Nucleic Acids Research, 1981, Vol. 9, No. 23 6505-6525
© 1981
MOLECULAR BIOLOGY |
Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis
Departments of Chemistry and Molecular Biophysics and Biochemistry, Yale University P.O.Box 6666, New Haven, CT 06511, USA
Received July 6, 1981.
We describe the use of gel electrophoresis in studies of equilibrium binding, site distribution, and kinetics of protein-DNA interactions. The method, which we call protein distribution analysis, is simple, sensitive and yields thermodynamically rigorous results. It is particularly well suited to studies of simultaneous binding of several proteins to a single nucleic acid. In studies of the lac repressor-operator interaction, we found that binding to the so-called third operator site (03) is 15–18 fold weaker than operator binding, and that the binding reactions with the first and third operators are uncoupled, implying that there is no communication between the sites. Pseudo-first order dissociation kinetics of the repressor-203 bp operator complex were found to be temperature sensitive, with 
E of 80 kcal mol1 above 29°C and 26 kcal mol–1 below. The half life of the complex (5 min at 21°C) is shorter than that reported for very high molecular weight operator-containing DNAs, but longer than values reported for much shorter fragments. The binding of lac repressor core to DNA could not be detected by this technique: the maximum binding constant consistent with this finding is 105 M–1.
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