The long-term goal of this project is to define the interactions within transcription elongation complexes and with regulators that cause and control pausing and termination by RNA polymerase. Pausing and premature termination underlie many aspects of gene regulation in prokaryotes and eukaryotes, including transcription through chromatin and linkages to RNA maturation and translation. 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 RNA polymerase involving mobile modules including the clamp, trigger loop, and lineage-specific insertions that must achieve particular conformations for efficient transcription. Understand- ing how regulators promote or inhibit these different conformations will provide key basic knowledge essential to guide the rational manipulation of regulators for antimicrobials or gene therapies. Knowledge gained about model bacterial systems also facilitates understanding of highly conserved mechanisms of transcription in humans. Additionally, bacterial RNA polymerase is a known target of antibiotics, and knowledge about its functional mechanisms will aid in identifying and characterizing new antibiotics. A combination of structural, biochemical, and genetic approaches will be used to characterize the interac- tions in the elongation complex that mediate regulation. New methods for transcription assay by cryo-electron microscopy, for single-molecule assay of RNA polymerase interactions with RNA structures, regulators, and ribosomes, and for genome-scale analysis of chromatin structure and elongation complex regulation will be developed. This combination of approaches will be used to understand connections among progress of the elongation complex during transcription, the structure of bacterial chromatin, RNA folding, and RNA translation. The work builds on recent discoveries of the structural basis by which RNA polymerase assists RNA folding, of the role of H-NS family nucleoprotein filaments in stimulation of pausing and termination during transcriptional silencing, and of interaction of RNA polymerase with the pioneering ribosome in a complex called the expressome.
The specific aims of the project are to (i) elucidate steps in pausing, termina- tion, and RNA folding and roles of key RNAP modules; (ii) define structures, patterns, and elongation complex interactions of H-NS family nucleoprotein filaments; and (iii) determine when the expressome forms and how it functions during transcript elongation. This integrated research will help build a new understanding of tran- scriptional regulation by defining how pause and termination signals change elongation complex structure and activity dynamically and how chromosome structure and translational coupling modulate elongation complex activity. The impact of these studies will be an improved understanding of elongation complex regulation, 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 aid in the quest for new antibiotics that can keep humankind a step ahead of microbial pathogens. The research also will elucidate complex connections between RNA synthesis by RNA polymerase, chromatin structure, RNA maturation, and RNA translation that are key to understanding the function of microbial communities in promoting both human health and disease.
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