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 2-3 x than the corresponding binding enthalpies to conDNA. Small angle neutron scattering measurements on the DNA-CRP/mutant complexes in D(2)O/H(2)O solutions exhibit an increase in the Rg of the CRP/mutant component from 22 to 27-31 A that can be attributed to a conformational change in the N-terminal domain of CRP. The Rg = 27 A 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 A 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.
Krueger
Susan
S
National Institute of Science and Technology Center for Neutron Research, Gaithersburg, Maryland 20899, USA.
Gregurick
Susan
S
Shi
Ying
Y
Wang
Shenglun
S
Wladkowski
Brian D
BD
Schwarz
Frederick P
FP
D002151:Calorimetry / Q000706:statistics & numerical data; D016384:Consensus Sequence; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry* / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D019277:Entropy; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D008024:Ligands; D008956:Models, Chemical; D008958:Models, Molecular; D009010:Monte Carlo Method; D009154:Mutation; D009502:Neutrons; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000235:genetics; D012542:Scattering, Radiation; D055672:Static Electricity
D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D008024:Ligands; D012321:DNA-Directed RNA Polymerases