Interaction between the cyclic AMP receptor protein and DNA. Conformational studies.
The binding of the cyclic adenosine 3',5' monophosphate receptor protein (CRP or CAP) of Escherichia coli to non-specific DNA and to a specific lac recognition sequence has been investigated by circular dichroism (c.d.) spectroscopy. The effect of cAMP and cGMP on the co-operative non-specific binding was also studied. For the non-specific binding in the absence of cAMP a c.d. change (decrease of the intensity of the positive band with a shift of its maximum to longer wavelength) indicates that the DNA undergoes a conformational change upon CRP binding. This change might reflect the formation of the solenoidal coil previously observed by electron microscopy. The amplitude of the c.d. change increases linearly with the degree of saturation of the DNA and does not depend on the size of the clusters of CRP bound. From the variation of the c.d. effect as a function of the ionic strength, the product K omega (K, the intrinsic binding constant and omega, the co-operativity parameter) could be determined. The number of ion pairs involved in complex formation between CRP and DNA was found to be six to seven. Experiments performed with several DNAs, including the alternating polymers poly[d(A-T)] and poly[d(G-C)], demonstrated that the conformational change does not depend on the DNA sequence. However, in the presence of cAMP the c.d. spectrum of the DNA shows only a small variation upon binding CRP. In contrast, in the presence of cGMP the conformational change of the DNA is similar to that observed when non-liganded CRP binds. For the specific lac operon binding, the c.d. change is different from those observed for non-specific binding in the presence or absence of cAMP. These results emphasize the high variability of the DNA structure upon binding the same protein.
Blazy
B
B
Laboratoire de Biophysique Interactions Moleculaires, Universite Paul Sabatier, Toulouse, France.
Culard
F
F
Maurizot
J C
JC
D002942:Circular Dichroism; D000242:Cyclic AMP / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D009690:Nucleic Acid Conformation / Q000187:drug effects; D011067:Poly dA-dT / Q000378:metabolism; D011089:Polydeoxyribonucleotides / Q000378:metabolism; D011953:Receptors, Cyclic AMP / Q000378:metabolism*
D011089:Polydeoxyribonucleotides; D011953:Receptors, Cyclic AMP; D011067:Poly dA-dT; C017650:polydeoxy(guanine-thymine) nucleotide; D004247:DNA; D000242:Cyclic AMP; D006152:Cyclic GMP