The goal of the proposed biophysical studies is to understand the physical mechanisms by which cooperative DNA-binding proteins regulate the transcription of genes. Thermodynamic binding studies of cI-repressor from phage lambda and gal-repressor form E. coli. to their respective operators will be conducted using the quantitative DNase footprint titration method. This method is unique in that it allows resolution of both the intrinsic and cooperative Gibbs free energies for proteins which bind to multiple, specific-sites on DNA. The Gibbs free energies will be used to test statistical mechanical models on the cooperative interactions. Analysis of both the cI- and gal-repressors will be conducted in order to identify common mechanisms of cooperative interactions in different regulatory systems. The cI-repressor binds to three adjacent sites on the DNA. The proposed studies will focus on determining the mechanism of the cooperative interactions among the three binding sites and determining if the contacts between the repressor and DNA affect the cooperative interactions. In contrast, gal-repressor binds to two specific-sites separated by many turns of the DNA-helix. Studies are proposed to determine if gal-repressor binds cooperatively to DNA and if so, to determine the mechanism. Thermodynamic characterization of the binding and cooperative interactions among gal-repressor, catabolite activator protein (CAP), and RNA polymerase as a function of the allosteric regulatory molecules galactose and cAMP is proposed in order to develop a comprehensive physical- chemical description of the regulation of the goal operon. Studies are also proposed to determined whether DNA-topology affects cooperative binding of both the gal- and cI-repressors. Further development of the footprint titration method is proposed in order to directly measure repressor binding to supercoiled DNA. Other issues to be considered include determining how the geometric relationship of the repressor binding affects interaction between sites, and if local changes in DNA structure play a role in mediating cooperativity.
|Effective start/end date||4/1/88 → 3/31/94|
- National Institute of General Medical Sciences
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