On the different binding affinities of CRP at thelac, galandmalT promoter regions

Abstract

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.