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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Sledjeski, Darren D
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Ohio State University
Schools of Arts and Sciences
United States
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Artsimovitch, Irina (2018) Rebuilding the bridge between transcription and translation. Mol Microbiol 108:467-472
Kang, Jin Young; Mooney, Rachel Anne; Nedialkov, Yuri et al. (2018) Structural Basis for Transcript Elongation Control by NusG Family Universal Regulators. Cell 173:1650-1662.e14
Lawson, Michael R; Ma, Wen; Bellecourt, Michael J et al. (2018) Mechanism for the Regulated Control of Bacterial Transcription Termination by a Universal Adaptor Protein. Mol Cell 71:911-922.e4
Nedialkov, Yuri; Svetlov, Dmitri; Belogurov, Georgiy A et al. (2018) Locking the nontemplate DNA to control transcription. Mol Microbiol 109:445-457
Janissen, Richard; Arens, Mathia M A; Vtyurina, Natalia N et al. (2018) Global DNA Compaction in Stationary-Phase Bacteria Does Not Affect Transcription. Cell 174:1188-1199.e14
Zuber, Philipp K; Artsimovitch, Irina; NandyMazumdar, Monali et al. (2018) The universally-conserved transcription factor RfaH is recruited to a hairpin structure of the non-template DNA strand. Elife 7:
Hu, Kuang; Artsimovitch, Irina (2017) A Screen for rfaH Suppressors Reveals a Key Role for a Connector Region of Termination Factor Rho. MBio 8:
Shi, Da; Svetlov, Dmitri; Abagyan, Ruben et al. (2017) Flipping states: a few key residues decide the winning conformation of the only universally conserved transcription factor. Nucleic Acids Res 45:8835-8843
Strobel, Eric J; Watters, Kyle E; Nedialkov, Yuri et al. (2017) Distributed biotin-streptavidin transcription roadblocks for mapping cotranscriptional RNA folding. Nucleic Acids Res 45:e109
Elgamal, Sara; Artsimovitch, Irina; Ibba, Michael (2016) Maintenance of Transcription-Translation Coupling by Elongation Factor P. MBio 7:

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