Understanding the mechanism by which proteins recognize, and bind to specific sequences of nucleic acids is an important goal of contemporary biophysics. Techniques such as x-ray crystallography, NMR and fluorescence spectroscopy and quantitative nuclease protection footprinting studies have provided detailed pictures of the beginning and end states of reactions that result in the formation of complexes of proteins and nucleic acids. For many sequence specific DNA-protein complexes, changes in DNA structure during the reaction is a critical part of the reaction mechanism. The goal is to detect and analyze at high resolution the intermediate steps in the formation of protein-DNA complexes. High energy x-ray beams provide the means by which the binding of proteins to specific DNA sequences can be followed with millisecond time resolution. The time-resolved x-ray footprinting techniques will bridge the gaps in the thermodynamic and structural analysis of protein-DNA interactions in three ways. (1) Direct experimental correlations between kinetic processes and DNA conformational changes will be obtained. Complex formation can be followed at single base pair resolution so that structural information will be available. (2) The ability to measure binding at each individual site when multiple binding-sites are present allows the contribution of cooperative interactions to be uniquely discerned. (3) Millisecond time resolutions will allow quantitative analysis of protein-protein reactions linked to the protein-DNA reactions. Time resolved x-ray footprinting will be developed and validated using several well characterized protein-DNA systems. The technique will then be extended to the analysis of protein required for the initiation of gene transcription in eukaryotes; the TATA binding protein (TBP) and a transcription factor with which it interacts, TFIIB. This approach will provide time-resolved quantitative structure-function correlations for the analysis of DNA structure changes applicable to a variety of important biomedical problems in cellular regulation.
