A.Transcriptional Regulation in Bacterial Growth and Stress Responses We have only recently begun to gain insights into how transcription machinery is organized in the genome in response to changes in growth conditions. Our study is the first that used fluorescent RNAP to image RNAP in E. coli and found that the distribution of RNAP is dynamic and sensitive to environmental cues. We discovered that E. coli contains nucleolus-like structure, where RNAP concentrates as predominant foci for active rRNA synthesis in fast-growing cells and showed that active rRNA synthesis is the driving force for the distribution of RNAP in the cell. By co-imaging of RNAP and the nucleoid (DNA) in the cell we have opened a new research frontier at the interface between transcription and nucleoid organization. This interface defines cell biology of RNAP and the nucleoid in E. coli and emerges as an active research area. Like its eukaryotic counterpart, the E. coli nucleoid must be densely compacted and yet organized for optimum functionality;however, the mechanisms by which this is accomplished remain unclear. The traditional view is that """"""""histone-like"""""""" nucleoid-associated proteins (NAPs) are primarily responsible for nucleoid compaction in growing cells. Our research suggests an alternative and/or complementary perspective: RNAP, as the predominant NAP and key transcription machinery, helps organize the nucleoid - in particular, the formation of the predominant transcription foci or hubs of transcription networks centered at the nucleolus-like structures is pivotal for nucleoid compaction during optimal growth. 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. By developing new tools and using super-resolution and microfluidic live-cell imaging systems, we continue to study cell biology of RNAP and the nucleoid to further our understanding on the relationship between transcription and the organization of the nucleoid during bacterial growth and stress responses. We are currently focused on Osmotic stress response in E. coli by analyzing the dynamic process of the response in time-course experiments. Our results show that RNAP dissociates from the nucleoid during initial high salt shock when the cytoplasmic K+ increases transiently, followed by RNAP re-association during the later adaptation phase when the K+ decreases. In parallel with the dissociation and re-association of RNAP, there are significant changes in nucleoid structure, consistent with a role of RNAP in remodeling the nucleoid. 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. We also 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. Currently, we study the role of RapA in RNAP recycling, with the focus on the structure-function analysis and the mechanism for the competition between RapA and sigma 70 in binding to Core RNAP. While we have focused on E. coli, we have also extended our basic research to human pathogenic bacteria. Our studies on the SpoT mediated stress responses in Helicobacter pylori, classified as a Group 1 carcinogen by WHO, showed that during serum starvation, SpoT mediates the accumulation of polyphosphate (polyP), which binds the principal sigma factor, sigma 80, forming highly stable complexes without promoting degradation. The target site for the polyP binding is the unique arginine-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. B. Transcription Fidelity We also study transcription fidelity, an important process in the cell that is understudied due to intrinsic difficulties in identifying the fidelity mutant phenotypes. This line of research is one of the focal points of interaction within GRCBL. We have developed 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. Biochemical analysis of the RNAP mutants validates the genetic schemes. 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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