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Related Experiment Videos

Arc repressor is tetrameric when bound to operator DNA.

B M Brown1, J U Bowie, R T Sauer

  • 1Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.

Biochemistry
|December 25, 1990
PubMed
Summary
This summary is machine-generated.

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The Arc repressor protein binds bacteriophage P22 operator DNA as a tetramer. This DNA-binding protein interaction is highly cooperative, involving dimer formation before tetramer assembly on the operator site.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Structural Biology

Background:

  • The Arc repressor from bacteriophage P22 is a DNA-binding protein crucial for regulating viral gene expression.
  • Arc belongs to a family of proteins that recognize DNA operator sites using N-terminal residues in a beta-sheet conformation.

Purpose of the Study:

  • To elucidate the stoichiometry and mechanism of Arc repressor binding to its operator DNA.
  • To characterize the cooperative binding and kinetic parameters of the Arc-DNA interaction.

Main Methods:

  • Gel retardation assays using mixtures of wild-type Arc and an active variant to determine binding stoichiometry.
  • Rapid dilution experiments to identify the kinetically significant DNA-binding species.
  • Kinetic analysis of association and dissociation rates to determine equilibrium constants.

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Main Results:

  • Arc binds to its 21-base-pair operator site as a tetramer, forming discrete complexes with varying ratios of wild-type and variant proteins.
  • The Arc-operator binding reaction is highly cooperative, with a Hill constant of 3.5, indicating significant protein-protein interactions.
  • Kinetic experiments revealed that Arc dimers are the primary DNA-binding species, with estimated equilibrium dissociation constants for dimerization and tetramer formation.

Conclusions:

  • Arc repressor functions as a tetramer at its operator site, with dimer formation preceding DNA binding.
  • The cooperative binding mechanism involves coupled equilibria, highlighting the complex regulation of bacteriophage P22 gene expression by Arc.