Molecular Dissection of Chromosome Folding in E coli

Duquesne University, Pittsburgh PA

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Abstract

DESCRIPTION: (provided by applicant): This proposal is concerned with the molecular analysis of the genes and proteins required for chromosome condensation in prokaryotic cells. The ultimate goal of the project is to thoroughly understand the molecular mechanisms of this complex, fundamental process. E. coli is used as the model system for these studies due to the extensive genetic and biochemical tools readily available for experimental use. The intent of the proposal is to study novel and critical cellular components that are required to fold the chromosome into a compact yet functional structure. The components and mechanisms identified in these studies will lead to a greater understanding of how this essential process is accomplished. Given that chromosome folding is essential for cell viability, it is likely that these studies will identify potential new targets for therapeutic agents. Using the novel genetic selection of resistance to the DNA unfolding agent camphor, a previously unknown condensing system containing three components, CrcA. CspE and CrcB, was identified. The central component of this system is CspE, a 69 amino acid, nucleic acid-binding protein. The experiments described in this proposal undertake a complete structural and functional analysis of CspE. Mutations that genetically separate the different phenotypes of cspE will be isolated using two, independent approaches. Pilot studies indicate that one of these approaches can be used successfully. The proteins produced from the wild-type gene and the mutated genes will be isolated and characterized in a series of in vitro assays. Prokaryotic cells contain several small, DNA-binding proteins that play a role in chromosome folding. All of these proteins are involved in many, different cellular processes. In this respect, CspE is yet another member of the class. What sets CspE apart from these others proteins and makes it the most amenable member of the class for the study of a direct role in chromosome folding is the ability to genetically separate its different functions by mutation. The characterization of the mutant CspE proteins described in this proposal will lead to a direct understanding of how this class of small, nucleic acid-binding proteins function in the essential process of chromosome folding.