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.