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TATA -binding protein recognition of DNA promoters and TFIIA interactions with TBP -DNA complexes: Kinetic and thermodynamic studies

JoDell E Whittington, University of Nebraska - Lincoln

Abstract

Binding of the TATA-binding protein (TBP) to promoter DNA is the first step required for RNA Polymerase II transcription initiation. The interactions of human and yeast TBP (hTBP and yTBP, respectively) with the consensus Adenovirus Major Late Promoter (AdMLP, TATAAAAG) and the G6-variant TATA (TATAAGAG) elements yield very different DNA structural changes and a wide range of transcription activities. Both time-resolved and steady-state fluorescence resonance energy transfer (FRET) were used to determine the marked differences in the kinetics and energetics of AdMLP binding between the hTBP and yTBP. The DNA bend angle in hTBP-AdMLP was markedly greater than for the corresponding yeast complex. The movement of the N-terminal domain of yTBP bound to AdMLP was also studied. Movement of the TBP N-terminal domain was synchronous with changes in DNA conformation. ^ The interaction of yTBP with the single base pair variant G6 TATA-sequence was also studied. Global analyses of the kinetic and thermodynamic data describing the yTBP+G6 reaction showed that the data were best described by the same two-intermediate pathway as for the yTBP+AdMLP reaction. A comparison of the energetic changes for the two promoters showed marked divergence after the initial binding step. The average bend angle of yTBP-G6 showed a temperature dependence not seen for yTBP-AdMLP. ^ As part of a continuing study of the kinetics and thermodynamics of the first steps in the assembly of the eukaryotic transcription complex, the interactions of yTBP, DNA, and TFIIA (Transcription Factor IIA) were studied. Binding of yTFIIA to yTBP increases the binding constant for AdMLP by 200-fold. ^

Subject Area

Chemistry, Biochemistry|Chemistry, Physical

Recommended Citation

Whittington, JoDell E, "TATA -binding protein recognition of DNA promoters and TFIIA interactions with TBP -DNA complexes: Kinetic and thermodynamic studies" (2008). ETD collection for University of Nebraska - Lincoln. AAI3297952.
http://digitalcommons.unl.edu/dissertations/AAI3297952

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