UNIVERSITY OF ALABAMA AT BIRMINGHAM
Grant: $219,719 - National Institutes of Health - Sep. 25, 2009
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Award Description: This award is a supplement 3R01GM033143-22S1 ARRA, which is entitled Long Range DNA Interactions in Mu and Bacterial DNA. The start date is 09/30/2009 and this project will end on 08/31/2011. The full amount to be distributed over 2 years is $219,719. The supplement will create one job for a person who will either have experience or be trained to carry out bacterial growth in fermentors. The new person will then carry out steps necessary to isolate at least 30 gyrase subunits in a state of high biochemical purity. The subunits will be used to reconstitute different forms of DNA gyrase and will be used to define the mechanism(s) that control supercoil density in bacterial chromosomes.
Project Description: Chromosome dynamics and the proteins that channel DNA movement in vivo are critical determinants of cell replication, gene expression, genetic recombination, and Darwinian evolution. The primary focus of our research is identifying critical genes and dissecting the mechanisms that control bacterial chromosome structure and supercoil movement. Three specific aims are: 1) Connect the activities of gyrase and the bacterial condensin, MukB, to nucleoid compaction. DNA gyrase subunits will be genetically modified to understand the E. coli/Salmonella species difference in supercoil density. Candidate ?Domainins? or proteins that control a segment of chromosome structure will be run through a gauntlet of 4 tests to measure domain behavior. 2) Connect domains and DNA movement to structure. We are testing a loop model of domain structure and will define the dynamic characteristics of highly transcribed ribosomal RNA operons. As cells grow rapidly in rich medium, 70% of all RNA synthesis is devoted to stable RNAs (ribosomal and tRNA genes). We will establish whether or not transcription in WT bacteria can generate ?waves of positive supercoils?. 3) Connect the average chromosome structure to single cell behavior using fluorescent cell technology. Results from the first year prove two critical and surprising points. 1) The gyrB gene is the primary controller of supercoil density in E. coli and Salmonella. 2) This gene differs between the species at only 28 of 841 residues. One (or a subset) of these differences controls supercoil density in living cells. We will create chimeric genes containing all single amino substitutions individually and in clusters to map the critical protein residues that control the supercoil set point. We will use ARRA support to hire/retain one technician whose role will be to synthesize, produce, and purify each new protein in the UAB fermentation center.
Jobs Summary: Project not Started (Total jobs reported: 0)
Project Status: Not Started
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