title: Active site constraints in the hydrolysis reaction catalyzed by bacterial RNase P: analysis of precursor tRNAs with a single 3'-S-phosphorothiolate internucleotide linkage.
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.
authors.affiliation: Medizinische Universität zu Lübeck, Institut für Biochemie, Ratzeburger Allee 160, D-23538 Lübeck, Germany.
authors.forename: J M
authors.initials: JM
authors.lastname: Warnecke
authors.forename: E J
authors.initials: EJ
authors.lastname: Sontheimer
authors.forename: J A
authors.initials: JA
authors.lastname: Piccirilli
authors.forename: R K
authors.initials: RK
authors.lastname: Hartmann
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism; D003596:Cytosine / Q000737:chemistry / Q000378:metabolism; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009843:Oligoribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009946:Organothiophosphorus Compounds / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D012322:RNA Precursors / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012343:RNA, Transfer / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043262:Ribonuclease P; D013379:Substrate Specificity
chemical_list: D002413:Cations, Divalent; D029968:Escherichia coli Proteins; D009711:Nucleotides; D009843:Oligoribonucleotides; D009946:Organothiophosphorus Compounds; D012322:RNA Precursors; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D003596:Cytosine; D012343:RNA, Transfer; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli