Entropic Nature of the Interaction between Promoter Bound CRP Mutants and RNA Polymerase

Abstract

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.

Fluorescence quenching and kinetic studies of conformational changes induced by DNA and cAMP binding to cAMP receptor protein from Escherichia coli

“…As this residue is located close to the activation region, AR2, which is responsible for the interaction with the RNA polymerase, it is tempting to suggest that the binding of CRP to the DNA promoter in solution involves a further conformational signal transduction from the C-terminal domain to the N-terminal domain of CRP, and the magnitude of this conformational transduction solely depends on the promoter DNA sequence responsible for this interaction. This suggestion is in agreement with small angle neutron scattering measurements of the CRP-DNA complex, which indicate that this structural change in the N-terminal domain of the protein occurs upon binding of DNA to the C-terminal domain of CRP [30]. Our fluorescence studies of CRP-DNA interactions presented here also agree with the results of Baichoo & Heyduk [31], which were obtained by protein footprinting techniques.…”

Prokaryotic genome regulation: multifactor promoters, multitarget regulators and hierarchic networks

“…Regulons under the control of global regulators (or multigene regulators) include a large number of genes or operons, in which the genes for other transcription factors are often included, together forming hierarchic networks of transcription factors. and is the best-characterized global regulator from E. coli (Kolb et al, 1993;Harman, 2001;Krueger et al, 2003). The total number of promoters that have been identified to be under the control of cAMP-CRP is now reaching to 100 (Gama-Castro et al, 2008), acting as activators or repressors depending on the position of CRP binding (Kolb et al, 1993).…”

Prokaryotic genome regulation: A revolutionary paradigm

“…cAMP receptor protein CRP, also called catabolite gene activator protein CAP, was the first purified transcription activator, 120) and is the best-characterized global regulator involved in the regulation of genes for transport and utilization of carbon sources. 121–123) CRP is a dual regulator, acting as an activator or a repressor depending on the position of CRP binding relative to promoters. 122) In the absence of glucose, cAMP is synthesized, which associates CRP for its conversion into the active regulator in transcription.…”