TATA -binding protein recognition of DNA promoters: Kinetic and thermodynamic studies

Binding of the TATA-binding protein (TBP) to promoter DNA is the requisite initial step in RNA polymerase II transcription initiation. The interactions of TBP with consensus (TATAa/tAa/tN) and diverse variant TATA elements yield dramatic DNA structural changes and a wide range of transcription activities. Our laboratory has been investigating the relationships among TATA sequence, TBP-TATA solution structure, recognition mechanisms and transcription activity. TBP complexes with two consensus promoters, adenovirus Major Later (AdMLP: TATAAAAG) and E4 (TATATATA), have similar DNA geometries and high transcription efficiencies. These two binary interactions have been modeled by global analysis of detailed kinetic and thermodynamic data obtained using fluorimetric and fluorometric techniques in conjunction with fluorescence resonance energy transfer. TBP recognizes both promoters via a linear two-intermediate mechanism with simultaneous DNA binding and bending. However, important step-wise kinetic and thermodynamic differences distinguish the mechanisms. In contrast, recognition by TBP of a variant sequence (C7: TATAAACG) is described by a three-step model with two branching pathways. One pathway proceeds through an intermediate having severely bent DNA, reminiscent of the consensus interactions, with the other branch yielding a unique conformer with shallowly bent DNA. The resulting TBP·C7 complex has a dramatically different solution conformation and diminished relative transcription efficiency. These mechanistic similarities and differences correlate explicitly with sequence-directed structural features inherent to each of these duplexes. The intermediate TBP·TATA species in these mechanisms are proposed to be biologically relevant, consistent with their energetic stability and abundance over the course of these reactions. Evidence presented suggests that these conformers nucleate assembly of the pre-initiation complex. Also, TBP displacement by regulatory proteins may occur from these species. Finally, time-resolved fluorescence emission responses are compared for FRET data obtained in the time- and frequency domains. Molecular distances can be precisely determined using both methods. However, the distributions of such distances arising from the dynamic motions of biomolecules are well determined for typical data obtained in the time-domain, but not in the frequency-domain