Using FRET to measure the angle at which a protein bends DNA: TBP binding a TATA box as a model system

An undergraduate biochemistry laboratory experiment that will teach the technique of fluorescence resonance energy transfer (FRET) while analyzing protein-induced DNA bending is described. The experiment uses the protein TATA binding protein (TBP), which is a general transcription factor that recognizes and binds specific DNA sequences known as TATA boxes. When TBP binds to a TATA box, it bends the DNA. Such bending will be detected using FRET to measure the distance between two fluorophores located on the ends of the DNA. When TBP binds and bends the DNA, the fluorophores move closer together, reflected by an increase in FRET. At the completion of the experiment, three parameters will be determined: 1) the efficiency of the FRET, 2) the end-to-end distance between the fluorophores, and 3) the angle at which TBP bends the DNA. In performing this experiment, students will be introduced to FRET, gain experience in quantitative biophysical measurements, and appreciate how a protein can induce a dramatic change in DNA conformation.