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. 2000 Feb 1;28(3):720-7. doi: 10.1093/nar/28.3.720.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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

Substances

  • Cations, Divalent
  • Escherichia coli Proteins
  • Nucleotides
  • Oligoribonucleotides
  • Organothiophosphorus Compounds
  • RNA Precursors
  • RNA, Bacterial
  • RNA, Catalytic
  • Cytosine
  • RNA, Transfer
  • Endoribonucleases
  • Ribonuclease P
  • ribonuclease P, E coli