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Related Experiment Videos

Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity.

Anastasia Khvorova1, Aurélie Lescoute, Eric Westhof

  • 1Amgen, Inc., One Amgen Center Drive, Thousand Oaks, California 91320, USA. khovorova.a@dharmacon.com

Nature Structural Biology
|July 26, 2003
PubMed
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Hammerhead ribozymes (HHRz) require additional sequences for intracellular activity. These elements stabilize HHRz, enabling efficient RNA cleavage in vivo at physiological conditions.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • RNA Catalysis

Background:

  • Hammerhead ribozymes (HHRz) are RNA enzymes capable of site-specific RNA cleavage.
  • The minimal HHRz motif functions at high Mg2+ but not at physiological intracellular concentrations.
  • Previous studies focused on the conserved catalytic core, overlooking essential accessory elements.

Purpose of the Study:

  • To investigate the requirements for hammerhead ribozyme activity in intracellular environments.
  • To identify non-conserved sequence elements crucial for HHRz function in vivo.
  • To elucidate the mechanism by which these elements enhance HHRz catalytic efficiency.

Main Methods:

  • Analysis of natural hammerhead ribozyme sequences.
  • Site-directed mutagenesis of HHRz constructs.

Related Experiment Videos

  • In vitro cleavage assays at varying Mg2+ concentrations.
  • Functional assays in living cells (in vivo).
  • Structural and molecular modeling analyses.

    • Main Results:

    • Natural HHRzs require non-conserved sequence elements beyond the minimal catalytic core for intracellular activity.
    • These additional elements stabilize the ribozyme through tertiary interactions.
    • Stabilized HHRzs exhibit efficient RNA cleavage at physiological Mg2+ concentrations.
    • HHRz variants with stabilizing elements are functional in vivo.

    Conclusions:

    • Intracellular hammerhead ribozyme activity necessitates specific non-conserved sequence elements for stabilization.
    • Tertiary interactions mediated by these elements are critical for achieving catalytic competence at physiological Mg2+ levels.
    • Understanding these stabilizing elements expands the potential of HHRz as a gene-silencing tool.