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Nucleic Acids Research, 1983, Vol. 11, No. 22 7833-7852
© 1983

MOLECULAR BIOLOGY

On the different binding affinities of CRP at the lac, gal and malT promoter regions

A. Kolb, A. Spassky, C. Chapon+, B. Blazy§ and H. Buc

Unité de Physicochimie des Macromolècules Biologiques 25 rue du Docteur Roux, 75724 Paris Cedex 15 +Unité de Génétique Moléculaire, Institut Pasteur, épartement de Biologie Moléculaire 25 rue du Docteur Roux, 75724 Paris Cedex 15 §Universitè Paul Sabatier 118, Route de Narbonne, 31062 Toulouse Cedex, France

Received August 26, 1983. Accepted October 14, 1983.

We have determined the stoichiometry of CRP binding to various DNA fragments carrying the lac, mall or gal promoters in the presence of cAMP, using a gel electrophoresis method. In each case, one dimer of CRP binds to the functional CRP site upstream of the transcription start. At the lac promoter, a second CRP dimer can bind to the operator region. Direct binding analysis and competition experiments performed at 200 µM cAMP allow us to measure the affinity of CRP for these different sites and to correlate them with variations in the consensus sequences, already proposed. The order is lac > mal?T > gal > lac operator > lac L8 > non specific sites. No strong coupling exists between the two lac sites when on the same fragment. Conversely, we have studied, at constant CRP concentrations, the cAMP levels required to obtain half maximal binding to a particular DNA site : the required cAMP level increases inversely as the affinity for CRP. These variations may account for the differential activation of various cAMP sensitive operons in vivo.

Anomalies in the migrations of the 1:1 complexes between CRP and DNA have been analysed and related to the size and to the position of the CRP site in the fragment. The electrophoretic mobility of the complexes depends not only on the size of the fragment but on the position of the CRP site : the mobility is lower when CRP binds near the center of the fragment. This effect is due to a clear change in the persistence length of the DNA Induced by CRP binding. We suggest that, upon binding, the protein Introduces a local bend (or a kink) in the DNA structure.


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