Entropic nature of the interaction between promoter bound CRP mutants and RNA polymerase

The interaction between CRP, T127L, S128A, and CRP* and RNA polymerase bound to a 104 bp synthetic promoter were determined by ITC at 298 K and ranges from a DeltaG(b)degrees = 1.4 +/- 0.8 kJ mol(-1) (cAMP-ligated S128A) to 4.5 +/- 0.3 kJ mol(-1) (cAMP-ligated double mutant CRP*) with endothermicities that range from 4 +/- 3 kJ mol(-1) (cAMP-ligated CRP) to 47 +/- 8 kJ mol(-1) (cGMP-ligated T127L). The interaction is, thus, entropically driven, exhibits enthalpy-entropy compensation, and increases the binding affinity of the RNA polymerase to the promoter by factors ranging from 1.7 +/- 0.1 (cAMP-ligated S128A) to 6.1 +/- 0.1 (cAMP-ligated CRP*). Although the binding affinities to the promoter alone, except for cAMP-ligated S128A, are the same as to a shorter 40 bp duplex containing the same CRP consensus binding site sequence (conDNA), the binding enthalpies of CRP/mutant to the promoter are lower by factors of 2-3x than the corresponding binding enthalpies to conDNA. Small angle neutron scattering measurements on the DNA-CRP/mutant complexes in D2O/H2O solutions exhibit an increase in the Rg of the CRP/mutant component from 22 to 27-31 Angstrom that can be attributed to a conformational change in the N-terminal domain of CRP. The Rg = 27 Angstrom for the bound conDNA can be attributed to a slight unwinding of the DNA in solution that would also enhance the activation of transcription. The Rg = 53 +/- 3 Angstrom for the bound promoter is attributed to bending of the promoter in solution that can be responsible for the lower CRP/mutant-promoter binding endothermicities.