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Kinetic analysis of delta ribozyme cleavage

S Mercure1, D Lafontaine, S Ananvoranich

  • 1Département de biochimie, Faculté de médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.

Biochemistry
|December 4, 1998
PubMed
Summary

Delta ribozyme efficiently cleaves RNA substrates, with catalytic efficiency influenced by temperature and cofactor presence. Substrate binding involves specific RNA interactions, not just downstream helix formation, suggesting a conformational transition is key.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Enzymology

Background:

  • Delta ribozymes are catalytic RNA molecules.
  • Understanding their kinetics and substrate binding is crucial for RNA catalysis research.

Purpose of the Study:

  • To investigate the catalytic mechanism of delta ribozyme on an 11-mer RNA substrate.
  • To determine the influence of cofactors (Mg2+, Ca2+), temperature, and substrate concentration on ribozyme activity.
  • To elucidate the specific interactions involved in substrate binding.

Main Methods:

  • Single- and multiple-turnover kinetic assays.
  • Temperature-dependent activity measurements.
  • Substrate inhibition studies.
  • Gel-shift assays and cleavage inhibition studies with modified substrates and ribozymes.

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Main Results:

  • Kinetic parameters showed minor differences with Mg2+ or Ca2+.
  • Catalytic efficiency (kcat/KM) was higher at 37°C than 56°C, with reaction steps limited by product release or chemical cleavage respectively.
  • Substrate inhibition was observed at high substrate concentrations.
  • L4 tetraloop stability did not significantly affect catalytic activity.
  • The 2'-hydroxyl group and J4/2 cytosine residue are critical for substrate binding.
  • Substrate binding is not solely dependent on a downstream helix.

Conclusions:

  • Delta ribozyme's catalytic activity is temperature-dependent, with distinct rate-limiting steps at different temperatures.
  • Specific RNA-ribozyme interactions, including the 2'-hydroxyl and J4/2 junction, are essential for substrate binding.
  • A conformational transition is likely required for active complex formation, expanding the understanding of ribozyme-substrate interactions beyond simple helix formation.