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.Keywords: FRET, TBP, protein-DNA interaction, binding, fluorescence.Proper transcription of protein-encoding genes (the synthesis of mRNA from a DNA template) is essential for cellular viability. In eukaryotes, mRNA transcription is catalyzed by the enzyme RNA polymerase II, which requires numerous accessory factors in order to synthesize mRNA in a gene-specific fashion. One such factor is the TATA binding protein (TBP), 1 which recognizes and binds A/T-rich sequences called TATA-boxes in the promoter regions of genes. The resulting TBP/DNA complex can serve as a platform to recruit other necessary factors and RNA polymerase II to the promoter [1].Crystal structures of the TBP/DNA complex show that TBP resembles a saddle sitting on DNA with a stirrup on each end ([2-5]; these references also contain websites that allow 3D models of the TBP/DNA complex to be viewed). Phenylalanine residues in each stirrup insert into the minor groove of the TATA DNA, which disrupts base stacking interactions, resulting in the DNA being bent away from TBP. Human TBP bends DNA $1008 in solution, as determined using a two-kink model that is described in detail later [6,7]. Biochemical studies have shown that the affinity of TBP for DNA is dictated by the conformational flexibility of the DNA sequence more so than sequence specific interactions [2,8,9]. TBP bends different DNA sequences at different angles, with larger bend angles in general correlating with higher affinity and greater transcriptional activity [7,10].The experiment described here uses fluorescence resonance energy transfer (FRET) to investigate the bending of DNA by human TBP. FRET is the radiationless transfer of energy from an excited donor fluorophore to a suitable acceptor fluorophore [11]. The excitation spectrum of the acceptor must overlap the fluorescence emission spectrum of the donor for FRET to occur, and so specific pairs of fluorophores need to be chosen for FRET studies. The efficiency with which FRET occurs relates to the distanc...