Transcription Control Currently, we focus on: (i) RNAP of E. coli and the RNAP-associated proteins RapA and SspA; and (ii) the mechanisms of the global change in the transcription pattern and RNAP distribution/ redistribution associated with nutrient starvation and other stress responses. In addition, we recently initiated a basic research on transcriptional regulation in H. pylori pathogenesis. 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 the distribution of RNAP is dynamic and influenced dramatically by environmental cues, such as growth conditions and nutrient starvation leading to the stringent response. Also, our results indicate that stable RNA synthesis is a driving force for the distribution of RNAP. For example, during rapid growth RNAP is concentrated into transcription foci or factories, a structure analogous to the eukaryotic nucleolus, where active transcription of rRNA operons occurs, thus, suggesting the importance of rRNA synthesis in affecting the structure of the nucleoid. From these studies, we proposed a working model coupling global gene regulation to chromosome structure in bacteria. Thus, our study not only sheds new light on the regulation of different stress responses including the stringent response, but also potentially opens a new avenue from which to study the effects of transcription and RNAP distribution /redistribution on the structure of the nucleoid and other cellular functions. Our current and future studies are to test different aspects of this 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 continuing to isolate and characterize these RNAP mutants. RNAP-Associated Proteins We have continued to study two RNAP associated proteins, RapA and SspA, with an emphasis on the former. 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. In collaboration with Dr. Xinhua Ji (MCL, NCI), recently the crystal structure of RapA was determined, which is also the first full-length structure for the entire Swi2/Snf2 family. Our kinetic template-switching assay shows that RapA facilitates the release of sequestered RNAP from a posttranscrption/posttermination complex (PTC) for transcription reinitiation, thus, demonstrating the role of RapA in mobilization of nucleic acid-protein complexes to facilitate gene expression. Our in vitro competition experiment indicates that RapA binds to core RNAP only but is readily displaceable by sigma 70. RapA is likely another general transcription factor, the structure of which provides a framework for future studies of this bacterial Swi2/Snf2 protein and its important roles in RNAP recycling during transcription. Conceivably, such studies may also shed lights on how othe Swi/Snf proteins function in general. Mechanisms of the global change in the transcription pattern and RNAP distribution/ redistribution associated with nutrient starvation and other stress responses 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 other stress responses on the distribution of RNAP inside the cell. Transcriptional regulation in the pathogenesis of H. pylori H. pylori is a Gram-negative bacterium responsible for one of the most common bacterial infections, affecting about 50% of the human population. H. pylori is a major causative agent of gastritis, gastric and duodenal ulcers, and gastric cancer, mainly in developing countries and socio-economically disadvantaged subpopulations in the United States. Thus, basic research of H. pylori, aimed at understanding H. pylori pathogenesis, including factors that affect establishment and persistence of infection, is of public health significance. The study of transcriptional regulation in H. pylori is a new initiative with high risk because H. pylori has an entirely different lifestyle from E. coli. Our initial goals are modest, aiming at learning the basic biology of H. pylori and establishing methodologies for studying the bacterium. Our long term goal is to understand transcription control of H. pylori pathogenesis including growth regulation, establishment and persistence of infection. Currently, we are focusing on the role of SpoT, a transcriptional factor, in these processes.
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