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Related Concept Videos

Ribozymes02:47

Ribozymes

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The term ribozyme is used for RNA that can act as an enzyme. Ribozymes are mainly found in selected viruses, bacteria, plant organelles, and lower eukaryotes. Ribozymes were first discovered in 1982 when Tom Cech’s laboratory observed Group I introns acting as enzymes. This was shortly followed by the discovery of another ribozyme, Ribonulcease P, by Sid Altman’s laboratory. Both Cech and Altman received the Nobel Prize in chemistry in 1989 for their work on ribozymes.
Ribozymes can...
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Cooperative Allosteric Transitions01:58

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Cooperative allosteric transitions can occur in multimeric proteins, where each subunit of the protein has its own ligand-binding site. When a ligand binds to any of these subunits, it triggers a conformational change that affects the binding sites in the other subunits; this can change the affinity of the other sites for their respective ligands. The ability of the protein to change the shape of its binding site is attributed to the presence of a mix of flexible and stable segments in the...
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Binding sites linkages can regulate a protein's function.  For example, enzyme activity is often regulated through a feedback mechanism where the end product of the biochemical process serves as an inhibitor.
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to  N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis...
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Engineering high-speed allosteric hammerhead ribozymes.

Kristian H Link1, Lixia Guo, Tyler D Ames

  • 1Howard Hughes Medical Institute, Yale University, New Haven, CT 06520-8103, USA.

Biological Chemistry
|July 28, 2007
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Summary
This summary is machine-generated.

Researchers engineered allosteric ribozymes for gene control. While showing high activity in vitro, they failed in cellular assays, suggesting differences in RNA folding between in vitro and in vivo environments.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Synthetic Biology

Background:

  • Hammerhead ribozymes are catalytic RNA molecules with potential for gene regulation.
  • Allosteric regulation offers a mechanism for precise control of biological processes.
  • Developing RNA-based genetic control systems requires efficient and responsive regulatory elements.

Purpose of the Study:

  • To engineer hammerhead ribozymes for allosteric regulation by theophylline.
  • To assess the kinetic properties and potential for gene control applications of selected ribozymes.
  • To investigate discrepancies between in vitro performance and in vivo activity.

Main Methods:

  • In vitro selection (SELEX) was employed over 15 rounds to evolve allosteric ribozymes.
  • Kinetic analysis was performed to determine observed rate constants (k obs) with and without theophylline.
  • Reporter gene assays were used to evaluate cellular gene control efficacy.

Main Results:

  • Selected ribozymes exhibited significant allosteric regulation by theophylline, with rate constants up to 8 min(-1).
  • Observed rate constant increases reached up to 285-fold in the presence of theophylline compared to its absence.
  • Engineered ribozymes demonstrated insufficient activity in cellular reporter gene assays.

Conclusions:

  • The engineered allosteric ribozymes possess promising kinetic characteristics for gene control.
  • Differences in in vitro and in vivo RNA folding likely explain the lack of cellular gene expression control.
  • Further studies are needed to reconcile in vitro and in vivo behavior for successful RNA-based gene regulation.