Intermediate species possessing bent DNA are present along the pathway to formation of a final TBP-TATA complex

Binding of the TATA-binding protein (TBP) to the "TATA" sequences present i n the promoters of eukaryotic class II genes is the first step in the seque ntial assembly of transcription pre-initiation complexes. Myriad structural changes, including severe bending of the DNA, accompany TBP-TATA complex f ormation. A detailed kinetic study has been conducted to elucidate the mech anistic details of TBP binding and DNA bending. The binding of Saccharomyce s cerevisiae TBP to the adenovirus major late promoter (AdMLP) was followed in real-time through a range of temperatures and TBP concentrations using fluorescence resonance energy transfer (FRET) and stopped-flow mixing. The results of association and relaxation kinetics and equilibrium binding expe riments were analyzed globally to obtain the complete kinetic and energetic profile of the reaction. This analysis reveals a complex mechanism with tw o intermediate species, with the DNA in the intermediates apparently bent s imilarly to the DNA in the final complex. TBP binding and DNA bending occur simultaneously through the multiple steps of the reaction. The first and t hird steps in this sequential process show nearly identical large increases in both enthalpy and entropy, whereas the middle step is highly exothermic and proceeds with a large decrease in entropy. The first intermediate is s ignificantly populated at equilibrium and resembles the final complex both structurally and energetically. It is postulated that both this intermediat e and the final complex bind transcription factor IIB in the second step of pol II pre-initiation complex assembly. A consequence of such a reactive i ntermediate is that the rate of assembly of transcriptionally competent pre -initiation complexes from bi-directionally bound TBP is greatly increased. (C) 1999 Academic Press.