PMID:10637323
| Citation |
Warnecke, JM, Sontheimer, EJ, Piccirilli, JA and Hartmann, RK (2000) Active site constraints in the hydrolysis reaction catalyzed by bacterial RNase P: analysis of precursor tRNAs with a single 3'-S-phosphorothiolate internucleotide linkage. Nucleic Acids Res. 28:720-7 |
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| Abstract |
Endonucleolytic processing of precursor tRNAs (ptRNAs) by RNase P yields 3'-OH and 5'-phosphate termini, and at least two metal ions are thought to be essential for catalysis. To determine if the hydrolysis reaction catalyzed by bacterial RNase P (RNAs) involves stabilization of the 3'-oxyanion leaving group by direct coordination to one of the catalytic metal ions, ptRNA substrates with single 3'- S -phosphorothiolate linkages at the RNase P cleavage site were synthesized. With a 3'- S -phosphorothiolate-modified ptRNA carrying a 7 nt 5'-flank, a complete shift of the cleavage site to the next unmodified phosphodiester in the 5'-direction was observed. Cleavage at the modified linkage was not restored in the presence of thiophilic metal ions, such as Mn(2+)or Cd(2+). To suppress aberrant cleavage, we also constructed a 3'- S -phosphorothiolate-modified ptRNA with a 1 nt 5'-flank. No detectable cleavage of this substrate was seen in reactions catalyzed by RNase P RNAs from Escherichia coli and Bacillus subtilis, independent of the presence of thiophilic metal ions. Ground state binding of modified ptRNAs was not impaired, suggesting that the 3'- S -phosphorothiolate modification specifically prevents formation of the transition state, possibly by excluding catalytic metal ions from the active site. |
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| Keywords |
Bacillus subtilis/enzymology; Bacillus subtilis/genetics; Base Sequence; Binding Sites; Cations, Divalent/metabolism; Cytosine/chemistry; Cytosine/metabolism; Endoribonucleases/chemistry; Endoribonucleases/genetics; Endoribonucleases/metabolism; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins; Hydrolysis; Kinetics; Models, Chemical; Molecular Weight; Nucleic Acid Conformation; Nucleotides/chemical synthesis; Nucleotides/chemistry; Nucleotides/genetics; Nucleotides/metabolism; Oligoribonucleotides/chemical synthesis; Oligoribonucleotides/chemistry; Oligoribonucleotides/genetics; Oligoribonucleotides/metabolism; Organothiophosphorus Compounds/chemical synthesis; Organothiophosphorus Compounds/chemistry; Organothiophosphorus Compounds/metabolism; RNA Precursors/chemical synthesis; RNA Precursors/chemistry; RNA Precursors/genetics; RNA Precursors/metabolism; RNA Processing, Post-Transcriptional; RNA, Bacterial/chemistry; RNA, Bacterial/genetics; RNA, Bacterial/metabolism; RNA, Catalytic/chemistry; RNA, Catalytic/genetics; RNA, Catalytic/metabolism; RNA, Transfer/chemical synthesis; RNA, Transfer/chemistry; RNA, Transfer/genetics; RNA, Transfer/metabolism; Ribonuclease P; Substrate Specificity |
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