No abstract

Using the binding of cI repressor protein to the lambda right and left operators as a model system, we have analyzed the two common experimental techniques for studying the interactions of genome regulatory proteins with multiple, specific sites on DNA. These are the quantitative DNase footprint titration technique [Brenowitz, M., Senear, D. F., Shea, M. A., & Ackers, G. K. (1986) Methods Enzymol. 130, 132-181] and the nitrocellulose filter binding assay [Riggs, A., Suzuki, H., & Bourgeois, S. (1970) J. Mol. Biol. 48, 67-83]. The footprint titration technique provides binding curves that separately represent the fractional saturation for each site. In principle, such data contain the information necessary to determine the thermodynamic constants for local site binding and cooperativity. We show that in practice, this is not possible for all values of the constants in multisite systems, such as the lambda operators. We show how these constants can nevertheless be uniquely determined by using additional binding data from a small number of mutant operators in which the number of binding sites has been reduced. The filter binding technique does not distinguish binding to the individual sites and yields only macroscopic binding parameters which are composite averages of the various local site and cooperativity constants. Moreover, the resolution of even macroscopic constants from filter binding data for multisite systems requires ad hoc assumptions as to a relationship between the number of ligands bound and the filter retention of the complex. Our results indicate that no such relationship exists. Hence, the technique does not permit determination of thermodynamically valid interaction constants (even macroscopic) in multisite systems.

A central issue in gene regulation is the mechanism, and biological function, of the cooperative binding of regulatory protein ligands to specific sites on DNA. To elucidate the physical-chemical basis of these interactions we have developed a thermodynamically rigorous method for conducting DNase I "footprint" (protection) titration experiments. The intrinsic binding constants and also those for cooperative interactions between various sites can be resolved from the individual-site binding curves determined by this technique. Experimental studies of cl-repressor-operator binding have demonstrated that the method provides an accurate representation of the fractional saturation of a binding site. We present individual-site binding curves for a X operator with two competent sites that demonstrate the presence of cooperative interactions between the sites. These curves set a lower limit to the magnitude of the cooperative free energy without comparison to single-site mutant operators.The regulation of many prokaryotic and eukaryotic genes is dependent on specific, and often cooperative, interactions that control the binding of regulatory proteins to multiple sites on DNA. To identify the sites of interaction numerous investigators are currently using various forms of "footprinting" or "protection mapping" using DNase I or other cleavage reagents (1-4). As a titration method, footprinting has also been used to estimate the affinity of proteins for DNA (5,8 (6)(7)(8)(9). This information is required to understand and predict (i.e., model) the biological roles of these interactions (8, 10).To ensure the thermodynamic validity of the footprint titration method we have developed (i) protocols for conducting the experiment and quantitating the data and (ii) a rigorous theory of individual-site binding that is necessary for analysis and correct interpretation of the resolved binding curves (7). Here we show that when these procedures are used the technique correctly measures the equilibrium distribution of occupied and vacant sites on the DNA. We will outline the experimental guidelines by which thermodynamically valid binding curves can be obtained. The footprint titration method is broadly applicable and can potentially be used to study control systems with different numbers of sites and control proteins. A more detailed description of the methods has been published elsewhere (9). METHODS

The effects of proton activity on the site-specific interactions of cI repressors with operator sites OR were studied by using DNase I footprint titration. Individual-site binding isotherms were obtained for the binding of repressor to each site of wild-type OR and of mutant operators in which binding to some sites is eliminated. The Gibbs energies for binding and for cooperativity (in every operator configuration) were determined at each pH (range 5-8). The proton-linked effects clearly account for a significant fraction of the difference in affinities for the three operator sites. The most dramatic effects on the repressor-operator binding interactions are at acid pH, and therefore do not involve the basic groups in the repressor N-terminal arm known to contact the DNA. Also, the proton-linked effects are different at the three operator sites as indicated by significantly different derivative relationships, partial derivative of ln k versus partial derivative of ln aH = net proton absorption (delta nu bar(H)). These results implicate ionizable repressor groups which may not contact the DNA and conformational differences between the three repressor-operator site complexes as being important components to the mechanism of site specificity. The extensive data base generated by these studies was also used to reevaluate the traditional models used to describe cooperativity in this system. The results confirm the lack of significant cooperative interaction between OR1 and OR3 at all conditions. However, the data for some experimental conditions are clearly inconsistent with the (selection) rule, that cooperative interaction between OR2 and OR3 is eliminated by ligation at OR1.

Cooperative binding of the bacteriophage lambda cI repressor dimer to specific sites of the phage operators OR and OL controls the developmental state of the phage. It has long been believed that cooperativity is mediated by self-assembly of repressor dimers to form tetramers which can then bind simultaneously to adjacent operator sites. As a first step in defining the individual energy contributions to binding cooperativity, sedimentation equilibrium and steady-state fluorescence anisotropy methods have been used to study the higher order assembly reactions of the free repressor in solution. Wild-type repressor with 5-hydroxytryptophan (5-OHTrp) substituted for the native tryptophan [Ross et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12023-12027] and two mutant repressor proteins that bind cooperatively to OR but have altered dimerization properties were also studied. We report here that the primary assembly mode of all four proteins is dimer to octamer. It is not dimer to tetramer as previously assumed. While tetramer does form as an assembly intermediate, dimer-octamer assembly is a concerted process so that tetramer is never a predominant species in solution. Sedimentation velocity experiments suggest that the octamer is highly asymmetric, consistent with an elongated shape. This conformation could allow octamers to bind simultaneously to all three operator sites at either OR or OL. Examination of tetramer and octamer concentrations suggests that both species could be involved in cooperative repressor-operator interactions. Our previous work used the unique spectral properties of 5-OHTrp to demonstrate that octamer binds single-operator DNA and is not dissociated to tetramer [Laue et al. (1993) Biochemistry 32, 2469-2472]. Taken together with the results presented here, octamers as well as tetramers must be considered in developing models to explain the cooperativity of lambda cI repressor binding to operator DNA.