Q000235:genetics; D003432:Cross-Linking Reagents / Q000378:metabolism; D004722:Endoribonucleases / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D006157:Guanosine Monophosphate / Q000138:chemical synthesis / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes; D012329:RNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000235:genetics; D043262:Ribonuclease P; D013873:Thionucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D014158:Transcription, Genetic
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D015345:Oligonucleotide Probes; D012329:RNA, Bacterial; D012360:RNA, Transfer, Phe; D013873:Thionucleotides; C066524:guanosine 5'-monophosphorothioate; D006157:Guanosine Monophosphate; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli