Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis

Michael Fried, Donald M. Crothers

Research output: Contribution to journalArticlepeer-review

1680 Scopus citations

Abstract

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 ΔE 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.

Original languageEnglish
Pages (from-to)6505-6525
Number of pages21
JournalNucleic Acids Research
Volume9
Issue number23
DOIs
StatePublished - Dec 11 1981

Bibliographical note

Funding Information:
CAP: the cyclic AMP receptor protein of _E. coli. r .. .: input molar ratio lac repressor tetramer: DNA fragment, n: number of repressor tetramers bound to a DNA fragment. BSA: bovine serum albumin ACKNOWLEDGEMENTS This work was supported Science Foundation.

ASJC Scopus subject areas

  • Genetics

Fingerprint

Dive into the research topics of 'Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis'. Together they form a unique fingerprint.

Cite this