trp repressor/trp operator interaction. Equilibrium and kinetic analysis of complex formation and stability.
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.
Hurlburt
B K
BK
Department of Biological Sciences, Stanford University, California 94305.
Yanofsky
C
C
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
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D046911:Macromolecular Substances; D012097:Repressor Proteins; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins