Fluorescence anisotropy assays implicate protein-protein interactions in regulating trp repressor DNA binding

Abstract

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.

Control of vitellogenin genes expression by sequences derived from transposable elements in rainbow trout

“…This technology is remarkably sensitive and robust for measuring protein-DNA equilibrium constants [36][37][38][39]. We carried out titration curves with purified recombinant hERα but not with rtER S , since we could not produce biologically active proteins, despite the use of several over-production and purification systems.…”

Characterization of the multidrug efflux regulator AcrR from Escherichia coli

“…In this study, we used fluorescence polarization assay [15][16][17] to examine interaction between AcrR and its predicted target DNA. We also used this method to determine the affinities of three different AcrR drugs, ethidium (Et), proflavin (Pf) and rhodamine 6G (R6G).…”

Quantitative approaches to monitor protein–nucleic acid interactions using fluorescent probes

“…FP is a useful technique to study the thermodynamic and kinetic properties of a protein-nucleic acid interaction (LeTilly and Royer 1993;Aviv et al 2003;Ryder and Williamson 2004). FP takes advantage of the change in the tumbling properties of a fluorescent ligand upon binding to a larger macromolecule: in our case, a fluorescent DNA or RNA probe and a nucleic acid-binding protein.…”

“…Due to the challenges associated with using radioisotopes, fluorescent probes have become a favorable alternative in many biochemical assays (Royer and Scarlata 2008;LeTilly and Royer 1993;Aviv et al 2003;Ryder and Williamson 2004;Hardin and Batey 2006;Besse et al 2009;Kohn et al 2010). With modern instrumentation, the detection of fluorescent probes now rivals that of 32 P-labeled probes.…”