The formation of unique ensembles of protein-DNA and protein-protein interactions determine which of three RNA polymerases transcribe a gene in eukaryotic organisms. Quantitative studies ar proposed to describe the thermodynamic driving forces and kinetic pathways of these interactions. A focus is the central role of the TATA binding protein (TBP).
Aim I seeks to correlate the energetics of DNA sequence-specific bindin by TBP with the atomic resolution 3D structures of TBP-DNA complexes that differ in their affinity for TBP. An analysis of TBP binding to DNA molecules of altered conformation will assess the contribution of DNA conformation to binding.
Aim II seeks to determine the kinetic mechanism describing formation of the TBP-DNA complexes using complementary stopped-flow fluorescence resonance energy transfer (FRET) and synchrotron x-ray hydroxyl radical footprinting techniques that monitor global and local changes in DNA conformation. The generality of the kinetic mechanism determined will be assessed by conducting selected kinetic studies using TBP from Homo sapiens an Arabidopsis thaliana.
Aim III proposes to determine the contributions of protein-protein and protein-DNA interactions and DNA conformation to the assembly of the minimal pre-initiation complexes specific for RNA polymerase I and III. Protein mutants that specifically affect the intrinsic and cooperativ interactions in the TBP-TFIIB-DNA ternary complex will be analyzed. The protein-protein association reactions linked to the cooperative formation of ternary complexes will be quantitatively analyzed by a new method of protein footprinting.
