COOPERATIVE BINDING TO DNA OF CATABOLITE ACTIVATOR PROTEIN OF ESCHERICHIA-COLI

Binding parameters are reported for the interaction of Escherichia coli catabolite activator protein (CAP) with nonspecific DNAs (primarily native calf thymus DNA, which presumably does not contain specific functional sites for CAP). The main experimental techniques used were circular dichroism and a thermodynamically rigorous centrifugation method which allows quantitative analysis of the binding. It was found that in the absence of cyclic adenosine 3',5'-monophosphate (cAMP) CAP binds cooperatively to double-helical DNAs, including poly[d(A-T)] and poly[d(I-C)]. The presence of cAMP eliminates the cooperativity, while the overall strength of the binding is increased. Circular dichroism spectra imply that the interaction of CAP with DNA causes a shift from the DNA B form to the C form; this occurs regardless of whether cAMP is present. Any conformational changes in the protein which may underlie the cooperative effect are not detectable by circular dichroism. It appears that CAP does not tend to melt double-helical DNA. The data show that a molecule of CAP covers 13 base pairs when bound to DNA. Values for other parameters of the cooperative interaction are reported for a range of ionic conditions (50-80 mM NaCl) at T = 22°C. The intrinsic affinity of the protein for DNA, K, decreases at higher [NaCl], while the cooperativity parameter, ω (which measures the probability that two CAP molecules on DNA will be bound at adjacent sites), has a value of about 100, independent of ionic strength. The data indicate that approximately six ionic interactions are involved in the binding of a CAP molecule to DNA. Extrapolation of the Kω results to more physiological ionic conditions implies that in vivo some CAP molecules may be nonspecifically bound to the E. coli chromosome even in the absence of cAMP. At the higher cAMP levels at which CAP actively promotes transcription it seems likely that nonspecific DNA binding may play an in vivo role in modulating the action of CAP. © 1979, American Chemical Society. All rights reserved.