Identification by modification-interference of purine N-7 and ribose 2'-OH groups critical for catalysis by bacterial ribonuclease P.
The RNA subunit of bacterial ribonuclease P is a catalytic RNA that cleaves precursor tRNAs to generate mature tRNA 5' ends. A self-cleaving RNase P RNA-substrate conjugate was used in modification-interference analysis to identify purine N-7 and ribose 2'-hydroxyl functional groups that are critical to catalysis. We identify six adenine N-7 groups and only one 2'-hydroxyl that, when substituted with 7-deazaadenine or 2'-deoxy analogues, respectively, reduce the RNase P catalytic rate approximately 10-fold at pH 8 and limiting concentration of magnesium. Two sites of low-level interference by phosphorothioate modification were detected in addition to the four sites of strong interference documented previously. These modification-interference results, the absolute phylogenetic conservation of these functional groups in bacterial RNase P RNA, their proximity to the substrate-phosphate in the tertiary structure of the ribozyme-substrate complex, and the importance of some of the sites for binding of catalytic magnesium all implicate these functional groups as components of the RNase P active site. Five of the 7-deazaadenine interferences are suppressed at pH 6, where the hydrolytic step is rate-limiting, or at saturating concentrations of magnesium. We propose, therefore, that these base functional groups are specifically engaged in the catalytic center of RNase P RNA, possibly by involvement in magnesium-dependent folding. One 7-deazaadenine interference and one 2'-deoxy-interference, although partially suppressed at pH 6, are not suppressed at saturating magnesium concentrations. This implicates these groups in magnesium-independent folding of the catalytic substructure of the ribozyme.
Department of Plant and Microbial Biology, University of California, Berkeley 94720-3102, USA.
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D001483:Base Sequence; D001665:Binding Sites; D003854:Deoxyribonucleotides / Q000737:chemistry; D004722:Endoribonucleases / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D006151:Guanosine / Q000031:analogs & derivatives / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D011687:Purines / Q000737:chemistry; D012322:RNA Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012329:RNA, Bacterial / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D043262:Ribonuclease P; D013873:Thionucleotides; D014372:Tubercidin / Q000737:chemistry
D003854:Deoxyribonucleotides; D011687:Purines; D012322:RNA Precursors; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D013873:Thionucleotides; D006151:Guanosine; C068218:7-deazaguanosine; D012343:RNA, Transfer; D004722:Endoribonucleases; D043262:Ribonuclease P; D008274:Magnesium; D014372:Tubercidin