E. coli RNAPDespite 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. Moreover, how RNAP distribution influences the structure of the bacterial chromosome called the nucleoid is virtually unknown. We initiated research on the cell biology of RNAP to visualize the RNAP in the cell under different physiological conditions. Our results show that RNAP is located exclusively in the nucleoid and its peripheries and the distribution of RNAP is dynamic and influenced dramatically by environmental cues. During optimal growth, concentrated RNAP is actively transcribing rRNA operons, as evidenced by the appearance of transcription foci, at a nucleolus-like structure. In contrast, during amino acid starvation or inhibition of transcription, RNAP is relatively homogenously distributed within the nucleoid. Our recent results also show that active rRNA synthesis is both a driving force for the distribution of RNAP and for the compaction of the nucleoid in the cell. Thus, RNAP (re)distribution has profound effects on nucleoid structure which is important for many vital cellular processes including DNA replication, recombination and cell division. From these studies, we propose a working model which couples RNAP (re)distribution to global gene regulation and the dynamic structure of the nucleoid. We are pursuing this new area of research using multidisciplinary approaches including superresolution imaging and genome conformation capture analyses. We also study transcription fidelity, an important but understudied process in vivo due to intrinsic difficulties of identifying transcriptional fidelity mutations. Taking advantage of the E. coli genetics, we have isolated and characterized RNAP mutants that exhibited an altered transcriptional slippage phenotype during elongation on DNA templates containing homopolymeric A/T runs.
Our aims are to identify the site(s) in RNAP important for transcriptional fidelity and to reveal the mechanism underling transcriptional slippage during elongation. This study is an active collaboration with other PIs in GRCBL including Drs. Strathern, Kashlev and Court.Transcriptional regulation in H. pylori pathogenesis 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.Extending our expertise on E. coli RNAP and the stringent response, we focused initially on the role of SpoT, which is the sole mediator for the stringent response in H. pylori. We previously found that SpoT mediates a serum starvation response, which not only restricts cell growth, but also prevents H. pylori from premature death. SpoT is also important for intracellular survival of H. pylori in macrophages during phagocytosis. Thus, SpoT plays an important role for the persistence of the pathogen in the host. Recently, we found that during a SpoT mediated serum starvation response in H. pylori, accumulated polyP forms a strong association with the major sigma factor. Such an interaction is critical for the bacterial persistence during nutrient depletion, a likely environment deep in human gastric mucus layer where the pathogen lives. A positively-charged-Lys-rich region at the NTD of the major sigma factor is identified as the binding region for polyP (region P), revealing a new element for sigma 70 family proteins. Putative """"""""region P"""""""" is present in primary sigma factors of other human pathogens including Bordetella pertussis and Coxiella burnetii, suggesting that the uncovered interaction might be a general strategy employed by other pathogens to cope with starvation/stress. Currently, we investigate how H. pylori uses this novel mechanism for global gene regulation and pathogenesis.
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