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 catalyzes the removal of pre-tRNA leader sequences to form the 5' end of mature tRNA. While several specific interactions between enzyme and substrate that direct this process have been determined, nucleotides on the ribozyme that interact directly with functional groups at the cleavage site are not well-defined. To identify individual nucleotides in the ribozyme that are in close proximity to the pre-tRNA cleavage site, we introduced the short-range photoaffinity cross-linking reagent 6-thioguanosine (s6G) at position +1 of tRNA and position -1 in a tRNA bearing a one-nucleotide leader sequence [tRNA(G-1)] and examined cross-linking in representatives of the two structural classes of bacterial RNase P RNA (from Escherichia coli and Bacillus subtilis). These photoagent-modified tRNAs bind with similar high affinity to both ribozymes, and the substrate bearing a single s6G upstream of the cleavage (-1) site is cleaved accurately. Interestingly, s6G 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 s6G 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 importantly, ribozymes cross-linked to position -1 of tRNA(G-1) accurately cleave the covalently attached substrate. These data indicate that the conserved 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 these nucleotides play a direct role in binding the substrate at the cleavage site.
Christian
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Center for RNA Molecular Biology, Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
McPheeters
D S
DS
Harris
M E
ME
D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D006151:Guanosine / Q000031:analogs & derivatives / Q000378:metabolism; D006868:Hydrolysis; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D019920:Photoaffinity Labels / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D043262:Ribonuclease P; D013872:Thionucleosides / Q000378:metabolism
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D009711:Nucleotides; D019920:Photoaffinity Labels; D012322:RNA Precursors; D016337:RNA, Catalytic; D013872:Thionucleosides; D006151:Guanosine; D012343:RNA, Transfer; C005783:6-thioguanosine; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli