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Related Experiment Videos

Sequence-dependent structural variations of hammerhead RNA enzymes.

H A Heus1, O C Uhlenbeck, A Pardi

  • 1Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.

Nucleic Acids Research
|March 11, 1990
PubMed
Summary

New RNA enzymes, or ribozymes, show sequence-specific cleavage. Structural studies reveal stable hairpins, and differing stabilities may explain varied catalytic rates by preventing enzyme-substrate complex formation.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • RNA Therapeutics

Background:

  • Hammerhead RNA self-cleaving domains exhibit in vivo catalytic activity.
  • This has enabled the creation of novel sequence-specific RNA endonucleases (ribozymes).

Purpose of the Study:

  • To synthesize and structurally characterize two novel ribozymes.
  • To investigate the structural basis for differences in catalytic activity between these ribozymes.

Main Methods:

  • In vitro transcription using T7 polymerase.
  • Optical spectroscopy, nuclear magnetic resonance (NMR), and nondenaturing gel electrophoresis.
  • Analysis of RNA enzyme structure and stability.

Main Results:

  • Both synthesized ribozymes feature a stable hairpin structure with a double helical stem and tetranucleotide loop.

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  • Additional, varying stabilities were observed in both RNA enzymes.
  • A 50-fold difference in substrate cleavage half-lives was previously established.

    • Conclusions:

    • The observed rate differences in ribozyme activity may stem from catalytically inactive conformations.
    • These inactive conformations likely hinder the formation of the enzyme-substrate complex, impacting catalytic efficiency.