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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM067153-07
Application #
7784532
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Tompkins, Laurie
Project Start
2003-02-01
Project End
2013-01-31
Budget Start
2010-02-01
Budget End
2011-01-31
Support Year
7
Fiscal Year
2010
Total Cost
$369,765
Indirect Cost
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
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
Elgamal, Sara; Artsimovitch, Irina; Ibba, Michael (2016) Maintenance of Transcription-Translation Coupling by Elongation Factor P. MBio 7:
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-?P(R) Promoter Complexes. Biochemistry 55:2174-86
Artsimovitch, Irina; Santangelo, Thomas J (2015) Preface. Bacterial transcriptional control. Methods Mol Biol 1276:v
Liu, Zhaokun; Artsimovitch, Irina (2015) Mapping the Escherichia coli transcription elongation complex with exonuclease III. Methods Mol Biol 1276:1-12
Belogurov, Georgiy A; Artsimovitch, Irina (2015) Regulation of Transcript Elongation. Annu Rev Microbiol 69:49-69
Ramírez-Sarmiento, César A; Noel, Jeffrey K; Valenzuela, Sandro L et al. (2015) Interdomain Contacts Control Native State Switching of RfaH on a Dual-Funneled Landscape. PLoS Comput Biol 11:e1004379
Svetlov, Vladimir; Artsimovitch, Irina (2015) Purification of bacterial RNA polymerase: tools and protocols. Methods Mol Biol 1276:13-29
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

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