Interaction of the Escherichia coli gal repressor protein with its DNA operators in vitro

Michael D. Brenowitz, Elizabeth Jamison, Alokes Majumdar, Sankar Adhya

Research output: Contribution to journalArticle

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Abstract

The binding of Escherichia coli Gal repressor to linear DNA fragments containing two binding sites (OE and OI) within the gal operon was analyzed in vitro with quantitative footprint and mobility-shift techniques. In vivo analysis of the regulation of the gal operon [Haber, R., & Adhya, S. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 9683-9687] has suggested the role of a regulatory "looped complex" mediated by the association of Gal repressor dimers bound at OE and OF. The binding of Gal repressor to a single site can be described by a model in which monomer and dimer are in equilibrium and only the dimer binds to DNA. At pH 7.0, 25 mM KCl, and 20 °C, the binding and dimerization free energies are comparable, suggesting that the equilibrium governing the formation of dimers may be important to regulation. The two intrinsic binding constants, ΔG1 and ΔGE, and a constant describing cooperativity, ΔGIE, were determined by footprint titration analysis as a function of pH, [KCl], and temperature. Only at 4 and 0 °C was ΔGIE negative, signifying cooperative binding. These results are thought to be due to a weak dimer to tetramer association interface. ΔGE and ΔGI had maximal values between pH 6 and pH 7. The dependence of these constants on [KCl] corresponded to the displacement of approximately 2 ion equiv. The temperature dependence could be described by a change in the heat capacity, ΔCP, of -2.3 kcal mol-1 deg-1. Mobility-shift titration experiments conducted at 20 and 0 °C yielded values for ΔGIE that were consistent with those resolved from the footprint analysis. Unique values of ΔGIE were determined by analysis of mobility-shift titrations of Gal repressor with wild-type operator subject to the constraint that ΔGE = ΔGI: a procedure that eliminates the need to simultaneously analyze wild-type titrations with titrations of OE- and OI- operators.

Original languageEnglish (US)
Pages (from-to)3374-3383
Number of pages10
JournalBiochemistry
Volume29
Issue number13
StatePublished - 1990

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Titration
Dimers
Escherichia coli
DNA
Operon
Temperature
Association reactions
Dimerization
Hot Temperature
Binding Sites
Free energy
Specific heat
Ions
Monomers
Galactose repressor proteins
In Vitro Techniques
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Interaction of the Escherichia coli gal repressor protein with its DNA operators in vitro. / Brenowitz, Michael D.; Jamison, Elizabeth; Majumdar, Alokes; Adhya, Sankar.

In: Biochemistry, Vol. 29, No. 13, 1990, p. 3374-3383.

Research output: Contribution to journalArticle

Brenowitz, MD, Jamison, E, Majumdar, A & Adhya, S 1990, 'Interaction of the Escherichia coli gal repressor protein with its DNA operators in vitro', Biochemistry, vol. 29, no. 13, pp. 3374-3383.
Brenowitz, Michael D. ; Jamison, Elizabeth ; Majumdar, Alokes ; Adhya, Sankar. / Interaction of the Escherichia coli gal repressor protein with its DNA operators in vitro. In: Biochemistry. 1990 ; Vol. 29, No. 13. pp. 3374-3383.
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AB - The binding of Escherichia coli Gal repressor to linear DNA fragments containing two binding sites (OE and OI) within the gal operon was analyzed in vitro with quantitative footprint and mobility-shift techniques. In vivo analysis of the regulation of the gal operon [Haber, R., & Adhya, S. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 9683-9687] has suggested the role of a regulatory "looped complex" mediated by the association of Gal repressor dimers bound at OE and OF. The binding of Gal repressor to a single site can be described by a model in which monomer and dimer are in equilibrium and only the dimer binds to DNA. At pH 7.0, 25 mM KCl, and 20 °C, the binding and dimerization free energies are comparable, suggesting that the equilibrium governing the formation of dimers may be important to regulation. The two intrinsic binding constants, ΔG1 and ΔGE, and a constant describing cooperativity, ΔGIE, were determined by footprint titration analysis as a function of pH, [KCl], and temperature. Only at 4 and 0 °C was ΔGIE negative, signifying cooperative binding. These results are thought to be due to a weak dimer to tetramer association interface. ΔGE and ΔGI had maximal values between pH 6 and pH 7. The dependence of these constants on [KCl] corresponded to the displacement of approximately 2 ion equiv. The temperature dependence could be described by a change in the heat capacity, ΔCP, of -2.3 kcal mol-1 deg-1. Mobility-shift titration experiments conducted at 20 and 0 °C yielded values for ΔGIE that were consistent with those resolved from the footprint analysis. Unique values of ΔGIE were determined by analysis of mobility-shift titrations of Gal repressor with wild-type operator subject to the constraint that ΔGE = ΔGI: a procedure that eliminates the need to simultaneously analyze wild-type titrations with titrations of OE- and OI- operators.

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