Cooperative interactions among proteins bound to specific-sites on the DNA play a key role in regulating transcription initiation, replication and recombination. The goals of the proposed studies are to describe the mechanisms by which cooperative protein-DNA interactions are regulated and determine the contributions of the component protein-protein, protein-DNA and protein-ligand interactions. The two model systems for transcriptional regulation to be studied, the Gal and Lac repressors of Escherichia coli, bind cooperatively to separated sites on the DNA. The studies of Gal repressor focus on understanding how cooperative protein-DN interactions between proteins bound to separated sites are specifically regulated through interaction with a """"""""co-factor"""""""" protein. Cooperative binding by Gal repressor requires the protein H1. Quantitative footprint and gel mobility-shift titration analysis will be used to characterize the functional properties of the H1-Gal repressor-DNA cooperative complex and determine the chemical driving forces that stabilize the complex. Quantitative footprint titrations conducted using chemical probes will determine the positions of the DNA-bound proteins and correlate DNA conformational changes with protein-DNA complex formation. The studies of Lac repressor focus on understanding how """"""""DNA looping"""""""" by proteins is specifically regulated. Cooperative binding by Lac repressor is specifically regulated by the binding of chloride and interaction with Catabolite Activator Protein (CAP), a protein that bends DNA. Analytical gel permeation chromatography will be used to quantitate directly the dimer-tetramer association reaction of Lac repressor. This protein-protein association reaction is responsible for the formation of the Lac repressor-mediated """"""""looped complex"""""""". The binding of DNA by the bidentate Lac repressor tetramer will be analyzed by quantitative mobility-shift analysis in order to determine the mechanism of its allosteric regulation by chloride. Circular permutation and quantitative interference assays will be conducted to determine how Lac repressor attenuates DNA-bending by CAP bound within the """"""""DNA loop"""""""" of the cooperative complex.
