Protein binding to DNA (linear and plasmid forms) and conformational changes of the DNA upon protein binding are being investigated via atomic force microscopy (AFM), analytical ultracentrifugation (AUC) and other biophysical approaches. This is a broad collaboration with the laboratory of Dr. Sankar Adhya of NCI and several members of his group. The project involves a number of bacterial proteins that are known to play an important gene regulatory role by binding with galactose repressor operators as well as by non-specific binding whereby it is believed that HU plays central role in DNA packaging in bacteria through an unknown mechanism. Detailed description of the relevant complexes would clarify fundamental aspects of gene regulation. We are developing the conditions for optimal visualization of DNA-protein complexes and using AUC to establish stoichiometry and to quantify the binding strength. Topological analysis of AFM images allows the localization of the binding site(s). We continue to develop surface modification protocols that will allow for optimal imaging conditions of the adsorbed DNA/protein complexes. So far, we have examined the binding of the histone-like protein HU to specific DNA plasmids containing the appropriate operators and have established the function of a mutant form of HU from a 2-nucleotide substitution whose expression results in a drastically altered E-coli phenotype. One of the hypotheses for HU function allows for a DNA packaging role of the protein and it was found that the mutant form binds very strongly and non-specifically to DNA causing it to collapse into a tightly packed nucleoid. By studying mutant HU binding to short linear DNA we found that DNA may actually wrap around a protein octamer incontrast to the wildtype whose tetramer simply binds DNA causing slight it to slightly bend. We continue the characterization of the mutant and are finding significant pH dependence of its self-association patterns, which is significant in its role of DNA packaging. We continue investigations of the ternary, GalR/HU/DNA, complex and of the binding of other regulatory proteins such as LacI and eiF to DNA. The introduction of new surface modification techniques will allow fast and optimally controlled imaging of the DNA/protein complexes and will have applicability to any number of other systems that intramural investigators are interested in studying. We plan to continue complementing the AFM studies with the AUC and with the newly acquired Raman spectroscopy. The combined expertise in all three techniques represents a powerful toolbox available to the intramural community.
|Roy, Siddhartha; Dimitriadis, Emilios K; Kar, Sudeshna et al. (2005) Gal repressor-operator-HU ternary complex: pathway of repressosome formation. Biochemistry 44:5373-80|