Fluorescence anisotropy assays implicate protein-protein interactions in regulating trp repressor DNA binding.
The study of interactions between proteins and nucleic acids is central to the understanding of the control of genetic expression. Fluorescence anisotropy has been used to measure, in solution, the equilibrium binding profiles of a bacterial repressor protein, the tryptophan repressor (TR), to a fluorescently labeled oligonucleotide containing one of its target operator sequences. Investigation of the effects of changing concentrations of corepressor, operator DNA, and protein implicate TR oligomers in the regulation of DNA binding. These studies also demonstrate that the relatively straightforward technique of fluorescence anisotropy can be applied to the study of the interactions between proteins and nucleic acids. The fluorescence technique exhibits sufficient sensitivity to replace radioactive methods of detection in most cases. In addition, since it is a solution-based methodology, it offers a true equilibrium measure of the protein-nucleic acid equilibria, and the effects of changes in solution conditions such as salt and ligand concentration, pH, and temperature can be readily evaluated. Data acquisition is relatively simple and rapid, and the data are of sufficient quality for detailed thermodynamic analyses of complex systems. Given these attributes, fluorescence anisotropy will find multiple applications in the area of genetic regulation.
LeTilly
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School of Pharmacy, University of Wisconsin-Madison 53706.
Royer
C A
CA
D001426:Bacterial Proteins; D001483:Base Sequence; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005454:Fluorescence Polarization; D008969:Molecular Sequence Data; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins