Thermodynamic and kinetic characterization of the binding of the TATA binding protein to the adenovirus E4 promoter.
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 degrees C. An unusually negative value of the apparent standard heat capacity change, delta Cp degrees = -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 degrees C (ka = 5.2 +/- 0.5) x 10(5) 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Petri
V
V
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Hsieh
M
M
Brenowitz
M
M
D017504:Adenovirus E4 Proteins / Q000235:genetics*; D001665:Binding Sites; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D011189:Potassium Chloride / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D035181:TATA-Box Binding Protein; D013816:Thermodynamics; D017186:Titrimetry; D014157:Transcription Factors / Q000378:metabolism*
D017504:Adenovirus E4 Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; D011189:Potassium Chloride