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 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.
Original language | English |
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Pages (from-to) | 19064-19070 |
Number of pages | 7 |
Journal | Journal of Biological Chemistry |
Volume | 277 |
Issue number | 21 |
DOIs | |
State | Published - May 24 2002 |
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology