A continuing long term goal of this project is to understand the assembly, control, and function of nucleoprotein complexes that promote Hin-catalyzed site-specific DNA inversion. Work during the past funding period established the overall structure of the tetrameric catalytic core of the Hin synaptic complex and provided strong evidence that recombination of DNA strands was mediated by translocating Hin subunits between dimers. Future work will entail an ensemble of genetic, biochemical, and structural approaches that are directed at obtaining higher resolution views of the structural changes that occur upon synapsis and the mechanism(s) by which Fis-activates DNA catalysis and exchange. The crucial yet varied roles of accessory chromatin proteins has emerged as a second prominent theme of this work. Fis (Factor for Inversion Stimulation) was discovered because of its essential regulatory role in the Hin and Gin DNA inversion reactions, but it is now known to function in a large number of DNA reactions. We will investigate how Fis collaborates with the Xis protein of phage lambda to stimulate phage excision, with the ultimate goal of obtaining a detailed understanding of binding cooperativity and DNA structural changes that control the directionality of lambda site-specific recombination. Cellular Fis levels vary enormously with respect to growth phase and growth rates: Fis is the most abundant transcriptional regulator in E. coli under rapid growth conditions, whereas it is virtually absent in stationary phase. We will profile genome-wide effects of Fis on gene expression and protein levels under different growth conditions and molecularly dissect potentially novel regulatory units. Fis activates transcription by specifically interacting with the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). X-ray crystal structures of Fis-DNA and Fis-alphaCTD-DNA complexes will be pursued. In addition to forming stable complexes at specific DNA binding sites, Fis also interacts nonspecifically with DNA at physiological concentrations and may play important roles in modulating chromosome structure because of its DNA-bending and looping activities. Fis effects on chromosome structure will be collaboratively addressed by single DNA molecule approaches as well as by bulk-phase in vitro and in vivo experiments.
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