Proteins interacting with supercoiled DNA: implications for nucleoid structure and genetic expression in Escherichia coli.

Our aim is to elucidate certain aspects of gene expression as it relates to the local nucleoid structure in E. coli. Our objectives are 1) to characterize two important nucleoid-associated proteins (H-NS and HU) and their interactions with supercoiled DNA plasmids in vitro, and 2) to determine to what extent changes in the structure of supercoiled E. coli DNA induced by these adhering proteins can affect transcription of a particular gene. Ultimately, we also hope to infer from these experiments, a better understanding of how transcription impacts on the dynamics of the supercoiled nucleoid. Interactions among the proteins (either H-NS or HU) will be characterized using osmotic pressure and light-scattering measurements. Their influence on the structure and dynamics of supercoiled plasmids will then be studied in vitro using dynamic light scattering. The data will be interpreted in terms of statistical physical theories for supercoils constrained by adhering proteins, that we will develop. In vivo modification of the nucleoid structure in the vicinity of an inducible gene will be visualized with the help of fluorescently labeled repressor proteins that bind to a long stretch of operator sequences, inserted into the chromosome, next to the inducible gene. The impact of H-NS and HU will be investigated by labeling these proteins. Precise measurements of the size of the fluorescent spots will be made using the novel technique of spatially modulated illumination (SMI) microscopy. Isolating the nucleoids using a gentle lysis procedure will allow us to further probe the nucleoid structure and thermodynamics by exposing them to various external buffers.