YEAST TATA-BINDING PROTEIN-INTERACTION WITH DNA - FLUORESCENCE DETERMINATION OF OLIGOMERIC STATE, EQUILIBRIUM BINDING, ON-RATE, AND DISSOCIATION KINETICS

A combination of steady-state, stopped-flow, and time-resolved fluores cence of intrinsic tryptophan and extrinsically labeled fluorescent DN A is utilized to examine the interaction of yeast TATA binding protein (TBP) with DNA. TBP is composed of two structural domains, the carbox y domain (residues 61-240), which is responsible for DNA binding and i nitiation of basal level transcription, and an amino terminal domain ( residues 1-60), whose function is currently unknown. The steady-state fluorescence emission spectrum of the single tryptophan in the amino t erminal domain of TBP undergoes a huge (30-40 nm) red-shift upon inter action with stoichiometric amounts of TATA box containing DNA. From ti me-resolved tryptophan fluorescence anisotropy studies, we demonstrate that, in the absence of DNA, the protein exists as a multimer in solu tion and it contains (at least) two primary conformations, one with th e amino terminus associated tightly with the protein(s) in a hydrophob ic environment and one with the amino terminus decoupled away from the rest of the protein and solvent-exposed. Upon binding DNA, the protei n dissociates into a monomeric complex, upon which only the solvent-ex posed amino terminus conformation remains. Kinetic and equilibrium bin ding studies were performed on TATA box containing DNA which was extri nsically labeled with a fluorescent probe Rhodamine-X at the 5'-end. T his ''fluorescent'' DNA allowed for the collection of quantitative spe ctroscopic binding, kinetic on-rate, and kinetic off-rate data at phys iological concentrations. Global analysis of equilibrium binding studi es performed from 500 pM to 50 nM DNA reveals a single dissociation co nstant (K-d) of approximately 5 nM. Global analysis of stopped-flow an isotropy on-rate experiments, with millisecond timing resolution and T BP concentrations ranging from 20 to 600 nM (20 nM DNA), can be perfec tly described by a single second-order rate constant of 1.66 x 10(5) M (-1) s(-1). These measurements represent the very first stopped flow a nisotropy study of a protein/DNA interaction. Stopped-flow anisotropy off-rate experiments reveal a single exponential k(off) of 4.3 x 10(-2 ) min(-1) (1/k(off) = 23 min). From the ratio of on-rate to off-rate, a predicted K-d of 4.3 nM is obtained, revealing that the kinetic and equilibrium studies are internally consistent. Deletion of the amino t erminal domain of TBP decreases the k(on) of TBP approximately 45-fold and eliminates classic second-order behavior.