Skip to main content
SpringerLink
Log in

Measuring Riboswitch Activity In Vitro and in Artificial Cells with Purified Transcription–Translation Machinery

  • Protocol
  • First Online:
Artificial Riboswitches

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1111))

Abstract

We present a simple method to measure the real-time activity of riboswitches with purified components in vitro and inside of artificial cells. Typically, riboswitch activity is measured in vivo by exploiting β-galactosidase encoding constructs with a putative riboswitch sequence in the untranslated region. Additional in vitro characterization often makes use of in-line probing to explore conformational changes induced by ligand binding to the mRNA or analyses of transcript lengths in the presence and absence of ligand. However, riboswitches ultimately control protein levels and often times require accessory factors. Therefore, an in vitro system capable of monitoring protein production with fully defined components that can be supplemented with accessory factors would greatly aid riboswitch studies. Herein we present a system that is amenable to such analyses. Further, since the described system can be easily reconstituted within compartments to build artificial, cellular mimics with sensing capability, protocols are provided for building sense-response systems within water-in-oil emulsion compartments and lipid vesicles. Only standard laboratory equipment and commercially available material are exploited for the described assays, including DNA, purified transcription–translation machinery, i.e., the PURE system, and a spectrofluorometer.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Soukup GA, Breaker RR (1999) Relationship between internucleotide linkage geometry and the stability of RNA. RNA 5:1308–1325

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  2. Nahvi A, Sudarsan N, Ebert MS, Zou X, Brown KL, Braker RR (2002) Genetic control by a metabolite-binding mRNA. Chem Biol 9:1043

    Article  CAS  PubMed  Google Scholar 

  3. Wakeman CA, Winkler WC (2009) Analysis of the RNA backbone: structural analysis of riboswitches by in-line probing and selective 2′-hydroxyl acylation and primer extension. In: Serganov A (ed) Riboswitches. Methods Mol Biol, vol 540, pp 173–191

    Google Scholar 

  4. Ellington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346:818–822

    Article  CAS  PubMed  Google Scholar 

  5. Serganov A, Yuan Y-R, Pikovskaya O, Polonskaia A, Malinina L, Phan AT, Hobartner C, Micura R, Breaker RR, Patel DJ (2004) Structural basis for discriminative regulation of gene expression by adenine- and guanine-sensing mRNAs. Chem Biol 11:1729–1741

    Article  CAS  PubMed  Google Scholar 

  6. Serganov A, Nudler E (2013) A decade of riboswitches. Cell 152:17–24

    Article  CAS  PubMed  Google Scholar 

  7. Desai SK, Gallivan JP (2004) Genetic screens and selections for small molecules based on a synthetic riboswitch that activates protein translation. J Am Chem Soc 126:13247–13254

    Article  CAS  PubMed  Google Scholar 

  8. Nomura Y, Yokobayashi Y (2007) Reengineering a natural riboswitch by dual genetic selection. J Am Chem Soc 129:13814–13815

    Article  CAS  PubMed  Google Scholar 

  9. Artsimovitch I, Henkin TM (2009) In vitro approaches to analysis of transcription termination. Methods 47:37–43

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. Mironov A, Epshtein V, Nudler E (2009) Transcriptional approaches to riboswitch studies. IN: Serganov A (Ed) Riboswitches. Methods Mol Biol, vol 540: 39-51, Humana Press, NY, USA

    Google Scholar 

  11. Muranaka N, Yokobayashi Y (2010) Posttranscriptional signal integration of engineered riboswitches yields band-pass output. Angew Chem Int Ed 49:4653–4655

    Article  CAS  Google Scholar 

  12. Topp S, Gallivan JP (2007) Guiding bacteria with small molecules and RNA. J Am Chem Soc 129:6807–6811

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  13. Soukup GA, Breaker RR (1999) Engineering precision RNA molecular switches. Proc Natl Acad Sci U S A 96:3584–3589

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Wickiser JK, Winkler WC, Breaker RR, Crothers DM (2005) The speed of RNA transcription and metabolite binding kinetics operate an FMN riboswitch. Mol Cell 18:49–60

    Article  CAS  PubMed  Google Scholar 

  15. Tawfik DS, Griffiths AD (1998) Man-made cell-like compartments for molecular evolution. Nat Biotechnol 16:652–656

    Article  CAS  PubMed  Google Scholar 

  16. Sunami T, Matsuura T, Suzuki H, Yomo T (2010) Synthesis of functional proteins within liposomes. Methods Mol Biol 607:243–256

    Article  CAS  PubMed  Google Scholar 

  17. Lynch SA, Gallivan JP (2009) A flow cytometry-based screen for synthetic riboswitches. Nucleic Acids Res 37:184–192

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Martini L, Mansy SS (2011) Cell-like systems with riboswitch controlled gene expression. Chem Commun 47:10734–10736

    Article  CAS  Google Scholar 

  19. Lemay J-F, Desnoyers G, Blouin S, Heppell B, Bastet L, St-Pierre P, Massé E, Lafontaine DA (2011) Comparative study between transcriptionally- and translationally-acting adenine riboswitches reveals key differences in riboswitch regulatory mechanisms. PLoS Genet 7:e1001278

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Lentini R, Forlin M, Martini L, Del Bianco C, Spencer AC, Torino D, Mansy SS (2013) Fluorescent proteins and in vitro genetic organization for cell-free synthetic biology. ACS Synth Biol 2:482–489

    PubMed  Google Scholar 

  21. Aiba H, Hanamura A, Yamano H (1991) Transcriptional terminator is a positive regulatory element in the expression of the Escherichia coli crp gene. J Biol Chem 266:1721–1727

    CAS  PubMed  Google Scholar 

  22. Sambrook J, Russel DW (2001) Commonly used techniques in molecular cloning. In: Molecular cloning. (Ed) Jan Argentine Cold Spring Harbor Laboratory Press, NY, USA

    Google Scholar 

  23. Sambrook J, Russell DW (2006) Standard ethanol precipitation of DNA in microcentrifuge tubes. Cold Spring Harb Protoc. doi:10.1101/pdb.prot4456

    Google Scholar 

  24. Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, Ueda T (2001) Cell-free translation reconstituted with purified components. Nat Biotechnol 19:752–755

    Article  Google Scholar 

  25. Zhou Y, Asahara H, Gaucher EA, Chong S (2012) Reconstitution of translation from Thermus thermophilus reveals a minimal set of components sufficient for protein synthesis at high temperatures and functional conservation of modern and ancient translation components. Nucleic Acids Res 40:7932–7945

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Forlin M, Lentini R, Mansy SS (2012) Cellular imitations. Curr Opin Chem Biol 16:586–592

    Article  CAS  PubMed  Google Scholar 

  27. Ichihashi N, Matsuura T, Kita H, Sunami T, Suzuki H, Yomo T (2010) Constructing partial models of cells. Cold Spring Harb Perspect Biol. doi:10.1101/cshperspect.a004945

    PubMed Central  PubMed  Google Scholar 

  28. Bianco CD, Mansy SS (2012) Non replicating protocells. Acc Chem Res 45:2125–2130

    Article  PubMed  Google Scholar 

  29. Colletier J-P, Chaize B, Winterhalter M, Fournier D (2002) Protein encapsulation in liposomes: efficiency depends on interaction between protein and phospholipid bilayer. BMC Biotechnol 2:9

    Article  PubMed Central  PubMed  Google Scholar 

  30. Davidson EA, Dlugosz PJ, Levy M, Ellington AD (2009) Directed evolution of proteins in vitro using compartmentalization in emulsions. Curr Protoc Mol Biol. doi:10.1002/0471142727.mb2406s87

    PubMed  Google Scholar 

  31. Lazzerini-Ospri L, Stano P, Luisi P, Marangoni R (2012) Characterization of the emergent properties of a synthetic quasi-cellular system. BMC Bioinformat 13(Suppl 4):S9

    Google Scholar 

  32. Nguyen AW, Daugherty PS (2005) Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat Biotechnol 23:355–360

    Article  CAS  PubMed  Google Scholar 

  33. Donoso P, O'Neill SC, Dilly KW, Negretti N, Eisner DA (1994) Comparison of the effects of caffeine and other methylxanthines on [Ca2+]i in rat ventricular myocytes. Br J Pharmacol 111:455–458

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Pereira de Souza T, Stano P, Luisi PL (2009) The minimal size of liposome-based model cells brings about a remarkably enhanced entrapment and protein synthesis. Chembiochem 10:1056–1063

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. CIBIO, University of Trento, Mattarello, Italy

    Laura Martini & Sheref S. Mansy

Authors
  1. Laura Martini
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sheref S. Mansy
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Ehime University, Matsuyama, Ehime, Japan

    Atsushi Ogawa

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media New York

About this protocol

Cite this protocol

Martini, L., Mansy, S.S. (2014). Measuring Riboswitch Activity In Vitro and in Artificial Cells with Purified Transcription–Translation Machinery. In: Ogawa, A. (eds) Artificial Riboswitches. Methods in Molecular Biology, vol 1111. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-755-6_11

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-755-6_11

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-754-9

  • Online ISBN: 978-1-62703-755-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation