The Escherichia coli Cyclic AMP Receptor Protein Forms a 2:2 Complex with RNA Polymerase Holoenzyme, in Vitro

Abstract

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 MgCl 2 , 0.1 mM dithiothreitol, 5% glycerol, 100 mM KCl) is 2 ؋ 10 20 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 M 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.

Interactions of Human O6-Alkylguanine-DNA Alkyltransferase (AGT) with Short Single-stranded DNAs

“…This approach has been used successfully for other protein-protein and protein-DNA interactions (42)(43)(44), although there are notable exceptions (45). Estimated with Equation 5, v P n D is ϳ0.73 for the complexes described here.…”

CRP-Cyclic AMP Dependent Inhibition of the Xylene-Responsive σ54-Promoter Pu in Escherichia coli

“…On the other hand, the recruitment of the Eσ 54 RNA polymerase to Pu requires the interaction of the αCTD with the UP-like elements at Pu [8], [9]. Moreover, CRP can interact directly with RNA polymerase in solution [23], [51] using a method described previously [52], although there are not any potential binding sites for CRP by analyzing statistically the Pu promoter. The " Pu phenomenon" observed in this article could also be compared with what has been observed for the dctA and glnA p2 promoter.…”

Protein-DNA Interactions Studied at Sedimentation Equilibrium

“…Buffer density was measured with a Mettler DA-300 density meter. A spread-sheet calculation was used to predict the buoyant molecular weights of CAP-DNA complexes using input molecular weights and previously determined values of the partial specific volumes of CAP (0.737)61 and DNA (0.55) 62. Binding densities (values shown to left of curves) were calculated from the CAP/DNA ratios that gave observed buoyant molecular weights.…”