A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system.
The use of gel electrophoresis for quantitative studies of DNA-protein interactions is described. This rapid and simple technique involves separation of free DNA from DNA-protein complexes based on differences in their electrophoretic mobilities in polyacrylamide gels. Under favorable conditions both unbound DNA and DNA associated with protein can be quantified. This gel method is applied to the study of the E. coli lactose operon regulatory system. At ionic strengths in the physiological range, the catabolite activator protein (CAP) is shown to form a long-lived complex with the wild type lac promotor, but not with a CAP-insensitive mutant. Formation of a stable "open" or "melted-in" complex of RNA polymerase with the wild type promoter requires the participation of CAP and cyclic AMP. Further, it is demonstrated that even when pre-formed in the presence of CAP-cAMP, the polymerase-promoter open complex becomes unstable if CAP is then selectively removed.
D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D007763:Lac Operon; D046911:Macromolecular Substances; D010957:Plasmids; D011953:Receptors, Cyclic AMP / Q000378:metabolism
D002352:Carrier Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D011953:Receptors, Cyclic AMP; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D004262:DNA Restriction Enzymes