RfaH
RfaH is a transcription/ translation coupling factor found in bacteria, specifically in Escherichia coli , that belongs to the NusG family of transcription elongation factors. As a specialized paralog of NusG, RfaH regulates the expression of long operons and has been extensively studied, particularly for its role in activating cell wall biosynthesis, conjugation, and virulence genes by inhibiting the Rho factor.[1][2] RfaH is characterized as a sequence-specific paralog, which preferentially enhances distal expression within operons that contain specific promoter-proximal ops DNA elements. The ops sequence facilitates the binding of RfaH to elongating RNA polymerase (RNAP), thereby restricting its functional influence to a limited number of operons within E. coli.[3]
RfaH Domain architecture (NTD and CTD)
RfaH has two main structural domain protein, N-terminal domain (NTD) and C-terminal domain (CTD) which are connected by a flexible linker.[4][1][5] The NTDs exhibit mixed α/β topology that comprised of a four-stranded antiparallel β sheet surrounded by two and one α helices on each sides.[2] The CTD has two long antiparallel α helices that form a coiled coil. RfaH CTD is closely linked to the NTD and takes on an all-α fold in the free state. When NTD bound to RNAP, the domains separate and the CTD transform into an all-β fold, while the NTD remains mostly intact. This existence of CTD in two distinct folded states makes RfaH a classic metamorphic or transformer protein.[2][6]
Structural State of RfaH CTD
The CTD of RfaH populates two completely different folded states depending on whether the protein is in its autoinhibited or active form.[4] In the closed, autoinhibited state, the CTD forms a compact two-helical α-hairpin arranged in an antiparallel topology that tightly packs against the NTD.[7] This arrangement masks the RNAP-binding interface on the NTD and keeps RfaH inactive until it encounters its specific DNA recruitment signal on the transcription complex. Structural studies show that this α-helical fold is not intrinsically stable on its own; instead, it is stabilized by extensive NTD–CTD interactions, including buried hydrophobic patches and a well-defined interface where both domains move as a single rigid body[4][2][8][9]
Upon recruitment of RfaH to the paused transcription elongation complex at the ops hairpin, the NTD engages RNAP and the CTD is forcibly displaced. Once released, the CTD undergoes a dramatic refolding event into a five-stranded β-barrel,[10] topologically equivalent to the NusG-CTD. NMR analyses of isolated CTD[4], along with computational modeling, show that the thermodynamically preferred state in the absence of interaction between NTD and CTD, is the β-barrel.[11][12][13][7] In this β-form, the CTD gains a new functional surface that is required for downstream interactions, particularly with ribosomal protein S10 where it facilitates translation when canonical ribosome recruitment elements is absent.[8][7]
Mechanism of CTD Metamorphosis
Activation: RNAP encounters an operon polarity suppressor (ops) sequence thereby exposing a DNA hairpin (non-template DNA strand) which serve as signal for the recruitment of RfaH to the paused elongation complex (EC).[14]
Domain dissociation: RfaH NTD binds to the RNAP leading to the release of CTD.[14]
Refolding of CTD (α → β): CTD dissociation triggers its metamorphic fold switching from all-α helical hairpin to an all-β five-stranded β-barrel thereby activating RfaH.[3]
Downstream processes: The activated RfaH: opsEC complex then moves downstream with RNAP, where the β-CTD recruits ribosomal protein S10, enabling RfaH to assemble a transcription–translation expressome and promote processive transcription of long operons.
Termination: Transcription is terminated when RNAP arrives at the operon terminal. This leads to the dissociation of RfaH from the complex, allowing the CTD to refold back into the α-helical state and rebind the NTD.[2]
Biological Importance and Implication
- Transcriptional regulation: RfaH helps to prevent premature termination of specific gene transcription by binding to RNAP. RfaH is a primary transcript elongation regulator through a process known as antitermination.[4][14]
- Translation factor: The refolded RfaH-CTD interacts with the 30S subunit and initiator tRNA, enabling a translation initiation complex to scan nascent mRNA for a start signal. If this recruitment and contact with ribosome is maintained, it enhances translation, reduces Rho-dependent termination by shielding mRNA and possibly preventing ribosome pausing and release.[4]
- Polarity suppression: It suppresses the "polarity" effect that can cause the transcription of distal genes in an operon to be significantly reduced compared to proximal genes. This is achieved by RfaH antipausing activity on RNAP and suppression of transcription factor Rho (ρ) which preferentially targets paused RNAPs in order to terminate transcription.[3][15]
- Enzyme stabilization: RfaH stabilizes the RNAP complex, ensuring it can continue moving along the DNA and finish transcribing target long operons.[3][1]
- Regulation of virulence and adaptation: RfaH is pivotal for bacterial virulence, host colonization, and survival in hostile environments by regulating the expression of genes involved in cell surface structures like lipopolysaccharide.[16][17]
- Fold-switching: RfaH CTD can switch between different three-dimensional shapes (all α helices in free state → β-barrel in activated state) to perform different functions, a property that allows it to be a key regulator in conditions requiring both transcription and translation.[16]
See also
External links
- Uniport entry for RfaH
References
- ↑ 1.0 1.1 1.2 Bailey, Marc J. A.; Hughes, Colin; Koronakis, Vassilis (1996). "Increased distal gene transcription by the elongation factor RfaH, a specialized homologue of NusG". Molecular Microbiology. 22 (4): 729–737. doi:10.1046/j.1365-2958.1996.d01-1726.x. ISSN 1365-2958. PMID 8951819.
- ↑ 2.0 2.1 2.2 2.3 2.4 2.5 2.6 Zuber, Philipp Konrad; Schweimer, Kristian; Rösch, Paul; Artsimovitch, Irina; Knauer, Stefan H. (11 February 2019). "Reversible fold-switching controls the functional cycle of the antitermination factor RfaH". Nature Communications. 10 (1). Bibcode:2019NatCo..10..702Z. doi:10.1038/s41467-019-08567-6. ISSN 2041-1723. PMC 6370827. PMID 30742024. Unknown parameter
|article-number=ignored (help) - ↑ 3.0 3.1 3.2 3.3 Sevostyanova, Anastasia; Belogurov, Georgiy A.; Mooney, Rachel A.; Landick, Robert; Artsimovitch, Irina (2011). "The β Subunit Gate Loop Is Required for RNA Polymerase Modification by RfaH and NusG". Molecular Cell. 43 (2): 253–262. doi:10.1016/j.molcel.2011.05.026. PMC 3142557. PMID 21777814.
- ↑ 4.0 4.1 4.2 4.3 4.4 4.5 Burmann, Björn M.; Knauer, Stefan H.; Sevostyanova, Anastasia; Schweimer, Kristian; Mooney, Rachel A.; Landick, Robert; Artsimovitch, Irina; Rösch, Paul (July 2012). "An α Helix to β Barrel Domain Switch Transforms the Transcription Factor RfaH into a Translation Factor". Cell. 150 (2): 291–303. doi:10.1016/j.cell.2012.05.042. PMC 3430373. PMID 22817892.
- ↑ Seifi, Bahman; Wallin, Stefan (March 2025). "Impact of N-Terminal Domain Conformation and Domain Interactions on RfaH Fold Switching". Proteins: Structure, Function, and Bioinformatics. 93 (3): 608–619. doi:10.1002/prot.26755. ISSN 0887-3585.
- ↑ Belogurov, Georgiy A.; Vassylyeva, Marina N.; Svetlov, Vladimir; Klyuyev, Sergiy; Grishin, Nick V.; Vassylyev, Dmitry G.; Artsimovitch, Irina (April 2007). "Structural Basis for Converting a General Transcription Factor into an Operon-Specific Virulence Regulator". Molecular Cell. 26 (1): 117–129. doi:10.1016/j.molcel.2007.02.021. PMC 3116145. PMID 17434131.
- ↑ 7.0 7.1 7.2 Li, Shanshan; Xiong, Bing; Xu, Yuan; Lu, Tao; Luo, Xiaomin; Luo, Cheng; Shen, Jingkang; Chen, Kaixian; Zheng, Mingyue; Jiang, Hualiang (10 June 2014). "Mechanism of the All-α to All-β Conformational Transition of RfaH-CTD: Molecular Dynamics Simulation and Markov State Model". Journal of Chemical Theory and Computation. 10 (6): 2255–2264. Bibcode:2014JCTC...10.2255L. doi:10.1021/ct5002279. ISSN 1549-9618. PMID 26580748.
- ↑ 8.0 8.1 Cai, Mengli; Agarwal, Nipanshu; Garrett, Daniel S.; Baber, James; Clore, G. Marius (20 August 2024). "A Transient, Excited Species of the Autoinhibited α-State of the Bacterial Transcription Factor RfaH May Represent an Early Intermediate on the Fold-Switching Pathway". Biochemistry. 63 (16): 2030–2039. doi:10.1021/acs.biochem.4c00258. ISSN 0006-2960. PMC 11345854 Check
|pmc=value (help). PMID 39088556 Check|pmid=value (help). - ↑ Galaz-Davison, Pablo; Román, Ernesto A.; Ramírez-Sarmiento, César A. (2021). "The N-terminal domain of RfaH plays an active role in protein fold-switching". PLOS Computational Biology. 17 (9): e1008882. Bibcode:2021PLSCB..17E8882G. doi:10.1371/journal.pcbi.1008882. ISSN 1553-7358. PMC 8454952 Check
|pmc=value (help). PMID 34478435 Check|pmid=value (help). - ↑ Sevostyanova, Anastasia; Belogurov, Georgiy A.; Mooney, Rachel A.; Landick, Robert; Artsimovitch, Irina (22 July 2011). "The β Subunit Gate Loop Is Required for RNA Polymerase Modification by RfaH and NusG". Molecular Cell. 43 (2): 253–262. doi:10.1016/j.molcel.2011.05.026. ISSN 1097-2765. PMC 3142557. PMID 21777814.
- ↑ Bernhardt, Nathan A.; Hansmann, Ulrich H. E. (8 February 2018). "Multifunnel Landscape of the Fold-Switching Protein RfaH-CTD". The Journal of Physical Chemistry B. 122 (5): 1600–1607. Bibcode:2018JPCB..122.1600B. doi:10.1021/acs.jpcb.7b11352. ISSN 1520-6106. PMC 5823028. PMID 29323497.
- ↑ GC, Jeevan B.; Bhandari, Yuba R.; Gerstman, Bernard S.; Chapagain, Prem P. (15 May 2014). "Molecular Dynamics Investigations of the α-Helix to β-Barrel Conformational Transformation in the RfaH Transcription Factor". The Journal of Physical Chemistry B. 118 (19): 5101–5108. Bibcode:2014JPCB..118.5101G. doi:10.1021/jp502193v. ISSN 1520-6106. PMID 24758259.
- ↑ Joseph, Jerelle A.; Chakraborty, Debayan; Wales, David J. (8 January 2019). "Energy Landscape for Fold-Switching in Regulatory Protein RfaH". Journal of Chemical Theory and Computation. 15 (1): 731–742. Bibcode:2019JCTC...15..731J. doi:10.1021/acs.jctc.8b00912. ISSN 1549-9618.
- ↑ 14.0 14.1 14.2 Artsimovitch, Irina; Landick, Robert (19 April 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. doi:10.1016/S0092-8674(02)00724-9. ISSN 0092-8674. PMID 12007406.
- ↑ Bailey, Marc J. A.; Hughes, Colin; Koronakis, Vassilis (November 1996). "Increased distal gene transcription by the elongation factor RfaH, a specialized homologue of NusG". Molecular Microbiology. 22 (4): 729–737. doi:10.1046/j.1365-2958.1996.d01-1726.x. ISSN 0950-382X. PMID 8951819.
- ↑ 16.0 16.1 Tomar, Sushil Kumar; Knauer, Stefan H.; NandyMazumdar, Monali; Rösch, Paul; Artsimovitch, Irina (1 December 2013). "Interdomain contacts control folding of transcription factor RfaH". Nucleic Acids Research. 41 (22): 10077–10085. doi:10.1093/nar/gkt779. ISSN 1362-4962. PMC 3905879. PMID 23990324.
- ↑ Waititu, Joram Kiriga; Nilsson, Kristina; Larrouy-Maumus, Gerald; Costa, Tiago R. D.; Avican, Kemal (29 September 2025). "RfaH is essential for virulence and adaptive responses in Yersinia pseudotuberculosis infection". mBio. 16 (11): e02122–25. doi:10.1128/mbio.02122-25. PMC 12607645 Check
|pmc=value (help). PMID 41020597 Check|pmid=value (help).
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