E. coli 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. Moreover, how RNAP distribution influences the structure of the bacterial chromosome called the nucleoid was virtually unknown. We initiated research on the cell biology of RNAP to visualize the RNAP in the cell, which was aimed at testing a hypothesis that RNAP redistribution is coupled to global gene regulation associated with the stringent response. Our results show that RNAP is located exclusively within and/or surrounding the nucleoid and the distribution of RNAP is dynamic and influenced dramatically by environmental cues. Remarkably, during optimal growth, concentrated RNAP is actively transcribing rRNA operons, as evidenced by the appearance of transcription foci, a structure analogous to the eukaryotic nucleolus. In contrast, during amino acid starvation leading to the stringent response, RNAP is relatively homogenously distributed within the nucleoid. Thus, for the first time, RNAP redistribution is visualized during global change in the transcription pattern associated with the stringent response. Transcription profiling of the stringent response in E. coli is consistent with the RNAP redistribution model. Also, our recent results 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. 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. We continue to study the mechanism of RNAP recycling in transcription by RapA. We identified RapA as a novel RNAP-associated protein that is also an ATPase. RapA is a member of the Swi2/Snf2 superfamily of proteins. ATP-dependent chromatin remodeling by these proteins is known to be an important aspect of transcriptional regulation in eukaryotes. RapA activates transcription by promoting RNAP recycling. We recently showed that RapA facilitates the release of sequestered RNAP from an undefined post-transcription/post-termination complex allowing for transcription reinitiation. This demonstrates the role of RapA in mobilization of nucleic acid-protein complexes to facilitate gene expression, and provides a new paradigm for the action of transcriptional activators. In collaboration with Dr. Xinhua Ji (CCR, NCI), the crystal structure of RapA was recently determined, and represents the first full-length structure for a member of the Swi2/Snf2 family. The structure of RapA provides a framework for future studies of this bacterial Swi2/Snf2 protein. Conceivably, these studies may also shed light on how other Swi/Snf proteins function in general. We also study transcription fidelity, an important but understudied process in vivo due to its intrinsic difficulties of the assay. 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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National Cancer Institute (NCI)
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Bubunenko, Mikhail G; Court, Carolyn B; Rattray, Alison J et al. (2017) A Cre Transcription Fidelity Reporter Identifies GreA as a Major RNA Proofreading Factor in Escherichia coli. Genetics 206:179-187
Jin, Ding Jun; Mata Martin, Carmen; Sun, Zhe et al. (2017) Nucleolus-like compartmentalization of the transcription machinery in fast-growing bacterial cells. Crit Rev Biochem Mol Biol 52:96-106
Zhou, Qingxuan; Zhou, Yan Ning; Jin, Ding Jun et al. (2017) Deacetylation of topoisomerase I is an important physiological function of E. coli CobB. Nucleic Acids Res 45:5349-5358
Jin, Ding J; Cagliero, Cedric; Martin, Carmen M et al. (2015) The dynamic nature and territory of transcriptional machinery in the bacterial chromosome. Front Microbiol 6:497
Cagliero, Cedric; Zhou, Yan Ning; Jin, Ding Jun (2014) Spatial organization of transcription machinery and its segregation from the replisome in fast-growing bacterial cells. Nucleic Acids Res 42:13696-705
Zhou, Yan Ning; Lubkowska, Lucyna; Hui, Monica et al. (2013) Isolation and characterization of RNA polymerase rpoB mutations that alter transcription slippage during elongation in Escherichia coli. J Biol Chem 288:2700-10
Strathern, Jeffrey; Malagon, Francisco; Irvin, Jordan et al. (2013) The fidelity of transcription: RPB1 (RPO21) mutations that increase transcriptional slippage in S. cerevisiae. J Biol Chem 288:2689-99
Cagliero, Cedric; Grand, Ralph S; Jones, M Beatrix et al. (2013) Genome conformation capture reveals that the Escherichia coli chromosome is organized by replication and transcription. Nucleic Acids Res 41:6058-71
Jin, Ding Jun; Cagliero, Cedric; Zhou, Yan Ning (2013) Role of RNA polymerase and transcription in the organization of the bacterial nucleoid. Chem Rev 113:8662-82
Cagliero, Cedric; Jin, Ding Jun (2013) Dissociation and re-association of RNA polymerase with DNA during osmotic stress response in Escherichia coli. Nucleic Acids Res 41:315-26

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