The long-term goal of this project is to define the interactions within transcription elongation complexes and with regulatory proteins that cause and control pausing and termination by RNA polymerase. Pausing and premature termination affect expression of bacterial genes and also of most human genes, which are regulated by switching elongation complexes from susceptibility to pausing or termination in the promoter-proximal region to an efficient state able to resist termination while transcribing through nucleosomes. A similar process operates during transcription of bacterial genes bound by the histone-like nucleoid structuring protein H-NS. In both bacteria and eukaryotes, specialized regulatory proteins modify the transcription complex to make it resistant to pausing and termination. Both the basic mechanisms of pausing and termination and the mechanisms by which regulators control pausing and termination depend on poorly understood changes to interactions within the elongation complex. Many of these interactions modulate conformational changes in RNAP polymerase that involve its clamp domain, bridge helix, and trigger loop, which must achieve particular conformations for efficient transcription. Understanding how regulators promote or inhibit these clamp, bridge-helix, and trigger-loop conformations will provide key basic knowledge essential to guide rational manipulation of regulators for antimicrobials or gene therapies. Additionally, bacterial RNA polymerase is a known target of antibiotics, and knowledge about its mechanism will aid in identifying and characterizing new antibiotics. A combination of biochemical, genetic, and biophysical approaches will be used to characterize the interactions in the elongation complex that mediate regulation. The ability of elongation complexes to transcribe nucleoprotein templates in which nucleoid-associated proteins are bound to DNA, as found within bacterial cells, will be studied.
The specific aims of the project are to (i) elucidate the mechanism of a consensus pause signal affecting both bacterial and eukaryotic RNA polymerases; (ii) determine the mechanisms by which proteins that combine with DNA to form bacterial chromatin inhibit transcript elongation by RNA polymerase; (iii) determine the mechanism by which the NusG transcription factor in mycobacteria enhances pausing and termination; and (iv) dissect the mechanism of promoter-proximal pausing in bacteria by defining how ribosomes and NusG are recruited to elongation complexes and determining the roles in promoter-proximal pausing of other transcription factors. The impact of these studies will be an improved understanding of how elongation complexes are regulated, with broad applications to biotechnology, human medicine, and both prokaryotic and eukaryotic molecular biology
This research will increase knowledge about the regulation of gene expression, which underlies virtually every aspect of human health. By improving understanding of RNA polymerase, which is an established target for effective antibiotics, the work will also aid in the quest for new antibiotics that can keep humankind a step ahead of microbial pathogens. Finally, the research will elucidate the mechanism of a key regulator of mycobacterial RNA polymerases, which will increase understanding of this pathogenic class of bacteria responsible for multiple infectious diseases including tuberculosis.
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