|Gene:||rfaH||Accession Numbers: EG10839 (EcoCyc), b3842, ECK3834|
Synonyms: hlyT, sfrB
RfaH acts as a transcriptional antiterminator for a subset of operons required for the production of extracellular components such as lipopolysaccharide [Beutin81, Leeds96, Bailey96]. It is an allosteric modulator of RNA polymerase (RNAP), reducing polymerase pausing at certain positions [Svetlov07]. RfaH, the β' clamp helices (β'CH) and the β subunit gate loop (β GL) of RNAP jointly form a clamp on the DNA that allows the RNAP to resist pausing and termination signals [Sevostyanova11].
RfaH is a so-called "transformer protein": its C-terminal domain can assume an α-helical or a β-barrel structure, and both conformations have different functions [Knauer12].
RfaH is a homolog of the essential protein NusG, which modulates general transcriptional pausing and termination [Bailey96]. RfaH and NusG compete for the same binding site on RNAP in vitro [Belogurov09]. RfaH interaction with RNAP abolishes sigma factor-dependent pausing, likely because it interacts with a site adjacent to the β' subunit clamp helix site that may interact with σ during elongation [Sevostyanova08]. RfaH is able to reverse a non-productive RNAP trigger loop conformation that stabilizes paused RNAP complexes [Nayak13] and stabilizes the closed clamp conformation of RNAP, inhibiting formation of an exit channel RNA duplex that would lead to RNAP pausing [Kolb14]. RfaH decreases ρ-mediated polarity [Sevostyanova11].
The so-called JUMPStart sequence which is present in the 5' UTRs of RfaH-enhanced operons is associated with transcriptional pausing [Leeds97]. The ops (operon polarity suppressor) element, a short conserved sequence, is a Class II pause signal [Artsimovitch00] that directs recruitment of RfaH into a paused RNAP complex that subsequently resists transcription termination [Bailey00, Artsimovitch02]. RfaH recognizes the ops sequence on the partially accessible nontemplate strand, binds to RNAP while inhibiting escape from the ops pause site, but subsequently suppresses pausing downstream [Artsimovitch02].
A crystal structure of RfaH has been solved [Vassylyeva06, Belogurov07]. The protein consists of a globular N-terminal domain (NTD) with similarity to NusG, and an α-helical C-terminal domain (CTD) that caps the hydrophobic cavity of the NTD. Cap opening is required for RfaH activity. The NTD mediates RNAP pausing, while the CTD is required for ops sequence specificity [Belogurov07]. Analysis of single-residue substitutions in the NTD allowed seperation of its functional regions for interaction with the ops site, pausing at ops sites, and subsequent anti-pausing activity in vitro [Belogurov10]. Interaction of the NTD with its ops DNA target releases the CTD, which then switches from its α-helical conformation to an all-β conformation that is similar to the NusG CTD. In the all-β conformation, the CTD can interact with S10 and activate translation [Burmann12]. The conformation of the CTD is determined by the availability and ability to interact with the NTD [Tomar13]. The conformational switch of the CTD has been investigated by molecular dynamics simulations [Gc14].
RfaH participates in controlling several genes involved in the biosynthesis, assembly, and export of the lipopolysaccharide core [Creeger84, Pradel91], F-factor sex pili [Beutin79], hemolysin [Bailey92, Leeds96], and indirectly (via MicA and σE) the FimB recombinase [Moores14]. Loss of RfaH attenuates virulence of pathogenic strains of E. coli [Nagy02, Nagy05]. An rfaH mutant has an increased-biofilm phenotype in both the nonpathogenic K-12 strain and in the uropathogenic strain 536 [Beloin06].
SfrB: "sex factor regulation" [Beutin79]
|Map Position: [4,022,356 <- 4,022,844] (86.69 centisomes, 312°)||Length: 489 bp / 162 aa|
Molecular Weight of Polypeptide: 18.34 kD (from nucleotide sequence), 18 kD (experimental) [Rehemtulla86 ]
Unification Links: ASAP:ABE-0012553 , CGSC:164 , EchoBASE:EB0832 , EcoGene:EG10839 , EcoliWiki:b3842 , ModBase:P0AFW0 , OU-Microarray:b3842 , PortEco:rfaH , PR:PRO_000023720 , Pride:P0AFW0 , Protein Model Portal:P0AFW0 , RefSeq:NP_418284 , RegulonDB:EG10839 , SMR:P0AFW0 , String:511145.b3842 , UniProt:P0AFW0
Relationship Links: InterPro:IN-FAMILY:IPR005824 , InterPro:IN-FAMILY:IPR006645 , InterPro:IN-FAMILY:IPR010215 , PDB:Structure:2OUG , Pfam:IN-FAMILY:PF02357 , Smart:IN-FAMILY:SM00738 , Smart:IN-FAMILY:SM00739
|Biological Process:||GO:0001124 - transcription elongation from bacterial-type RNA polymerase promoter
GO:0031564 - transcription antitermination [UniProtGOA11a, GOA06, Pradel91, Bailey96]
GO:0045727 - positive regulation of translation [Burmann12]
GO:0006351 - transcription, DNA-templated [UniProtGOA11a]
GO:0006355 - regulation of transcription, DNA-templated [UniProtGOA11a, GOA01a]
|Molecular Function:||GO:0001000 - bacterial-type RNA polymerase core enzyme binding
GO:0001073 - DNA binding transcription antitermination factor activity [GOA06, Artsimovitch02]
GO:0005515 - protein binding [Burmann12]
GO:0008494 - translation activator activity [Burmann12]
GO:0003677 - DNA binding [UniProtGOA11a, GOA06]
|Cellular Component:||GO:0005829 - cytosol [DiazMejia09]|
|MultiFun Terms:||information transfer → RNA related → Transcription related|
|metabolism → biosynthesis of macromolecules (cellular constituents) → fimbri, pili|
|metabolism → biosynthesis of macromolecules (cellular constituents) → lipopolysaccharide → core region|
|regulation → type of regulation → transcriptional level → activator|
|Growth Medium||Growth?||T (°C)||O2||pH||Osm/L||Growth Observations|
|LB enriched||Yes||37||Aerobic||6.95||Yes [Gerdes03, Comment 1]|
10/20/97 Gene b3842 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG10839; confirmed by SwissProt match.
Artsimovitch00: Artsimovitch I, Landick R (2000). "Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals." Proc Natl Acad Sci U S A 97(13);7090-5. PMID: 10860976
Artsimovitch02: Artsimovitch I, Landick R (2002). "The transcriptional regulator RfaH stimulates RNA chain synthesis after recruitment to elongation complexes by the exposed nontemplate DNA strand." Cell 109(2);193-203. PMID: 12007406
Bailey00: Bailey MJ, Hughes C, Koronakis V (2000). "In vitro recruitment of the RfaH regulatory protein into a specialised transcription complex, directed by the nucleic acid ops element." Mol Gen Genet 262(6);1052-9. PMID: 10660066
Bailey92: Bailey MJ, Koronakis V, Schmoll T, Hughes C (1992). "Escherichia coli HlyT protein, a transcriptional activator of haemolysin synthesis and secretion, is encoded by the rfaH (sfrB) locus required for expression of sex factor and lipopolysaccharide genes." Mol Microbiol 6(8);1003-12. PMID: 1584020
Belogurov07: Belogurov GA, Vassylyeva MN, Svetlov V, Klyuyev S, Grishin NV, Vassylyev DG, Artsimovitch I (2007). "Structural basis for converting a general transcription factor into an operon-specific virulence regulator." Mol Cell 26(1);117-29. PMID: 17434131
Beloin06: Beloin C, Michaelis K, Lindner K, Landini P, Hacker J, Ghigo JM, Dobrindt U (2006). "The Transcriptional Antiterminator RfaH Represses Biofilm Formation in Escherichia coli." J Bacteriol 188(4);1316-1331. PMID: 16452414
Burmann12: Burmann BM, Knauer SH, Sevostyanova A, Schweimer K, Mooney RA, Landick R, Artsimovitch I, Rosch P (2012). "An α helix to β barrel domain switch transforms the transcription factor RfaH into a translation factor." Cell 150(2);291-303. PMID: 22817892
Creeger84: Creeger ES, Schulte T, Rothfield LI (1984). "Regulation of membrane glycosyltransferases by the sfrB and rfaH genes of Escherichia coli and Salmonella typhimurium." J Biol Chem 259(5);3064-9. PMID: 6230355
DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114
Gc14: Gc JB, Bhandari YR, Gerstman BS, Chapagain PP (2014). "Molecular dynamics investigations of the α-helix to β-barrel conformational transformation in the RfaH transcription factor." J Phys Chem B 118(19);5101-8. PMID: 24758259
Gerdes03: Gerdes SY, Scholle MD, Campbell JW, Balazsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabasi AL, Oltvai ZN, Osterman AL (2003). "Experimental determination and system level analysis of essential genes in Escherichia coli MG1655." J Bacteriol 185(19);5673-84. PMID: 13129938
Hartzog11: Hartzog GA, Kaplan CD (2011). "Competing for the clamp: promoting RNA polymerase processivity and managing the transition from initiation to elongation." Mol Cell 43(2);161-3. PMID: 21777806
Kolb14: Kolb KE, Hein PP, Landick R (2014). "Antisense oligonucleotide-stimulated transcriptional pausing reveals RNA exit channel specificity of RNA polymerase and mechanistic contributions of NusA and RfaH." J Biol Chem 289(2);1151-63. PMID: 24275665
Leeds97: Leeds JA, Welch RA (1997). "Enhancing transcription through the Escherichia coli hemolysin operon, hlyCABD: RfaH and upstream JUMPStart DNA sequences function together via a postinitiation mechanism." J Bacteriol 179(11);3519-27. PMID: 9171395
Moores14: Moores A, Chipper-Keating S, Sun L, McVicker G, Wales L, Gashi K, Blomfield IC (2014). "RfaH Suppresses Small RNA MicA Inhibition of fimB Expression in Escherichia coli K-12." J Bacteriol 196(1);148-56. PMID: 24163336
Nagy02: Nagy G, Dobrindt U, Schneider G, Khan AS, Hacker J, Emody L (2002). "Loss of regulatory protein RfaH attenuates virulence of uropathogenic Escherichia coli." Infect Immun 70(8);4406-13. PMID: 12117951
Nagy05: Nagy G, Dobrindt U, Grozdanov L, Hacker J, Emody L (2005). "Transcriptional regulation through RfaH contributes to intestinal colonization by Escherichia coli." FEMS Microbiol Lett 244(1);173-80. PMID: 15727837
Nieto96: Nieto JM, Bailey MJ, Hughes C, Koronakis V (1996). "Suppression of transcription polarity in the Escherichia coli haemolysin operon by a short upstream element shared by polysaccharide and DNA transfer determinants." Mol Microbiol 19(4);705-13. PMID: 8820641
Sevostyanova08: Sevostyanova A, Svetlov V, Vassylyev DG, Artsimovitch I (2008). "The elongation factor RfaH and the initiation factor sigma bind to the same site on the transcription elongation complex." Proc Natl Acad Sci U S A 105(3);865-70. PMID: 18195372
Sevostyanova11: Sevostyanova A, Belogurov GA, Mooney RA, Landick R, Artsimovitch I (2011). "The β subunit gate loop is required for RNA polymerase modification by RfaH and NusG." Mol Cell 43(2);253-62. PMID: 21777814
Stevens94: Stevens MP, Hanfling P, Jann B, Jann K, Roberts IS (1994). "Regulation of Escherichia coli K5 capsular polysaccharide expression: evidence for involvement of RfaH in the expression of group II capsules." FEMS Microbiol Lett 124(1);93-8. PMID: 8001774
Svetlov07: Svetlov V, Belogurov GA, Shabrova E, Vassylyev DG, Artsimovitch I (2007). "Allosteric control of the RNA polymerase by the elongation factor RfaH." Nucleic Acids Res 35(17);5694-705. PMID: 17711918
Vassylyeva06: Vassylyeva MN, Svetlov V, Klyuyev S, Devedjiev YD, Artsimovitch I, Vassylyev DG (2006). "Crystallization and preliminary crystallographic analysis of the transcriptional regulator RfaH from Escherichia coli and its complex with ops DNA." Acta Crystallogr Sect F Struct Biol Cryst Commun 62(Pt 10);1027-30. PMID: 17012804
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