title: Application of fluorescence energy transfer and polarization to monitor Escherichia coli cAMP receptor protein and lac promoter interaction.
abstract: A fluorescence method was developed to study DNA-protein interactions in solution. A 32-base-pair (bp) DNA fragment of the lac promoter containing the primary binding site for Escherichia coli cAMP receptor protein (CRP) was chemically synthesized and labeled specifically at the 5' end with fluorescent probe. Binding of cAMP receptor protein to this fragment can be conveniently followed by measuring changes in polarization of fluorescence of the labeled DNA or by measuring fluorescence energy transfer from protein tryptophan residues to the DNA label. Formation of protein-DNA complex was monitored as a function of cAMP concentration. Various equilibrium constants can be resolved to characterize the binding of cAMP to CRP and the subsequent binding of CRP-cAMP and CRP-(cAMP)2 to DNA. These binding studies showed that the two ligated forms of CRP have significantly different affinities for specific-site DNA. These results show that, in principle, the fluorescence technique can yield thermodynamically valid equilibrium constants under essentially any solution conditions. This technique also has the potential of providing information regarding the structure of protein-DNA complexes.
authors.affiliation: E. A. Doisy Department of Biochemistry, Saint Louis University School of Medicine, MO 63104.
authors.forename: T
authors.initials: T
authors.lastname: Heyduk
authors.forename: J C
authors.initials: JC
authors.lastname: Lee
mesh_terms: D001483:Base Sequence; D000242:Cyclic AMP / Q000494:pharmacology; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism*; D004735:Energy Transfer; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D007785:Lactose / Q000378:metabolism*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D011401:Promoter Regions, Genetic; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D002373:Cyclic AMP Receptor Protein; D000242:Cyclic AMP; D007785:Lactose