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 (T BP) from Saccharomyces cerevisiae to the adenovirus E4 promoter was co nducted using quantitative DNase I ''footprint'' titration techniques. These studies were conducted to provide a foundation for studies of T BP structure-function relations and its assembly into transcription pr einitiation complexes. The binding of TBP to the E4 promoter is well d escribed by the Langmuir binding polynomial, suggesting that no linked equilibria contribute to the binding reaction under the conditions ex amined. Van't Hoff analysis yielded a nonlinear dependence on temperat ure with the TBP-E4 promoter interaction displaying maximal affinity a t 30 degrees C. An unusually negative value of the apparent standard h eat capacity change, Delta C-p(o) = -3.5 +/- 0.5 kcal/mol . K, was det ermined from these data. The dependence of the TBP-E4 promoter interac tion on [KCl] indicates that 3.6 +/- 0.3 K+ ions are displaced upon co mplex formation. Within experimental error, no linkage of proton bindi ng with the TBP-E4 promoter interaction is detectable between pH 5.9 a nd 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 ''foo tprinting'' 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 (k(a) = (5.2 +/- 0.5) x 10(5) M(-1) s(-1)) confirms the results obtain ed by Hawley and co-workers [Hoopes, B. C., LeBlanc, J. F., and Hawley , D. K. (1992) J. Biol. Chem. 267, 11539-11547] and extends them throu gh TBP concentrations of 636 nM. The Arrhenius plot of the rate of TBP association is also nonlinear, yielding Delta C-p(o) = -3.2 +/- 0.3 k cal/mol . K. Thus, the Delta C-p(o) associated with the TBP-E4 promote r 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-pr omoter complex formation are discussed.