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 not only of bacterial genes, as long appreciated, but 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. 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 regulatory proteins control pausing and termination depend on changes to interactions within the elongation complex that are poorly understood. A central target of these interactions is folding of the trigger loop in RNA polymerase into the trigger helices, which is required to catalyze addition of nucleotides to the growing RNA chain. Pausing interferes with trigger loop folding. Understanding how regulatory proteins and nascent RNA structures influence trigger-loop folding and regulate the activity of elongation complexes will provide key basic knowledge about gene regulation, which underlies virtually every aspect of human health. Further, bacterial RNA polymerase is a known target of antibiotics, and knowledge about how it works aids 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. Additionally, 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) test a specific model of elongation complex regulation known as the bridge-helix/trigger-loop model and to define specific changes in RNA polymerase that underlie elongation complex regulation;(ii) discover how a module that inserts in the trigger loop of some bacterial RNA polymerases, called SI3, participates in trigger-loop function and modulates the activity of elongation complexes;(iii) determine the mechanism of transcription termination and the specific roles of the trigger loop and two regulatory proteins called NusA and NusG;and (iv) determine the interplay among transcription, pausing, and termination caused by a regulatory protein called Rho, and nucleoid-associated proteins. 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.

Public Health Relevance

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 efficacious 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 functions of lineage-specific parts of RNA polymerase that will aid in understanding transcription in the diverse bacteria found in the human microbiome.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM038660-28
Application #
8691849
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Sledjeski, Darren D
Project Start
1987-07-01
Project End
2015-06-30
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
28
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Earth Sciences/Resources
DUNS #
City
Madison
State
WI
Country
United States
Zip Code
53715
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