Thermodynamic and kinetic characterization of the binding of the TATA binding protein to the adenovirus E4 promoter

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Abstract

A thermodynamic analysis of the binding of the TATA binding protein (TBP) from Saccharomyces cerevisiae to the adenovirus E4 promoter was conducted using quantitative DNase I "footprint" titration techniques. These studies were conducted to provide a foundation for studies of TBP structure-function relations and its assembly into transcription preinitiation complexes. The binding of TBP to the E4 promoter is well described by the Langmuir binding polynomial, suggesting that no linked equilibria contribute to the binding reaction under the conditions examined. Van't Hoff analysis yielded a nonlinear dependence on temperature with the TBP-E4 promoter interaction displaying maximal affinity at 30 °C. An unusually negative value of the apparent standard heat capacity change, ΔCP ° = -3.5 ± 0.5 kcal/mol·K, was determined from these data. The dependence of the TBP-E4 promoter interaction on [KCl] indicates that 3.6 ± 0.3 K+ ions are displaced upon complex formation. Within experimental error, no linkage of proton binding with the TBP-E4 promoter interaction is detectable between pH 5.9 and 8.7. Rates of association of TBP for the E4 promoter were obtained using a novel implementation of a quench-flow device and DNase I "footprinting" techniques. The value determined for the second-order rate constant at pH 7.4, 100 mM KCl, 5 mM MgCl2, 1 mM CaCl2, 30 °C (ka = (5.2 ± 0.5) × 105 M-1 s-1) confirms the results obtained by Hawley and co-workers [Hoopes, B. C., LeBlanc, J. F., & Hawley, D. K. (1992) J. Biol. Chem. 267, 11539-11547] and extends them through TBP concentrations of 636 nM. The Arrhenius plot of the rate of TBP association is also nonlinear, yielding ΔCP ° = -3.2 ± 0.3 kcal/mol·K. Thus, the ΔCP ° associated with the TBP-E4 promoter interaction appears associated principally, if not exclusively, with the rate-limiting step of binding. The consequences of these results for the interpretation of structural and biochemical studies of TBP-promoter complex formation are discussed.

Original languageEnglish (US)
Pages (from-to)9977-9984
Number of pages8
JournalBiochemistry
Volume34
Issue number31
StatePublished - 1995

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TATA-Box Binding Protein
Thermodynamics
Adenoviridae
Kinetics
Deoxyribonuclease I
Association reactions
Arrhenius plots
Magnesium Chloride
Transcription
Titration
Yeast
Specific heat
Saccharomyces cerevisiae
Protons
Rate constants
Hot Temperature

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Thermodynamic and kinetic characterization of the binding of the TATA binding protein to the adenovirus E4 promoter",
abstract = "A thermodynamic analysis of the binding of the TATA binding protein (TBP) from Saccharomyces cerevisiae to the adenovirus E4 promoter was conducted using quantitative DNase I {"}footprint{"} titration techniques. These studies were conducted to provide a foundation for studies of TBP structure-function relations and its assembly into transcription preinitiation complexes. The binding of TBP to the E4 promoter is well described by the Langmuir binding polynomial, suggesting that no linked equilibria contribute to the binding reaction under the conditions examined. Van't Hoff analysis yielded a nonlinear dependence on temperature with the TBP-E4 promoter interaction displaying maximal affinity at 30 °C. An unusually negative value of the apparent standard heat capacity change, ΔCP ° = -3.5 ± 0.5 kcal/mol·K, was determined from these data. The dependence of the TBP-E4 promoter interaction on [KCl] indicates that 3.6 ± 0.3 K+ ions are displaced upon complex formation. Within experimental error, no linkage of proton binding with the TBP-E4 promoter interaction is detectable between pH 5.9 and 8.7. Rates of association of TBP for the E4 promoter were obtained using a novel implementation of a quench-flow device and DNase I {"}footprinting{"} techniques. The value determined for the second-order rate constant at pH 7.4, 100 mM KCl, 5 mM MgCl2, 1 mM CaCl2, 30 °C (ka = (5.2 ± 0.5) × 105 M-1 s-1) confirms the results obtained by Hawley and co-workers [Hoopes, B. C., LeBlanc, J. F., & Hawley, D. K. (1992) J. Biol. Chem. 267, 11539-11547] and extends them through TBP concentrations of 636 nM. The Arrhenius plot of the rate of TBP association is also nonlinear, yielding ΔCP ° = -3.2 ± 0.3 kcal/mol·K. Thus, the ΔCP ° associated with the TBP-E4 promoter interaction appears associated principally, if not exclusively, with the rate-limiting step of binding. The consequences of these results for the interpretation of structural and biochemical studies of TBP-promoter complex formation are discussed.",
author = "Brenowitz, {Michael D.}",
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journal = "Biochemistry",
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TY - JOUR

T1 - Thermodynamic and kinetic characterization of the binding of the TATA binding protein to the adenovirus E4 promoter

AU - Brenowitz, Michael D.

PY - 1995

Y1 - 1995

N2 - A thermodynamic analysis of the binding of the TATA binding protein (TBP) from Saccharomyces cerevisiae to the adenovirus E4 promoter was conducted using quantitative DNase I "footprint" titration techniques. These studies were conducted to provide a foundation for studies of TBP structure-function relations and its assembly into transcription preinitiation complexes. The binding of TBP to the E4 promoter is well described by the Langmuir binding polynomial, suggesting that no linked equilibria contribute to the binding reaction under the conditions examined. Van't Hoff analysis yielded a nonlinear dependence on temperature with the TBP-E4 promoter interaction displaying maximal affinity at 30 °C. An unusually negative value of the apparent standard heat capacity change, ΔCP ° = -3.5 ± 0.5 kcal/mol·K, was determined from these data. The dependence of the TBP-E4 promoter interaction on [KCl] indicates that 3.6 ± 0.3 K+ ions are displaced upon complex formation. Within experimental error, no linkage of proton binding with the TBP-E4 promoter interaction is detectable between pH 5.9 and 8.7. Rates of association of TBP for the E4 promoter were obtained using a novel implementation of a quench-flow device and DNase I "footprinting" techniques. The value determined for the second-order rate constant at pH 7.4, 100 mM KCl, 5 mM MgCl2, 1 mM CaCl2, 30 °C (ka = (5.2 ± 0.5) × 105 M-1 s-1) confirms the results obtained by Hawley and co-workers [Hoopes, B. C., LeBlanc, J. F., & Hawley, D. K. (1992) J. Biol. Chem. 267, 11539-11547] and extends them through TBP concentrations of 636 nM. The Arrhenius plot of the rate of TBP association is also nonlinear, yielding ΔCP ° = -3.2 ± 0.3 kcal/mol·K. Thus, the ΔCP ° associated with the TBP-E4 promoter interaction appears associated principally, if not exclusively, with the rate-limiting step of binding. The consequences of these results for the interpretation of structural and biochemical studies of TBP-promoter complex formation are discussed.

AB - A thermodynamic analysis of the binding of the TATA binding protein (TBP) from Saccharomyces cerevisiae to the adenovirus E4 promoter was conducted using quantitative DNase I "footprint" titration techniques. These studies were conducted to provide a foundation for studies of TBP structure-function relations and its assembly into transcription preinitiation complexes. The binding of TBP to the E4 promoter is well described by the Langmuir binding polynomial, suggesting that no linked equilibria contribute to the binding reaction under the conditions examined. Van't Hoff analysis yielded a nonlinear dependence on temperature with the TBP-E4 promoter interaction displaying maximal affinity at 30 °C. An unusually negative value of the apparent standard heat capacity change, ΔCP ° = -3.5 ± 0.5 kcal/mol·K, was determined from these data. The dependence of the TBP-E4 promoter interaction on [KCl] indicates that 3.6 ± 0.3 K+ ions are displaced upon complex formation. Within experimental error, no linkage of proton binding with the TBP-E4 promoter interaction is detectable between pH 5.9 and 8.7. Rates of association of TBP for the E4 promoter were obtained using a novel implementation of a quench-flow device and DNase I "footprinting" techniques. The value determined for the second-order rate constant at pH 7.4, 100 mM KCl, 5 mM MgCl2, 1 mM CaCl2, 30 °C (ka = (5.2 ± 0.5) × 105 M-1 s-1) confirms the results obtained by Hawley and co-workers [Hoopes, B. C., LeBlanc, J. F., & Hawley, D. K. (1992) J. Biol. Chem. 267, 11539-11547] and extends them through TBP concentrations of 636 nM. The Arrhenius plot of the rate of TBP association is also nonlinear, yielding ΔCP ° = -3.2 ± 0.3 kcal/mol·K. Thus, the ΔCP ° associated with the TBP-E4 promoter interaction appears associated principally, if not exclusively, with the rate-limiting step of binding. The consequences of these results for the interpretation of structural and biochemical studies of TBP-promoter complex formation are discussed.

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