Specific binding of cyclic-AMP receptor protein to DNA. Effect of the sequence and of the introduction of a nick in the binding site.
The binding of Escherichia coli Cyclic AMP Receptor Protein (CRP) to several DNA fragments of about 45 base pairs, bearing the natural lactose or galactose sites, as well as several synthetic related sites, was investigated using fluorescence spectroscopy and gel retardation experiments. The salt dependence of the equilibrium binding constant indicates that CRP makes an identical number of ion pairs with the lac, lacL8 and gal sites although the binding constants are drastically different. However increasing the symmetry of the gal site leads to an increase of the number of ion pairs between the protein and the DNA. A single strand nick was introduced at the centre of a symmetrized gal site and this reduces the binding energy of CRP by about 0.6 Kcal. These results are discussed with respect to the bending constraints imposed on the DNA by the binding of CRP. The results are in agreement with the recently published crystal structure of the CRP complexed with DNA [Schutz, S.C., Shields, G.C. and Steitz, T.A., Science 253, 1001-1007 (1991)] showing that the 90 degrees bending of the DNA in the complex results from two kinks.
Giraud-Panis
M J
MJ
Centre de Biophysique Moléculaire, Orléans, France.
Toulmé
F
F
Maurizot
J C
JC
Culard
F
F
D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific; D005690:Galactose / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D013056:Spectrophotometry, Ultraviolet
D002373:Cyclic AMP Receptor Protein; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases; D005690:Galactose