On the different binding affinities of CRP at the lac, gal and malT promoter regions.
We have determined the stoichiometry of CRP binding to various DNA fragments carrying the lac, malT 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 microM 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 greater than malT greater than gal greater than lac operator greater than lac L8 much greater than 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.
D001483:Base Sequence; D001667:Binding, Competitive; D000242:Cyclic AMP / Q000378:metabolism; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D007763:Lac Operon; D009876:Operon; D011953:Receptors, Cyclic AMP / Q000378:metabolism*
D004269:DNA, Bacterial; D011953:Receptors, Cyclic AMP; D000242:Cyclic AMP; D004262:DNA Restriction Enzymes