Transcription Control Currently, we focus on: (i) RNAP of E. coli and the RNAP-associated proteins RapA and SspA; and (ii) the mechanism of the global change in the transcription pattern associated with nutrient starvation known as the stringent response. In addition, we recently initiated a basic research project to study transcriptional regulation in the pathogenesis of H. pylori, which is an important risk factor for gastric cancer. RNAP Despite extensive genetic, biochemical and structural studies on RNAP, little was known about the location and distribution of RNAP in E. coli under different physiological conditions. We initiated a cell biology approach to visualize the RNAP in E. coli. We constructed a functional rpoC-gfp gene fusion on the chromosome and used fluorescence techniques to image RNAP. Our results show that RNAP is located either within or surrounding the nucleoid; and that the distribution of RNAP is dynamic and influenced dramatically by environmental cues, such as growth conditions and nutrient starvation. Moreover, our results indicate that stable RNA synthesis is a driving force for the distribution of RNAP and plays an important role in the compactness of the nucleoid in vivo . From these studies, we proposed a working model coupling global gene regulation to chromosome condensation in bacteria. Thus, our study not only sheds new light on the regulation of the stringent response, but also potentially opens a new avenue from which to study the effect of transcription on other cellular functions. Our current and future studies are to test different aspects of the working model. Transcription fidelity is an important but understudied process. We have continued to identify sites in RNAP which are important for transcriptional fidelity. We have developed genetic systems to isolate and characterize RNAP mutants with an altered transcriptional fidelity phenotype in active collaborations with other PIs in GRCBL including Drs. Court, Kashlev and Strathern. Preliminary results showed that mutations in RNAP form E. coli and those in the eukaryotic RNAP, Pol II from yeast, are located at similar or identical positions in the structures. Currently, we are isolating additional RNAP mutants. RNAP-Associated Proteins We have continued to study two RNAP associated proteins, RapA and SspA. RapA is an ATPase, which is stimulated by the interaction with RNAP. RapA activates transcription by stimulating RNAP recycling. Interestingly, RapA is a member of the SWI/SNF superfamily of helicase-like proteins, which was first identified in yeast and is conserved from bacteria to humans. ATP-dependent chromatin remodeling by these proteins is an important aspect of transcriptional regulation of many genes. Defects in these proteins are associated with a variety of human diseases and tumors. The tumor suppressor proteins Rb and BRCA1 have been shown to be present in complexes with the SWI/SNF proteins. Mutations in the BRG1 (a SNF2 homologue) or hSNF5/INI1 genes are found in many human cancers. Our finding that RapA is a SWI2/SNF2 related protein in E. coli, and associated with RNAP provides us with a unique opportunity to study the basic mechanism of this protein in highly tractable genetic and biochemically defined systems. SspA is highly conserved among Gram-negative bacteria and its homologs have been shown to play a role in pathogenesis. However, the function of SspA in transcription remains unclear. Our study found that SspA acts as a transcriptional activator for phage P1 late genes. Also, SspA functions as a global regulator, which derepresses multiple stress defense systems including those for acid tolerance in stationary phase, by down-regulation of H-NS in E. coli. In addition, our results showed that SspA plays a critical role in the pathogenesis of E. coli O157:H7. In collaboration with Dr. Xinhua Ji (MCL, NCI), the crystal structure of SspA was determined. Currently, we are focusing on the identification of the SspA targets both in RNAP and in the cell. Mechanisms of the Stringent Response Understanding the mechanism of the global change in the transcription pattern associated with the stringent response (nutrient starvation) has been a challenging issue in E. coli biology. Previously, by studying RNAP mutants that altered the stringent response we proposed that more free RNAP will be available along the genome allowing increased number of genes to express during the stringent response, a novel concept for the global gene regulation at the time. We have been testing this RNAP redistribution hypothesis using multiple approaches including: (a) isolating and characterizing more RNAP mutants and/or other mutants that alter the stringent response; (b) analyzing transcription profiles during the stringent response; and (c) determining the effects of the stringent response and different growth conditions on the distribution of RNAP inside the cell
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