TY - JOUR
T1 - The Escherichia coli cyclic AMP receptor protein forms a 2:2 complex with RNA polymerase holoenzyme, in vitro
AU - Dyckman, Damian
AU - Fried, Michael G.
PY - 2002/5/24
Y1 - 2002/5/24
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.M110554200
DO - 10.1074/jbc.M110554200
M3 - Article
C2 - 11904295
AN - SCOPUS:0037166272
SN - 0021-9258
VL - 277
SP - 19064
EP - 19070
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 21
ER -