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 #
3R01GM067153-09S1
Application #
8720361
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Sledjeski, Darren D
Project Start
2003-02-01
Project End
2014-01-31
Budget Start
2012-02-01
Budget End
2014-01-31
Support Year
9
Fiscal Year
2013
Total Cost
$124,937
Indirect Cost
$43,589
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
Miropolskaya, Nataliya; Esyunina, Daria; Klimasauskas, Saulius et al. (2014) Interplay between the trigger loop and the F loop during RNA polymerase catalysis. Nucleic Acids Res 42:544-52
Malinen, Anssi M; Nandymazumdar, Monali; Turtola, Matti et al. (2014) CBR antimicrobials alter coupling between the bridge helix and the ? subunit in RNA polymerase. Nat Commun 5:3408
Tomar, Sushil Kumar; Artsimovitch, Irina (2013) NusG-Spt5 proteins-Universal tools for transcription modification and communication. Chem Rev 113:8604-19
Furman, Ran; Sevostyanova, Anastasiya; Artsimovitch, Irina (2012) Transcription initiation factor DksA has diverse effects on RNA chain elongation. Nucleic Acids Res 40:3392-402
Deaconescu, Alexandra M; Sevostyanova, Anastasia; Artsimovitch, Irina et al. (2012) Nucleotide excision repair (NER) machinery recruitment by the transcription-repair coupling factor involves unmasking of a conserved intramolecular interface. Proc Natl Acad Sci U S A 109:3353-8
Perdrizet 2nd, George A; Artsimovitch, Irina; Furman, Ran et al. (2012) Transcriptional pausing coordinates folding of the aptamer domain and the expression platform of a riboswitch. Proc Natl Acad Sci U S A 109:3323-8
Santangelo, Thomas J; Artsimovitch, Irina (2011) Termination and antitermination: RNA polymerase runs a stop sign. Nat Rev Microbiol 9:319-29
Sevostyanova, Anastasia; Belogurov, Georgiy A; Mooney, Rachel A et al. (2011) The ýý subunit gate loop is required for RNA polymerase modification by RfaH and NusG. Mol Cell 43:253-62
Sevostyanova, Anastasiya; Artsimovitch, Irina (2010) Functional analysis of Thermus thermophilus transcription factor NusG. Nucleic Acids Res 38:7432-45
Pupov, Danil; Miropolskaya, Nataliya; Sevostyanova, Anastasiya et al. (2010) Multiple roles of the RNA polymerase {beta}' SW2 region in transcription initiation, promoter escape, and RNA elongation. Nucleic Acids Res 38:5784-96

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