The Escherichia coli cyclic AMP receptor protein forms a 2:2 complex with RNA polymerase holoenzyme, in vitro

Sedimentation equilibrium studies show that the Escherichia coli cyclic AMP receptor protein (CAP) and RNA polymerase holoenzyme associate to form a 2:2 complex in vitro. No complexes of lower stoichiometry (1:1, 2:1, 1:2) were detected over a wide range of CAP and RNA polymerase concentrations, suggesting that the interaction is highly cooperative. The absence of higher stoichiometry complexes, even in the limit of high [protein], suggests that the 2:2 species represents binding saturation for this system. The 2:2 pattern of complex formation is robust. A lower-limit estimate of the formation constant in our standard buffer (40 mM Tris (pH 7.9), 10 mM MgCl₂, 0.1 mM dithiothreitol, 5% glycerol, 100 mM KCl) is 2 × 10²⁰ M^-3. The qualitative pattern of association is unchanged over the temperature range 4°C ≤ T ≤ 20°C, by substitution of glutamate for chloride as the dominant anion, or on addition of 20 μM cAMP to the reaction mix. These results limit the possible mechanisms of CAP-polymerase association. In addition, they support the idea that CAP binding may influence the availability of the monomeric form of RNA polymerase that mediates transcription at many promoters.