Identification of individual nucleotides in the bacterial ribonuclease P ribozyme adjacent to the pre-tRNA cleavage site by short-range photo-cross-linking

The bacterial RNase P ribozyme is a site-specific endonuclease that catalyz es the removal of pre-tRNA leader sequences to form the 5' end of mature tR NA. While several specific interactions between enzyme and substrate that d irect this process have been determined, nucleotides on the ribozyme that i nteract directly with functional groups at the cleavage site are not well-d efined. To identify individual nucleotides in the ribozyme that are in clos e proximity to the pre-tRNA cleavage site, we introduced the short-range ph otoaffinity cross-linking reagent 6-thioguanosine (s(6)G) at position +1 of tRNA and position -1 in a tRNA bearing a one-nucleotide leader sequence [t RNA(G-1)] and examined cross-linking in representatives of the two structur al classes of bacterial RNase P RNA (from Escherichia coli and Bacillus sub tilis). These photoagent-modified tRNAs bind with similar high affinity to both ribozymes, and the substrate bearing a single s(6)G upstream of the cl eavage (-1) site is cleaved accurately. Interestingly, s(6)G at position +1 of tRNA cross-links with high efficiency to homologous positions in J5/15 in both E. coli and B. subtilis RNase P RNAs, while s(6)G at position -1 of tRNA(G-1) cross-links to homologous nucleotides in J18/2. Both cross-links are detected over a range of ribozyme and substrate concentrations, and im portantly, ribozymes cross-linked to position -1 of tRNA(G-1) accurately cl eave the covalently attached substrate. These data indicate that the conser ved guanosine at the 5' end of tRNA is adjacent to A248 (E. coli) of J5/15, while the base upstream of the substrate phosphate is adjacent to G332 (E. coli) of J18/2 and, along with available biochemical data, suggest that th ese nucleotides play a direct role in binding the substrate at the cleavage site.