title: FRep: a fluorescent protein-based bioprobe for in vivo detection of protein-DNA interactions.
abstract: We describe a bacterial reporter system, FRep, for rapid and facile detection of protein-DNA recognition. The bioprobe reporter comprises genes of two fluorescent proteins (FPs) separated by a potential DNA target. If a coexpressed transcription factor binds the DNA target, transcription of the second FP is impeded, resulting in loss of FRET partner. Using ratiometric FRET, we show that evaluation of protein-DNA recognition can be reliably made on bZIP and bHLHZ transcription factors and their DNA targets. FRep displays similar thresholds of detection regarding protein-DNA binding affinities, as compared to well-established electrophoretic and yeast assays, although we observed variations in the intensity of fluorescence signals and detection thresholds that may depend on differences between DNA-binding protein production levels and/or stability in the cell, or the expressed bioprobe linker between the two FPs. FRep can potentially be applied to high-throughput searches of both protein and DNA libraries; in a mock library screen, binding and nonbinding complexes can even be distinguished by visual inspection of colonies on plates. FRep presents notable advantages over existing technologies when applied to assessing protein-DNA interactions in vivo, and this approach has the potential for applications in assaying protein-protein interactions and screening molecules that influence specific macromolecular interactions.
authors.affiliation: Department of Chemical and Physical Sciences, University of Toronto at Mississauga, Mississauga, Ontario, Canada.
authors.forename: S Hesam
authors.initials: SH
authors.lastname: Shahravan
authors.forename: Isaac T S
authors.initials: IT
authors.lastname: Li
authors.forename: Kevin
authors.initials: K
authors.lastname: Truong
authors.forename: Jumi A
authors.initials: JA
authors.lastname: Shin
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D015374:Biosensing Techniques; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D004247:DNA