In all domains of life, efficient synthesis of long RNAs requires accessory proteins that modify RNA polymerase into a highly processive state. Bacterial transcription factor RfaH, which belongs to a universally conserved family of NusG proteins, activates expression of horizontally-transferred operons encoding cell wall and capsule components, antibiotics, and virulence factors, all of which are subject to strong Rho-mediated polarity. RfaH action depends on a DNA sequence called ops that mediates RfaH recruitment to RNA polymerase during elongation. We carried out detailed analysis of RfaH effects on transcription and identified determinants for its interactions with RNA polymerase and the ops DNA. These studies allowed us to propose a model for RfaH interactions with the transcription elongation complex and suggested that RfaH acts as a processivity clamp which stabilizes RNA polymerase contacts with DNA and RNA. Although studies in other groups confirmed that this mechanism appears to be ancient and ubiquitous, our results revealed that it makes a relatively small contribution to a dramatic, hundred-fold activation of gene expression by RfaH. The main effect of RfaH appears to rely on blocking Rho-dependent termination by competing with a Rho cofactor NusG and recruiting the ribosome to the nascent mRNA. The second mode of action is mediated by interactions between RfaH and ribosomal protein S10, which require a complete refolding of the C-terminal domain of RfaH from an ?-helical hairpin into ?-barrel. This metamorphosis is as dramatic as the one proposed to occur during formation of an infectious form of prions. In this proposal, we will use a combination of biochemical, genetic, and structural approaches to pursue these unexpected findings. First, we will study conformational transitions of RfaH C-terminal domain during its life cycle, from synthesis on the ribosome to hypothetical recycling upon dissociation from RNA polymerase at the end of an operon. Second, we will follow the events that trigger RfaH domain dissociation, a prelude to refolding, during recruitment at the ops site. Third, we will begin to elucidate the mechanism by which RfaH activates translation of mRNAs that lack functional Shine- Dalgarno elements.

Public Health Relevance

This project aims to elucidate the mechanism by which Escherichia coli RfaH regulates gene expression. The rfaH genes are present in many human pathogens and RfaH is essential for virulence in animal models. Our recent studies argue that RfaH activates expression of its target virulence operons by promoting transcription elongation and translation initiation. The mechanism of transcription regulation by RfaH is likely conserved in humans, where defects in mRNA chain elongation have been linked to cardiovascular, neurological, and autoimmune diseases, as well as to cancer. The mechanism of translation regulation is novel, and is the subject of the proposed studies. We will also study RfaH refolding, which is required for its interactions with the translational machinery and is strikingly similar t that of prions, which cause fatal brain diseases transmissible from cattle to humans.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM067153-10A1
Application #
8639155
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Sledjeski, Darren D
Project Start
2003-02-01
Project End
2018-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
10
Fiscal Year
2014
Total Cost
$374,616
Indirect Cost
$122,616
Name
Ohio State University
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
832127323
City
Columbus
State
OH
Country
United States
Zip Code
43210
Sreenivasan, Raashi; Heitkamp, Sara; Chhabra, Munish et al. (2016) Fluorescence Resonance Energy Transfer Characterization of DNA Wrapping in Closed and Open Escherichia coli RNA Polymerase-λP(R) Promoter Complexes. Biochemistry 55:2174-86
Elgamal, Sara; Artsimovitch, Irina; Ibba, Michael (2016) Maintenance of Transcription-Translation Coupling by Elongation Factor P. MBio 7:
Belogurov, Georgiy A; Artsimovitch, Irina (2015) Regulation of Transcript Elongation. Annu Rev Microbiol 69:49-69
Artsimovitch, Irina; Santangelo, Thomas J (2015) Preface. Bacterial transcriptional control. Methods Mol Biol 1276:v
Artsimovitch, Irina; Belogurov, Georgi A (2015) Creative Math of RNA Polymerase III Termination: Sense Plus Antisense Makes More Sense. Mol Cell 58:974-6
Furman, Ran; Danhart, Eric M; NandyMazumdar, Monali et al. (2015) pH dependence of the stress regulator DksA. PLoS One 10:e0120746
Ruff, Emily F; Drennan, Amanda C; Capp, Michael W et al. (2015) E. coli RNA Polymerase Determinants of Open Complex Lifetime and Structure. J Mol Biol 427:2435-50
Ruff, Emily F; Record Jr, M Thomas; Artsimovitch, Irina (2015) Initial events in bacterial transcription initiation. Biomolecules 5:1035-62
Svetlov, Vladimir; Artsimovitch, Irina (2015) Purification of bacterial RNA polymerase: tools and protocols. Methods Mol Biol 1276:13-29
NandyMazumdar, Monali; Artsimovitch, Irina (2015) Ubiquitous transcription factors display structural plasticity and diverse functions: NusG proteins - Shifting shapes and paradigms. Bioessays 37:324-34

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