PMID:11904295
| Citation |
Dyckman, D and Fried, MG (2002) The Escherichia coli cyclic AMP receptor protein forms a 2:2 complex with RNA polymerase holoenzyme, in vitro. J. Biol. Chem. 277:19064-70 |
|---|---|
| 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 x 10(20) m(-3). The qualitative pattern of association is unchanged over the temperature range 4 degrees C < or = T < or = 20 degrees C, by substitution of glutamate for chloride as the dominant anion, or on addition of 20 microm 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. |
| Links |
PubMed Online version:10.1074/jbc.M110554200 |
| Keywords |
Buffers; Cyclic AMP Receptor Protein/metabolism; DNA-Directed RNA Polymerases/metabolism; Dimerization; Escherichia coli/metabolism; Molecular Weight; Potassium Chloride |
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