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Equilibrium binding of inducer to lac repressor.operator DNA complex.

1980; Elsevier BV; Volume: 255; Issue: 21 Linguagem: Inglês

10.1016/s0021-9258(19)70434-7

1083-351X

Ronald B. O'Gorman, John M. Rosenberg, Olga B. Kallai, Richard E. Dickerson, Keiichi Itakura, Arthur D. Riggs, Kathleen S. Matthews,

RNA Interference and Gene Delivery

The characteristics of inducer binding to lactose repressor protein in the presence of a small (29 base pair) operator DNA fragment have been examined. The presence of operator DNA fragments decreases the affinity of the protein for inducer molecules. The maximum change observed is a 20-fold increase in the concentration of inducer necessary for half-saturation of the protein. The non-linearity of the Scatchard type plots and slopes of the Hill plots of the binding data indicate that inducer binding exhibits cooperative behavior in the presence of operator DNA fragments, while free repressor binds inducer in a noncooperative fashion. The experimental data for the binding of inducer to repressor both in the presence and absence of saturating amounts of operator DNA fragments were compared to curves predicted by several different models. Using the limits placed by the measured values for repressor-operator DNA-inducer interaction, it was possible to discriminate between the various models. The models which accurately predicted the inducer binding curves and conformed to the measured values for the other parameters were Monod-Wyman-Changeux and Koshland type models with the following features: 1) positive cooperativity in the binding of inducer to the repressor . operator DNA complex; 2) two sites for the binding of the operator DNA fragment to repressor protein; 3) effect of binding of each operator DNA fragment on all four subunits of the repressor. These models are consistent with all of the available data, and they indicate that the binding of the first two inducer molecules accounts for greater than or equal to 60% of the difference in affinities between free and induced repressor for operator DNA; in vivo this difference should be sufficient for induction to occur.