Kinetic studies of inducer binding to lac repressor.operator complex.
The rates of binding of inducer molecules to the lactose repressor protein are significantly affected by the presence of bound operator DNA fragments. The association rate is decreased from 4.6 x 10(4) M-1S-1 to 1.0 x 10(4) M-1S-1 by the presence of saturating amounts of operator DNA fragments. The inducer dissociation rate was measured by dilution of repressor . operator . inducer complexes and by displacement of the sugar molecule from repressor by the binding of operator DNA. The value for the dissociation rate in the presence of bound DNA was 0.8 S-1; this is 4-fold greater than the inducer dissociation rate measured in the absence of operator. These kinetic results suggest that repressor should exhibit a 20-fold lower affinity for inducer when operator DNA fragments are bound; this prediction is in agreement with previous equilibrium measurements. Measurement of the rate of operator fragment dissociation from repressor using a nitrocellulose filter assay yields a value of 0.04 S-1 in the absence of inducer; this dissociation rate is too rapid to measure in the presence of bound inducer molecules. Calculations assuming that the DNA-protein association rate is unaffected by sugar binding suggest that the rate of dissociation of DNA from repressor containing 4 bound inducer molecules may be as high as 40 S-1. Determination of the apparent association rate for inducer binding to operator DNA fragments indicates that DNA molecules do dissociate from the protein . operator . inducer complex at intermediate stages during the sugar binding reaction. The exact point at which dissociation occurs at a complex function of the concentrations of operator and inducer and the relationship between the rates for sugar binding and for operator fragment dissociation which are comparable for some of the protein species.
D000494:Allosteric Regulation; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D009876:Operon; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
D004269:DNA, Bacterial; D046911:Macromolecular Substances; D012097:Repressor Proteins; D014157:Transcription Factors