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.

The binding of adenosine cyclic 3',5'-monophosphate (cAMP) and guanosine cyclic 3',5'-monophosphate (cGMP) to the adenosine cyclic 3',5'-monophosphate receptor protein (CRP) from Escherichia coli was investigated by equilibrium dialysis at pH 8.0 and 20 degrees C at different ionic strengths (0.05--0.60 M). Both cAMP and cGMP bind to CRP with a negative cooperativity that is progressively changed to positive as the ionic strength is increased. The binding data were analyzed with an interactive model for two identical sites and site/site interactions with the interaction free energy--RT ln alpha, and the intrinsic binding constant K and cooperativity parameter alpha were computed. Double-label experiments showed that cGMP is strictly competitive with cAMP, and its binding parameters K and alpha are not very different from that for cAMP. Since two binding sites exist for each of the cyclic nucleotides in dimeric CRP and no change in the quaternary structure of the protein is observed on binding the ligands, it is proposed that the cooperativity originates in ligand/ligand interactions. When bound to double-stranded deoxyribonucleic acid (dsDNA), CRP binds cAMP more efficiently, and the cooperativity is positive even in conditions of low ionic strength where it is negative for the free protein. By contrast, cGMP binding properties remained unperturbed in dsDNA-bound CRP. Neither the intrinsic binding constant K nor the cooperativity parameter alpha was found to be very sensitive to changes of pH between 6.0 and 8.0 at 0.2 M ionic strength and 20 degrees C. For these conditions, the intrinsic free energy and entropy of binding of cAMP are delta H degree = -1.7 kcal . mol-1 and delta S degree = 15.6 eu, respectively.

No abstract

The cyclic adenosine 3',5'-monophosphate receptor protein of Escherichia coli (CRP) binds cooperatively to single- and double-stranded DNA. Binding data could be fitted to the model of McGhee and von Hippel (1) and show that neither strandedness of DNA, nor the effectors cAMP and cGMP or the ionic strength (KCl) do change appreciably the cooperativity parameter omega (omega approximately or equal to 100), and site size of DNA. Instead, distinctly different slopes were observed for the linear decrease of log K omega (a measure of the overall affinity) as a function of log (K+). From these double-log plots (2), the number of cations released and the non-electrostatic contributions to the binding free energy could be determined. Binding of CRP to single-stranded DNA is slightly favored under physiological ionic conditions (0.15-0.20 M), but such a preferential binding is almost abolished in the presence of cAMP which increases the strength of the interaction of the protein with both forms of DNA. CGMP does not change the binding properties and interactions of CRP with DNA. These observations do not support the proposal that the cAMP-CRP complex could stimulate transcription via some "melting" property unless its interactions be dramatically changed when it binds specifically to promoter DNA.

No abstract

No abstract

The regulatory protein CRP (or CAP) from E. coli is shown to display two distinct patterns of binding interactions with DNA-dependent RNA polymerase. The free core enzyme, and both the core and the holo polymerase when bound to single-stranded DNA, can bind CRP in a cAMP-independent association reaction. Instead, the binding of CRP to free holoenzyme and to holo or core polymerase bound to native DNA was undetectable in the absence of cAMP. The specific ligand of CRP (cAMP) strengthens distinctively this class of interactions. In no case could any release of sigma-factor be demonstrated. Estimates of the dissociation constants were obtained for the various binding reactions which were investigated under quasi-physiological ionic conditions. These, together with the known values of the in vivo concentrations of CRP and RNA polymerase, suggest that the interactions described may have a functional significance.

No abstract