Equilibrium studies of the cyclic AMP receptor protein-DNA interaction

The binding of the Escherichia coli cyclic AMP receptor protein (CAP) to restriction fragments containing the lac promoter-operator region has been investigated as a function of cAMP concentration, using a sensitive gel electrophoresis assay. Under standard conditions (13 mM ionic strength), the equilibrium constant for CAP binding to its primary site on a 203 base-pair lac promoter fragment is 6·3×10⁸M^-1 at 0·2 μM-cAMP, and increases to 8·4×10¹⁰ M^-1 at 5·0 μM-cAMP. The latter is about 10⁵ times larger than the equilibrium constant for binding to an isolated, non-specific site. The L8 mutation, which renders the lac promoter unresponsive to CAP in vivo, lowers this binding affinity by five- to tenfold. Analysis of the cAMP dependency of binding over the concentration range of 0·2 μM to 10 μM reveals that uptake of a single equivalent of cAMP is required for site-specific binding. Similarly, the transfer of CAP from a non-specific DNA site to a specific site requires the net uptake of a single molecule of cAMP. In contrast, co-operative non-specific binding to DNA was found to be independent of cAMP concentration with an equilibrium binding constant of 6×10⁶ M^-1. We conclude that the cAMP affinity of the two CAP subunits in the specific promoter complex is not equal, and that the complex structure therefore deviates significantly from twofold symmetry. A model for the regulation of the lac promoter by the intracellular cAMP concentration is proposed on the basis of the equilibrium binding results.