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Assembly of the Arc repressor-operator complex: cooperative interactions between DNA-bound dimers
1Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Biochemistry
|February 9, 1993
Summary
Arc repressor dimers bind operator half-sites, acting as intermediates for tetramer assembly. Cooperative binding significantly enhances DNA affinity through extended dimer half-lives.
Area of Science:
- Molecular Biology
- Biochemistry
- Structural Biology
Background:
- Arc repressor is a beta-ribbon DNA-binding protein.
- It typically binds its operator DNA as a tetramer.
Purpose of the Study:
- To investigate the binding of Arc dimers to operator half-sites.
- To determine the equilibrium and kinetic constants for dimer-DNA interactions.
- To elucidate the mechanism of Arc tetramer-operator complex assembly.
Main Methods:
- Electrophoretic mobility shift assays (EMSAs) or similar techniques to study DNA binding.
- Determination of equilibrium binding constants (Kd).
- Kinetic analysis to measure association and dissociation rates (kon, koff).
Main Results:
- Arc dimers specifically bind DNA fragments containing operator half-sites.
- Equilibrium and kinetic parameters for dimer-half-site interactions were determined.
- DNA-bound dimers are transient intermediates in tetramer formation.
- Sequential addition of dimers to operator half-sites facilitates tetramer assembly.
- Cooperative binding of the second dimer to an occupied half-site increases affinity ~5900-fold (ΔΔG = -5.1 kcal/mol).
- This affinity increase is primarily due to a dramatic increase in complex half-life (from seconds to >1 hour).
Conclusions:
- Arc tetramer-operator complex assembly proceeds via sequential dimer addition.
- Cooperative interactions between bound dimers are crucial for high-affinity binding.
- The extended half-life of cooperatively bound dimers stabilizes the final complex.