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
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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