Specific binding of the cAMP receptor protein of Escherichia coli to the lactose operon promoter.
The nitrocellulose filter binding assay has been used to study effects of pH, temperature, ionic strength and magnesium ions on the specific binding of the cyclic adenosine 3',5'-monophosphate (cAMP) receptor protein (CAP) to the promoter of the lactose (lac) operon of Escherichia coli. The pH has a significant effect on binding with the greatest amount of specific binding appearing at pHs near 7 with a gradual decrease in binding as the pH is increased to 8. Specific binding was observed at temperatures of 22 degrees C and 37 degrees C but not at 4 degrees C. The specific binding was also found to be a function of the concentration of magnesium acetate and potassium chloride, being dependent on the specific cation present, the total ionic strength, and the concentration of the CAP protein. All binding decreases as the ionic strength, increases, but this decrease occurs at a lower ionic strength in magnesium acetate than in potassium chloride. In a double label experiment the filter assay demonstrates that the cAMP-CAP complex preferentially binds to the wild-type lac promoter in the presence of a lac promoter mutated at the CAP binding site. Based on these results and comparisons with other experiments reported in the literature, buffer conditions that approximate the physiological state of a cell appear to be best for studying the interaction between CAP and the lactose promoter in vitro.
Koop
A H
AH
Staprans
S I
SI
Bourgeois
S
S
D002412:Cations; D000242:Cyclic AMP / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005990:Glycerol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007763:Lac Operon; D008274:Magnesium / Q000494:pharmacology; D009994:Osmolar Concentration; D011188:Potassium / Q000494:pharmacology; D011953:Receptors, Cyclic AMP / Q000187:drug effects / Q000378:metabolism*; D013696:Temperature
D002412:Cations; D011953:Receptors, Cyclic AMP; D000242:Cyclic AMP; D008274:Magnesium; D005990:Glycerol; D011188:Potassium; D004121:Dimethyl Sulfoxide