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Stoichiometry of lac repressor binding to nonspecific DNA: three different complexes form
1Department of Chemistry, University of Denver, Colorado 80208.
Biochemistry
|July 28, 1987
Summary
Lac repressor binding to DNA was studied using fluorescence and spectroscopy. Results suggest a novel mechanism for one-dimensional diffusion of repressor along DNA.
Area of Science:
- Molecular Biology
- Biophysics
Background:
- The lac repressor protein regulates gene expression by binding to DNA.
- Understanding the stoichiometry of repressor-DNA interactions is crucial for elucidating gene regulation mechanisms.
Purpose of the Study:
- To investigate the stoichiometry of lac repressor binding to nonspecific DNA using multiple techniques.
- To determine the number of repressor molecules that bind to a specific length of DNA under varying conditions.
Main Methods:
- Fluorescence spectroscopy using the probe 5,5'-bis(8-anilino-1-naphthalenesulfonate) [bis(ANS)].
- Boundary sedimentation velocity experiments.
- Circular dichroism spectroscopy.
Main Results:
- DNA binding displaced bis(ANS) from the repressor, allowing for fluorescence-based titration.
- Complexes formed with approximately one, two, or four repressors per 28 base pairs, depending on bis(ANS) concentration and ionic strength.
- Sedimentation and circular dichroism experiments confirmed the formation of these complexes and suggested that all repressor subunits contact DNA at high repressor density.
Conclusions:
- The study reveals distinct repressor-DNA stoichiometries, challenging previous assumptions.
- A novel mechanism for one-dimensional diffusion of lac repressor along DNA is proposed, involving differential subunit contact with DNA.