Wikipedia

Draft:RfaH

  • Comment: Still no secondary sources even though the LLM-generated content has been rewritten. GTrang (talk) 05:31, 27 December 2025 (UTC)
  • Comment: I will refrain from reviewing the article twice, but I will note this version is greatly improved over the previous version. WeirdNAnnoyed (talk) 12:42, 19 December 2025 (UTC)
  • Comment: I'm not certain this individual protein is notable based on the cited sources. All sources appear to be primary to me, and we typically require secondary sourcing (primary sources are fine for verification but not notability). I also suspect the article is partly LLM-written, which is allowed as long as the output is vetted by a human, but please see WP:MOS because there are some formatting issues that might raise alarm. Specifically, the last two sections should be converted to prose (and shortened, as they are too detailed). But my major reason for declining at this time is the lack of secondary sourcing. WeirdNAnnoyed (talk) 23:22, 8 December 2025 (UTC)

RfaH is a specialized paralog of NusG family of transcription elongation factors found in bacteria, specifically in Escherichia coli.[1] It regulates the expression of long operons and has been extensively studied[2][3], particularly for its role in activating cell wall biosynthesis, conjugation, and virulence genes by inhibiting the Rho factor.[4][5] RfaH preferentially enhances distal expression within operons that contain specific promoter-proximal operon polarity suppressor (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.[6] RfaH is a well-studied example of a metamorphic protein.[1][7][2][8]

RfaH Domain architecture (NTD and CTD)

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RfaH has two main structural domain protein, N-terminal domain (NTD) and C-terminal domain (CTD) which are connected by a flexible linker.[1][9][4][10] 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.[5] 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.[1][2][5][11][7]

Structural State of RfaH CTD

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Rfa CTD transformation from an α-hairpin to a β-barrel.[5]

The CTD of RfaH populates two completely different folded states depending on whether the protein is in its autoinhibited or active form.[9] 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.[1][12] 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 amino acids and a well-defined interface where both domains move as a single rigid body[9][5][13][14]

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,[15] topologically equivalent to the NusG-CTD. NMR analyses of isolated CTD[9], along with computational modeling, show that the thermodynamically preferred state in the absence of interaction between NTD and CTD, is the β-barrel.[16][17][18][12] 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.[13][12]

Mechanism of CTD Metamorphosis

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The process by which the CTD switches from an α-hairpin to a β-barrel—essentially its “metamorphosis”—is driven by activation of RfaH at the ops-paused transcription complex.​ When the NTD recognizes the ops sequence, the normally tight interface between the NTD and CTD loosens. This is the key event that “releases” the CTD from its inhibited state. Once released, the α-helical hairpin is no longer stable on its own, so it begins to unfold from an all-α helical hairpin to an all-β strand. This new state enables RfaH to interact with translation machinery[5][2]

When RNAP encounters an operon polarity suppressor (ops) sequence it exposes a DNA hairpin (non-template DNA strand) which serve as signal for the recruitment of RfaH to the paused elongation complex (EC). RfaH NTD then binds to the RNAP leading to the release of CTD.[2][19][20] CTD dissociation triggers its metamorphic fold switching from all-α helical hairpin to an all-β five-stranded β-barrel thereby activating RfaH.[6] 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. 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][5]

Biological Importance and Implication

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RfaH is a primary transcription elongation factor that prevents premature termination of specific operons by binding to the transcription elongation complex and enhancing RNAP processivity. It prevent RNAP pausing and counteracts Rho-dependent termination, thereby suppressing transcriptional polarity and allowing efficient expression of distal genes within long operons.[6][9][20][21] RfaH can also interact with the ribosome 30S subunit and components of the translation initiation machinery, enabling translation initiation complex formation to scan the nascent mRNA.[2] Due to its C-terminal domain fold switching from an all-α helices in autoinhibited state to a β-barrel in the active state, it underlies the transition from a transcription-focused factor to a translation factor.[1][2][5] [22]

RfaH in important for bacterial virulence, host colonization, and environmental adaptation by upregulating operons involved in cell-surface structures such as lipopolysaccharide and capsules.[22][23]

See also

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References

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  1. 1 2 3 4 5 6 Chakravarty, Devlina; Porter, Lauren L. (18 November 2025). "Fold-Switching Proteins". Annual Review of Biophysics. 55 (1): 17–37. doi:10.1146/annurev-biophys-022924-012038. ISSN 1936-122X. PMC 12629603. PMID 41252592.
  2. 1 2 3 4 5 6 7 8 Artsimovitch, Irina; Ramírez-Sarmiento, César A. (2022). "Metamorphic proteins under a computational microscope: Lessons from a fold-switching RfaH protein". Computational and Structural Biotechnology Journal. 20: 5824–5837. doi:10.1016/j.csbj.2022.10.024. PMC 9630627. PMID 36382197.
  3. ↑ Bailey, Marc J. A.; Hughes, Colin; Koronakis, Vassilis (December 1997). "RfaH and the ops element, components of a novel system controlling bacterial transcription elongation". Molecular Microbiology. 26 (5): 845–851. doi:10.1046/j.1365-2958.1997.6432014.x. ISSN 0950-382X. PMID 9426123.
  4. 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.
  5. 1 2 3 4 5 6 7 8 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) 702. Bibcode:2019NatCo..10..702Z. doi:10.1038/s41467-019-08567-6. ISSN 2041-1723. PMC 6370827. PMID 30742024.
  6. 1 2 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.
  7. 1 2 Porter, Lauren L.; Artsimovitch, Irina; Ramírez-Sarmiento, César A. (June 2024). "Metamorphic proteins and how to find them". Current Opinion in Structural Biology. 86 102807. doi:10.1016/j.sbi.2024.102807. PMC 11102287. PMID 38537533.
  8. ↑ Dishman, Acacia F.; Volkman, Brian F. (15 June 2018). "Unfolding the Mysteries of Protein Metamorphosis". ACS Chemical Biology. 13 (6): 1438–1446. doi:10.1021/acschembio.8b00276. ISSN 1554-8929. PMC 6007232. PMID 29787234.
  9. 1 2 3 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.
  10. ↑ 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. PMID 39400465.
  11. ↑ 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.
  12. 1 2 3 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.
  13. 1 2 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. PMID 39088556.
  14. ↑ 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. PMID 34478435.{{cite journal}}: CS1 maint: article number as page number (link)
  15. ↑ 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.
  16. ↑ 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.
  17. ↑ 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.
  18. ↑ 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. PMID 30537824.
  19. ↑ Santangelo, Thomas J; Roberts, Jeffrey W (April 2002). "RfaH, a Bacterial Transcription Antiterminator". Molecular Cell. 9 (4): 698–700. doi:10.1016/S1097-2765(02)00516-6. PMID 11983161.
  20. 1 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.
  21. ↑ 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.
  22. 1 2 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.
  23. ↑ 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. PMID 41020597.
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  • Uniport entry for RfaH
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