A combination of steady-state, stopped-flow, and time-resolved fluorescence of intrinsic tryptophan and extrinsically labeled fluorescent DNA is utilized to examine the interaction of yeast TATA binding protein (TBP) with DNA. TBP is composed of two structural domains, the carboxy domain (residues 61-240), which is responsible for DNA binding and initiation 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 terminal domain of TBP undergoes a huge (30-40 nm) red-shift upon interaction with stoichiometric amounts of TATA box containing DNA. From time-resolved tryptophan fluorescence anisotropy studies, we demonstrate that, in the absence of DNA, the protein exists as a multimer in solution and it contains (at least) two primary conformations, one with the amino terminus associated tightly with the protein(s) in a hydrophobic environment and one with the amino terminus decoupled away from the rest of the protein and solvent-exposed. Upon binding DNA, the protein dissociates into a monomeric complex, upon which only the solvent-exposed amino terminus conformation remains. Kinetic and equilibrium binding studies were performed on TATA box containing DNA which was extrinsically labeled with a fluorescent probe Rhodamine-X at the 5'-end. This "fluorescent" DNA allowed for the collection of quantitative spectroscopic binding, kinetic on-rate, and kinetic off-rate data at physiological concentrations. Global analysis of equilibrium binding studies performed from 500 pM to 50 nM DNA reveals a single dissociation constant (Kd) of approximately 5 nM. Global analysis of stopped-flow anisotropy on-rate experiments, with millisecond timing resolution and TBP concentrations ranging from 20 to 600 nM (20 nM DNA), can be perfectly 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 anisotropy 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 Kd of 4.3 nM is obtained, revealing that the kinetic and equilibrium studies are internally consistent. Deletion of the amino terminal domain of TBP decreases the k(on) of TBP approximately 45-fold and eliminates classic second-order behavior.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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