Mechanisms of Cooperative Protein-DNA Interactions

Project: Research project

Project Details


DESCRIPTION (provided by applicant): The TATA Binding Protein (TBP) plays a central role in the initiation of gene transcription in eukaryotes. In addition, unusual structural features of TBP and its complexes with DNA and regulatory proteins are reflected in equally unusual thermodynamics and kinetics of DNA binding. This concordance of unusual structural and functional properties permits fundamental questions of biophysical chemistry to be addressed in a system of high biological significance. The proposed studies apply both novel and well-established approaches to the study of protein and nucleic acid structure and the interaction of these macromolecules. Specific Aim I seeks to determine whether interdomain interactions within native TATA Binding Protein (TBP) regulate its sequence-specific DNA binding and if so, how. Radiolytic (hydroxyl radical) "protein footprinting" and intrinsic protein fluorescence will be used to map in solution the structures of the nonconserved N-terminal and conserved C-terminal domains of TBP and their interaction. Specific Aim II explores the mechanism(s) do the proteins NC2 and Mot1 positively and negatively regulate S. cerevisiae TBP-promoter interactions. Mot1 is unusual in that it uses the energy derived from ATP hydrolysis to dissociate the long-lived TBP-TATA Box complex from promoters. Radiolytic footprinting, analytical ultracentrifugation and equilibrium and quench-flow DNase I footprinting will be used to explore these cooperative regulatory interactions. Specific Aim III seeks to determine the mechanism by which the protein complex that specifies transcription by RNA Polymerase IIII, TFIIIB (i.e., TBP-TFIIIB70-TFIIIB90), achieve unusual stability when bound to DNA compared to its homolog in the RNA Polymerase II system, TBP-TFIIB. The long-term goal of this research program is to obtain a comprehensive understanding of the physical principles underlying the initiation of gene transcription and its regulation.
Effective start/end date7/1/9411/30/07


  • National Institute of General Medical Sciences: $375,750.00
  • National Institute of General Medical Sciences: $366,920.00
  • National Institute of General Medical Sciences: $375,750.00
  • National Institute of General Medical Sciences: $372,400.00


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