Ribonuclease P catalysis requires Mg2+ coordinated to the pro-RP oxygen of the scissile bond.
Ribonuclease P (RNase P) is an essential enzyme whose action produces the mature 5' termini of all cellular and organellar transfer RNA molecules. In bacteria, the catalytic subunit of RNase P is an RNA molecule which by itself can bind substrate pre-tRNA, select and hydrolyze the correct phosphodiester bond, and release product tRNA. The simple requirements of the reaction-a monovalent cation such as K+ or NH4+ and the divalent cation Mg2+ (or Mn2+)-have prompted proposals that all aspects of phosphodiester bond hydrolysis might be accomplished by one or more divalent metal cations coordinated to the enzyme or substrate. To precisely localize the ligands of catalytically-involved Mg2+, we assayed cleavage by Escherichia coli RNase P RNA of pre-tRNA in which specific pro-Rp phosphate oxygens were replaced with sulfur. RNase P cleavage was targeted to that bond, at or nearest to the normal cleavage site, at which Mg2+ or Mn2+ could be coordinated. Single-turnover kinetics demonstrated that the apparent rate constant for the hydrolysis event was determined quantitatively by the affinity of the divalent cation (Mg2+ or Mn2+) for the atom (O or S) at the pro-Rp position of the scissile phosphodiester bond. We propose a model for pre-tRNA cleavage in which an essential Mg2+ ion is coordinated directly to the pro-Rp phosphate oxygen and indirectly to two other ligands near the scissile bond: the upstream ribose 2'-hydroxyl and the downstream purine N7. This catalytic Mg2+ ion most likely positions and deprotonates a water molecule for in-line nucleophilic attack on the scissile bond phosphorus.
Chen
Y
Y
Department of Biochemistry, Molecular Genetics Program, University of Kansas, Lawrence 66045-2106, USA.
Li
X
X
Gegenheimer
P
P
D002384:Catalysis; D004722:Endoribonucleases / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins; D007700:Kinetics; D008274:Magnesium; D008345:Manganese / Q000502:physiology; D010100:Oxygen; D010758:Phosphorus; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000737:chemistry / Q000235:genetics; D043262:Ribonuclease P; D013455:Sulfur; D013873:Thionucleotides / Q000737:chemistry*
D029968:Escherichia coli Proteins; D012322:RNA Precursors; D016337:RNA, Catalytic; D012360:RNA, Transfer, Phe; D013873:Thionucleotides; D010758:Phosphorus; D008345:Manganese; D013455:Sulfur; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli; D008274:Magnesium; D010100:Oxygen