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A small metalloribozyme with a two-step mechanism

T Pan1, O C Uhlenbeck

  • 1Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309.

Nature
|August 13, 1992
PubMed

Insights

Lead ions (Pb2+) rapidly cleave specific RNA molecules with an internal loop, forming a 3' phosphomonoester. This RNA-catalyzed reaction mimics protein ribonuclease mechanisms previously unobserved in RNA.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • RNA Catalysis

Background:

  • Ribonucleases (RNases) are enzymes that catalyze the degradation of RNA.
  • Many protein RNases employ a two-step catalytic mechanism involving a cyclic phosphodiester intermediate.
  • RNA molecules themselves can act as catalysts (ribozymes), but their catalytic mechanisms are diverse and still being discovered.

Purpose of the Study:

  • To investigate the catalytic activity of a specific RNA molecule with an asymmetric internal loop.
  • To elucidate the reaction mechanism of RNA-mediated cleavage.
  • To determine if RNA catalysis can mimic protein ribonuclease mechanisms.

Main Methods:

  • In vitro cleavage assays using a synthetic RNA molecule containing an asymmetric internal loop.
  • Characterization of cleavage products using gel electrophoresis and mass spectrometry.
  • Kinetic analysis of the cleavage reaction in the presence of lead (Pb2+) and magnesium (Mg2+) ions.

Main Results:

  • The RNA molecule was rapidly and specifically cleaved by Pb2+ in the presence of Mg2+.
  • The 5' cleavage product consistently terminated with a 3' phosphomonoester.
  • A 2',3'-cyclic phosphodiester reaction intermediate was identified, indicating a two-step mechanism.
  • This mechanism mirrors that of many protein ribonucleases.

Conclusions:

  • RNA molecules can catalyze specific RNA cleavage via a mechanism previously thought to be exclusive to protein enzymes.
  • The identified RNA molecule acts as a self-cleaving ribozyme with a novel catalytic mechanism.
  • This finding expands the known repertoire of ribozyme functions and catalytic strategies.

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