Elongation is the longest part of transcription cycle during which RNA polymerase movement along the template is hindered by many roadblocks DNA-bound proteins, DNA lesions, termination signals, etc. Factors that allow RNA polymerase to bypass these barriers are required for efficient synthesis of long RNAs in all domains of life. Bacterial protein RfaH regulates expression of the cell wall and capsule components, antibiotics, and virulence factors by increasing the RNA polymerase processivity. RfaH action depends on a DNA sequence called ops that mediates RfaH recruitment to RNA polymerase during elongation. In the first granting period, we obtained the X-ray structure of RfaH, identified its binding site on transcription complex, characterized RfaH effects at different regulatory sites and on enzymes with altered elongation properties, and showed that RfaH acts by preventing pausing rather than by increasing the rate of nucleotide addition. This mechanism is likely fundamentally conserved in other antiterminators. In this proposal, we will use a combination of biochemical, genetic, and structural approaches to address several aspects of RfaH action. The first goal of this project is to study the mechanism of RfaH action. We will use a combination of genetic, biochemical, and structural analyses to dissect interactions of the N-terminal domain (which is sufficient for RfaH anti-pausing activity) with the transcription elongation complex and to elucidate the confomational changes triggered by these interactions. The second goal of this project is to elucidate the role of the ops element in recruitment of RfaH. We propose that ops not only establishes base-specific contacts with RfaH but also induces a specialized scrunched DNA conformation that is required for RfaH binding. The third goal of this project is to test if the """"""""modulatory"""""""" C-terminal domain changes its structure after RfaH recruitment and is involved in cross-talk with the translation apparatus. The fourth goal of this project is to characterize the RfaH regulon by identifying the RfaH-associated proteins and genes by in vivo crosslinking and chromatin immuno-precipitation, respectively. We will also analyze selected RfaH operons by quantitative RT PCR.

Public Health Relevance

This project aims to elucidate the mechanism by which transcription factor RfaH regulates gene expression. The rfaH genes are present in human, insect, and plant pathogens;moreover, RfaH is essential for virulence in animal models. These studies will reveal the mechanism of RfaH action, elucidate the unique role of its DNA target site in transcriptional control, and identify cellular RfaH targets which may be uncharacterized virulence factors.

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
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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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