Marked stepwise differences within a common kinetic mechanism characterizeTATA-binding protein interactions with two consensus promoters

Binding of the TATA-binding protein (TBP) to promoter DNA bearing the TATA sequence is an obligatory initial step in RNA polymerase II transcription i nitiation. The interactions. of Saccharomyces cerevisiae TBP with the E4 (T ATATATA) and adenovirus major late (TATAAAAG) promoters have been modeled v ia global analysis of kinetic and thermodynamic data obtained using fluores cence resonance energy transfer. A linear two-intermediate kinetic mechanis m describes the reaction of both of these consensus strong promoters with T BP. Qualitative features common to both interactions include tightly bound TBP-DNA complexes with similar solution geometries, simultaneous DNA bindin g and bending, and the presence of intermediate TBP-DNA conformers at high mole fraction throughout most of the reaction and at equilibrium. Despite v ery similar energetic changes overall, the stepwise entropic and enthalpic compensations along the two pathways differ markedly following the initial binding/bending event. Furthermore, TBP-E4 dissociation ensues from both re placement and displacement processes, in contrast to replacement alone for TBP-adenovirus major late promoter. A model is proposed that explicitly cor relates these similarities and differences with the sequence-specific struc tural properties inherent to each promoter. This detailed mechanistic compa rison of two strong promoters interacting with TBP provides a foundation fo r subsequent comparison between consensus and variant promoter sequences re acting with TBP.