Linkage of multiequilibria in DNA recognition by the D53H Escherichia coli cAMP receptor protein

Shwu Hwa Lin, James Lee

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

The transcription factor cyclic AMP receptor protein, CRP, regulates the operons that encode proteins involved in translocation and metabolism of carbohydrates in Escherichia coli. The structure of the CRP-cAMP complex reveals the presence of two sets of cAMP binding sites. Solution biophysical studies show that there are two high-affinity and two low-affinity binding sites, to which the binding of cAMP is characterized by varying degrees of cooperativity. A stoichiometry of four implies that potentially CRP can exist in five conformers with different numbers of bound cAMP. These conformers may exhibit differential affinities for specific DNA sequences. In this study, the affinity between DNA and each conformer of D53H CRP was defined through a dissection of the thermodynamic linkage scheme that included all the conformers. Loading of the high- and low-affinity sites with cAMP leads to high and low affinity for DNA, respectively. The specific magnitude of the binding constants of these conformers is DNA sequence dependent. The various association constants defined by the present study provide a solution to address an enigma of the CRP system, namely, the 3 orders of magnitude difference between the cAMP binding constants determined by in vitro studies and the cAMP concentration regime to which the bacteria respond. Under physiological conditions, the apo-CRP-DNA complex is the dominant species. As a consequence of the 1000-fold stronger affinity of cAMP to the apo-CRP-DNA complex than that to CRP, the relevant reaction is the binding of cAMP to this DNA - protein complex. The binding constant is of the order of 107 M-1, the same concentration regime as that of cellular concentration of cAMP. In addition, under physiological conditions the species that binds to the lac and gal operons is predicted to be CRP-(cAMP)1. A comparison of parameters between the wild type and the mutant CRP shows that the mutation apparently shifts the various thermodynamically linked equilibria without a change in the basic mechanism that governs CRP activities. Thus, the conclusions derived from a study of the mutant are relevant to wild-type CRP. A dissection of the individual binding constants in this multiequilibria reaction scheme leads to a definition of the mechanism of action of this transcription factor.

Original languageEnglish (US)
Pages (from-to)14935-14943
Number of pages9
JournalBiochemistry
Volume41
Issue number50
DOIs
StatePublished - Dec 17 2002

Fingerprint

Cyclic AMP Receptor Protein
Escherichia coli Proteins
Escherichia coli
DNA
Dissection
DNA sequences
Transcription Factors
Binding Sites
Lac Operon
Carbohydrate Metabolism
Operon
Thermodynamics
Metabolism
Stoichiometry
Bacteria
Proteins
Carbohydrates
Association reactions
Mutation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Linkage of multiequilibria in DNA recognition by the D53H Escherichia coli cAMP receptor protein. / Lin, Shwu Hwa; Lee, James.

In: Biochemistry, Vol. 41, No. 50, 17.12.2002, p. 14935-14943.

Research output: Contribution to journalArticle

Lin, Shwu Hwa ; Lee, James. / Linkage of multiequilibria in DNA recognition by the D53H Escherichia coli cAMP receptor protein. In: Biochemistry. 2002 ; Vol. 41, No. 50. pp. 14935-14943.
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abstract = "The transcription factor cyclic AMP receptor protein, CRP, regulates the operons that encode proteins involved in translocation and metabolism of carbohydrates in Escherichia coli. The structure of the CRP-cAMP complex reveals the presence of two sets of cAMP binding sites. Solution biophysical studies show that there are two high-affinity and two low-affinity binding sites, to which the binding of cAMP is characterized by varying degrees of cooperativity. A stoichiometry of four implies that potentially CRP can exist in five conformers with different numbers of bound cAMP. These conformers may exhibit differential affinities for specific DNA sequences. In this study, the affinity between DNA and each conformer of D53H CRP was defined through a dissection of the thermodynamic linkage scheme that included all the conformers. Loading of the high- and low-affinity sites with cAMP leads to high and low affinity for DNA, respectively. The specific magnitude of the binding constants of these conformers is DNA sequence dependent. The various association constants defined by the present study provide a solution to address an enigma of the CRP system, namely, the 3 orders of magnitude difference between the cAMP binding constants determined by in vitro studies and the cAMP concentration regime to which the bacteria respond. Under physiological conditions, the apo-CRP-DNA complex is the dominant species. As a consequence of the 1000-fold stronger affinity of cAMP to the apo-CRP-DNA complex than that to CRP, the relevant reaction is the binding of cAMP to this DNA - protein complex. The binding constant is of the order of 107 M-1, the same concentration regime as that of cellular concentration of cAMP. In addition, under physiological conditions the species that binds to the lac and gal operons is predicted to be CRP-(cAMP)1. A comparison of parameters between the wild type and the mutant CRP shows that the mutation apparently shifts the various thermodynamically linked equilibria without a change in the basic mechanism that governs CRP activities. Thus, the conclusions derived from a study of the mutant are relevant to wild-type CRP. A dissection of the individual binding constants in this multiequilibria reaction scheme leads to a definition of the mechanism of action of this transcription factor.",
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