A. Spatial and functional organization of transcription machinery in fast-growing cells. We have only recently begun to gain insights into how transcription machinery is organized in the genome in response to changes in growth conditions. Using fluorescent RNAP to image RNAP in E. coli our results show that the distribution of RNAP is dynamic and sensitive to environmental cues. In fast-growing cells most RNAP molecules are concentrated, forming foci at the clustering of rrn, which resembles the eukaryotic nucleolus. By co-imaging of RNAP and the nucleoid (DNA) in the cell using super-resolution structured illumination microscopy, we found that RNAP foci are located at the periphery or surroundings of the nucleoid. Such a spatial organization has logistical advantages, including coupling of rRNA synthesis with rRNA processing and ribosome assembly, as well as reducing the traffic jams with other cellular functions. We performed the first E. coli genome conformation capture analysis and the results reveal that the nucleoid is organized by both replication via SeqA-mediated interactions and transcription through gene clustering. Our recent studies show that the major transcription machinery and replisome are mostly located in different chromosome territories or spatially segregated in the nucleoid, which could explain why the two major cellular functions remain in harmony in fast-growing cells. By developing new imaging tools and using super-resolution imaging techniques, we continue to study the spatial and functional organization of transcription machinery in the nucleoid during bacterial growth and stress responses. B. Osmotic stress response. We are currently studying osmotic stress response in E. coli. Our results show that RNAP dissociates from the nucleoid during initial high salt shock when the cytoplasmic K+ increases transiently, and, concomitantly, the nucleoid becomes hyper-condensed. Subsequently, during the osmoadaptation phase when the cytoplasmic K+ levels decrease, the free RNAP re-associates with DNA and initially forms a ring at the periphery of the nucleoid, and the nucleoid gradually expands to a size approaching that prior to the salt shock. The ring of RNAP surrounding the hyper-condensed nucleoid during the early osmoadaptation phase is proposed to be the location of DNA loops for the expression of responsive genes. We continue to study the regulation of global gene expression and the genome-wide binding of RNAP during the osmotic stress response. The effects of other stresses will be examined similarly. C. SpoT mediated stress responses in Helicobacter pylori. While we have focused on E. coli, we have also extended our basic research to human pathogenic bacteria. Our studies show that the SpoT plays a critical role in stress responses in Helicobacter pylori, classified as a Group 1 carcinogen by WHO. During serum starvation, SpoT mediates the accumulation of polyphosphate (polyP), which binds the principal sigma factor, sigma 80, forming highly stable complexes. The target site for the polyP binding is the unique lysine-rich N-terminal region (named P region) of sigma 80. The putative P region is also found in the major sigma factors of other human pathogens, suggesting a new paradigm for the regulation of pathogenesis. We continue to study the interaction between sigma 80 and polyP, the enzyme(s) involved in the metabolism of polyP and other SpoT-mediated stress responses including bacterial survival in macrophages. In addition, we have found that SspA is required for the expression of the pathogenicity island in the enterohemorrhagic E. coli. D. Collaborative researches on RapA and transcription fidelity. We continue the structure-function study of E. coli RapA, a bacterial Swi2/Snf2 protein, in collaboration with colleagues at the NCI-Frederick campus. Swi2/Snf2 proteins mediate chromatin remodeling in eukaryotes. We previously found that RapA is an RNAP-associated protein and an ATPase, promoting RNAP recycling in transcription; its full-length crystal structure is solved. RapA also competes with sigma 70 in binding to core RNAP; the nature of the competition remains to be determined. In addition, we continue the study of transcription fidelity which is GRCBL-wide collaboration. We have played a major role in developing robust genetic systems in E. coli, allowing direct isolation and characterization of the first set of RNAP mutants that exhibit altered transcriptional slippage phenotypes during elongation on DNA templates containing homopolymeric A/T runs. Our results indicate that the fork domain of RNA polymerase controls slippage. We continue to study the mechanisms of maintenance of RNA/DNA register and fidelity during transcription using mutational and biochemical analyses.
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