Effects of anions on the binding of the cAMP receptor protein to the lactose promoter.
The DNA binding affinities of several gene-regulatory proteins, restriction endonucleases and the Escherichia coli RNA polymerase have previously been found to be dependent on the nature of the dominant buffer anion. To discover whether the E. coli cAMP receptor protein (CAP) exhibits a similar dependency, we measured its affinity for its primary lactose promoter binding site (lac site 1) in buffers in which the principal anion was chloride, phosphate, sulfate, acetate, or glutamate. We found that the affinity of CAP for lac site 1 is affected only slightly by changes in the dominant buffer anion. The binding of cAMP is similarly insensitive to buffer anion type, indicating that specific protein-anion interactions, if they occur, must be similar for the free and cAMP-bound forms of the protein. The effect of anion substitution on the ability of acrylamide to quench the intrinsic fluorescence of tryptophanyl residues of CAP is also small, suggesting that changes in buffer anion composition have minimal effect on the conformation of tryptophan-proximal regions of CAP. This conclusion is extended by the finding that anion substitution has a relatively small effect on the urea-concentration dependence of CAP denaturation. Taken together, these results support the notion that neither CAP nor CAP.cAMP nor the CAP.cAMP complex with lac promoter DNA interact selectively with anions present in the surrounding buffer. A possible role for this anion-insensitivity in the in vivo function of CAP is suggested.
Department of Chemistry, University of Houston, Texas.
J M
JM
Hudson
M
M
Crowe
M G
MG
Fried
D000838:Anions / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D005453:Fluorescence; D007785:Lactose / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011489:Protein Denaturation; D011953:Receptors, Cyclic AMP / Q000378:metabolism*
D000838:Anions; D011953:Receptors, Cyclic AMP; D004247:DNA; D000242:Cyclic AMP; D007785:Lactose