Thermodynamic and kinetic characterization of the interaction of Saccharomyces cerevisiae TATA-binding protein and transcription factor TFIIB with DNA promoters

Abstract

The thermodynamics and association kinetics of the interaction of the TATA binding protein (TBP) with TATA containing DNA promoters, and of transcription factor IIB (TFIIB) with the TBP-TATA complex have been monitored using DNase I quantitative 'footprinting' techniques. The dependence of these interactions on solution conditions has been assessed in order to characterize the chemical driving forces and the mechanism of the reactions and to understand the role the DNA sequence may play in modulating them. The TBP-TATA box interaction and the interaction of TFIIB with the TBP-DNA complex play a central role in the assembly of the pre-initiation complex on genes transcribed by RNA polymerase II. The interaction of TBP with the TATA box of adenovirus E4 and Major Late promoters exhibits an identical dependence on (KCl). The comparable electrostatic contribution to the interaction of TBP with the DNA backbone suggests that the two interfaces are structurally similar, consistent with the identity of TBP-DNA complexes solved by X-ray crystallography. In contrast, the temperature dependence of the interaction of TBP with the two promoters is markedly different. Given the severe distortion TBP imposes on the structure of DNA within the TATA box, the observed temperature dependent effect could reflect differences in the relative flexibility of the two promoters. The ensuing difference in the energetic contributions--{dollar}\rm\Delta H\sp\circ{dollar} and T{dollar}\rm\Delta S\sp\circ{dollar}--would manifest itself as different temperature dependencies. Interestingly, for both promoters, most of the salt dependent effect observed under equilibrium conditions can be accounted for in the association kinetics. This finding, corroborated with the magnitude of the temperature effect on the kinetics of TBP association with the two promoters obtained in parallel studies, identical to that observed under equilibrium conditions, suggests that TBP-DNA interaction follows a common pathway. The transition state for the reaction appears to have most of the structural and energetic features of the final complex. The results obtained in these, and other studies carried out in the laboratory, are consistent with the model proposed for the mechanism of TBP-DNA interaction: the slow kinetics observed for the association of TBP with TATA promoters is the consequence of the unfavorable formation of the diffusion limited TBP-TATA 'encounter complexes'. Consequently, only a small fraction of the DNA is capable of proceeding along the reaction pathway (Parkhurst, K. M., Brenowitz, M. & Parkhurst, L. J. (1996) Biochemistry 35, 7459-7465). The cooperative effect TFIIB exerts on the interaction of TBP with the TATA box of Major Late promoter is relatively modest, consistent with the lability of the TFIIB-TBP-DNA complex inferred from transcriptional studies. In the range of 25 to 100mM KCl, cooperativity is independent of salt concentration. The absence of notable cooperative effects on the association kinetics of TBP under the conditions investigated suggests that the primary function of TFIIB is to stabilize the TBP-DNA complex. Increased stability can be envisaged by inspection of a ternary DNA-TBP-TFIIB complex whose structure has been solved by x-ray crystallography. The small increase in the rate of TBP association in the presence of TFIIB under conditions that also promote TFIIB-DNA interactions, and the more pronounced effect observed at 50mM KCl, point to the issues of DNA sequence and local ionic strength as possible means to fine tune the assembly of RNA polymerase II preinitiation complex.