Monitoring RNA Unwinding by the Transcription Termination Factor Rho from Mycobacterium tuberculosis

  • François D’Heygère
  • Annie Schwartz
  • Franck Coste
  • Bertrand Castaing
  • Marc BoudvillainEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1259)


Transcription termination factor Rho is a ring-shaped, homo-hexamieric RNA translocase that dissociates transcription elongation complexes and transcriptional RNA–DNA duplexes (R-loops) in bacteria. The molecular mechanisms underlying these biological functions have been essentially studied with Rho enzymes from Escherichia coli or close Gram-negative relatives. However, phylo-divergent Rho factors may have distinct properties. Here, we describe methods for the preparation and in vitro characterization (ATPase and helicase activities) of the Rho factor from Mycobacterium tuberculosis, a specimen with uncharacteristic molecular and enzymatic features. These methods set the stage for future studies aimed at better defining the diversity of enzymatic properties of Rho across the bacterial kingdom.

Key words

Rho Transcription Termination Ring shaped Helicase Hexamer RNA Molecular motor 



This work was supported by a joint Ph.D. scholarship from the University of Orléans and Région Centre to F.d.H and by CNRS core funding to M.B.


  1. 1.
    Roberts JW (1969) Termination factor for RNA synthesis. Nature 224:1168–1174PubMedCrossRefGoogle Scholar
  2. 2.
    Boudvillain M, Figueroa-Bossi N, Bossi L (2013) Terminator still moving forward: expanding roles for Rho factor. Curr Opin Microbiol 16:118–124PubMedCrossRefGoogle Scholar
  3. 3.
    Peters JM, Vangeloff AD, Landick R (2011) Bacterial transcription terminators: the RNA 3′-end chronicles. J Mol Biol 412:793–813. doi: 10.1016/j.jmb.2011.03.036 PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    D’Heygere F, Rabhi M, Boudvillain M (2013) Phyletic distribution and conservation of the bacterial transcription termination factor Rho. Microbiology 159:1423–1436. doi: 10.1099/mic.0.067462-0 PubMedCrossRefGoogle Scholar
  5. 5.
    Boudvillain M, Nollmann M, Margeat E (2010) Keeping up to speed with the transcription termination factor Rho motor. Transcription 1:70–75. doi: 10.4161/trns.1.2.12232 PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Skordalakes E, Berger JM (2003) Structure of the Rho transcription terminator: mechanism of mRNA recognition and helicase loading. Cell 114:135–146PubMedCrossRefGoogle Scholar
  7. 7.
    Thomsen ND, Berger JM (2009) Running in reverse: the structural basis for translocation polarity in hexameric helicases. Cell 139:523–534. doi: 10.1016/j.cell.2009.08.043 PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Kalarickal NC, Ranjan A, Kalyani BS et al (2010) A bacterial transcription terminator with inefficient molecular motor action but with a robust transcription termination function. J Mol Biol 395:966–982PubMedCrossRefGoogle Scholar
  9. 9.
    Nowatzke WL, Richardson JP (1996) Characterization of an unusual Rho factor from the high G + C gram-positive bacterium Micrococcus luteus. J Biol Chem 271:742–747PubMedCrossRefGoogle Scholar
  10. 10.
    Nowatzke WL, Burns CM, Richardson JP (1997) Function of the novel subdomain in the RNA binding domain of transcription termination factor Rho from micrococcus luteus. J Biol Chem 272:2207–2211PubMedCrossRefGoogle Scholar
  11. 11.
    Boudvillain M, Walmacq C, Schwartz A, Jacquinot F (2010) Simple enzymatic assays for the in vitro motor activity of transcription termination factor Rho from Escherichia coli. Methods Mol Biol 587:137–154. doi: 10.1007/978-1-60327-355-8_10 PubMedCrossRefGoogle Scholar
  12. 12.
    Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234PubMedCrossRefGoogle Scholar
  13. 13.
    Liu F, Putnam A, Jankowsky E (2008) ATP hydrolysis is required for DEAD-box protein recycling but not for duplex unwinding. Proc Natl Acad Sci U S A 105:20209–20214. doi: 10.1073/pnas.0811115106 PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Miwa Y, Horiguchi T, Shigesada K (1995) Structural and functional dissections of transcription termination factor rho by random mutagenesis. J Mol Biol 254:815–837PubMedCrossRefGoogle Scholar
  15. 15.
    Xu Y, Kohn H, Widger WR (2002) Mutations in the rho transcription termination factor that affect RNA tracking. J Biol Chem 277:30023–30030PubMedCrossRefGoogle Scholar
  16. 16.
    Rabhi M, Rahmouni AR, Boudvillain M (2010) Transcription termination factor Rho: a ring-shaped RNA helicase from bacteria. In: Jankowsky E (ed) RNA helicases, vol 19. RSC Publishing, Cambridge, UK, pp 243–271CrossRefGoogle Scholar
  17. 17.
    Fiorini F, Boudvillain M, Le Hir H (2013) Tight intramolecular regulation of the human Upf1 helicase by its N- and C-terminal domains. Nucleic Acids Res 41:2404–2415. doi: 10.1093/nar/gks1320 PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Bradley MJ, De La Cruz EM (2012) Analyzing ATP utilization by DEAD-Box RNA helicases using kinetic and equilibrium methods. Methods Enzymol 511:29–63. doi: 10.1016/B978-0-12-396546-2.00002-4 PubMedCrossRefGoogle Scholar
  19. 19.
    Rabhi M, Gocheva V, Jacquinot F et al (2011) Mutagenesis-based evidence for an asymmetric configuration of the ring-shaped transcription termination factor Rho. J Mol Biol 405:497–518. doi: 10.1016/j.jmb.2010.11.006 PubMedCrossRefGoogle Scholar
  20. 20.
    Cummins LL, Owens SR, Risen LM et al (1995) Characterization of fully 2′-modified oligoribonucleotide hetero- and homoduplex hybridization and nuclease sensitivity. Nucleic Acids Res 23:2019–2024PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Walmacq C, Rahmouni AR, Boudvillain M (2004) Influence of substrate composition on the helicase activity of transcription termination factor Rho: reduced processivity of Rho hexamers during unwinding of RNA-DNA hybrid regions. J Mol Biol 342:403–420. doi: 10.1016/j.jmb.2004.07.026 PubMedCrossRefGoogle Scholar
  22. 22.
    Geiselmann J, Yager T, Gill S et al (1992) Physical properties of the Escherichia coli transcription termination factor rho. 1. Association states and geometry of the rho hexamer. Biochemistry 31:111–121PubMedCrossRefGoogle Scholar
  23. 23.
    Kao C, Rudisser S, Zheng M (2001) A simple and efficient method to transcribe RNAs with reduced 3′ heterogeneity. Methods 23:201–205PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • François D’Heygère
    • 1
    • 2
  • Annie Schwartz
    • 1
  • Franck Coste
    • 1
  • Bertrand Castaing
    • 1
  • Marc Boudvillain
    • 1
    Email author
  1. 1.Centre de Biophysique MoléculaireCentre National de la Recherche Scientifique (CNRS)Orléans, cedex 2France
  2. 2.Ecole doctorale Santé, Sciences Biologiques et Chimie du Vivant (ED 549)Université d’OrléansOrléansFrance

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