title: trp repressor/trp operator interaction. Equilibrium and kinetic analysis of complex formation and stability.
abstract: The trp repressor of Escherichia coli regulates transcription initiation in the trp operon by binding at an operator located within the trp promoter region. We have used a filter binding assay to analyze the interaction between purified trp repressor and a synthetic 43-base pair DNA fragment containing the natural trp promoter-operator region. In equilibrium binding experiments, the KD of high affinity binding of trp repressor to this DNA fragment was determined to be 2 x 10(-10) M. Low affinity binding was observed at repressor concentrations above 10 nM. In kinetic experiments with various input ratios of repressor to operator, trp repressor-operator complexes dissociated with equivalent, first-order kinetics. Instantaneous reduction of the tryptophan concentration resulted in increased rates of complex dissociation, indicating that loss of one or both tryptophan molecules from the repressor-operator complex destabilizes the complex. A heterodimeric repressor with a single tryptophan binding site was constructed and its affinity for operator was compared with that of ligand free aporepressor and tryptophan saturated repressor. The heterodimeric repressor had a 20-25-fold higher affinity for operator than did the aporepressor, and it had a 20-25-fold lower affinity for operator than did the tryptophan-saturated repressor.
authors.affiliation: Department of Biological Sciences, Stanford University, California 94305.
authors.forename: B K
authors.initials: BK
authors.lastname: Hurlburt
authors.forename: C
authors.initials: C
authors.lastname: Yanofsky
mesh_terms: D001426:Bacterial Proteins; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D009876:Operon; D011485:Protein Binding; D012097:Repressor Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013997:Time Factors; D014364:Tryptophan / Q000235:genetics; D029968:Escherichia coli Proteins
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D046911:Macromolecular Substances; D012097:Repressor Proteins; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins