Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Engineered riboregulators enable post-transcriptional control of gene expression

Nature Biotechnology volume 22, pages 841–847 (2004)Cite this article

Abstract

Recent studies have demonstrated the important enzymatic, structural and regulatory roles of RNA in the cell. Here we present a post-transcriptional regulation system in Escherichia coli that uses RNA to both silence and activate gene expression. We inserted a complementary cis sequence directly upstream of the ribosome binding site in a target gene. Upon transcription, this cis-repressive sequence causes a stem-loop structure to form at the 5′–untranslated region of the mRNA. The stem-loop structure interferes with ribosome binding, silencing gene expression. A small noncoding RNA that is expressed in trans targets the cis-repressed RNA with high specificity, causing an alteration in the stem-loop structure that activates expression. Such engineered riboregulators may lend insight into mechanistic actions of endogenous RNA-based processes and could serve as scalable components of biological networks, able to function with any promoter or gene to directly control gene expression.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The artificial riboregulator system used to control post-transcriptional gene regulation.
Figure 2: Results of cis repression of crRNA variants: crRL (red), crR7 (orange), crR10 (green), crRB (light blue) and control (dark blue).
Figure 3: Trans-activation mechanism and results.
Figure 4: Transient and steady-state responses and specificity results of riboregulator systems.

Similar content being viewed by others

References

  1. Gesteland, R.F., Cech, T.R. & Atkins, J.F. The RNA World, edn. 2 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999).

    Google Scholar 

  2. Joyce, G.F. The antiquity of RNA-based evolution. Nature 418, 214–221 (2002).

    Article  CAS  PubMed  Google Scholar 

  3. Eddy, S.R. Non-coding RNA genes and the modern RNA world. Nat. Rev. Genet. 2, 919–929 (2001).

    Article  CAS  PubMed  Google Scholar 

  4. Kruger, K. et al. Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena. Cell 31, 147–157 (1982).

    Article  CAS  PubMed  Google Scholar 

  5. Guerrier-Takada, C., Gardiner, K., Marsh, T., Pace, N. & Altman, S. The TNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell 35, 849–857 (1983).

    Article  CAS  PubMed  Google Scholar 

  6. Doudna, J.A. & Cech, T.R. The chemical repertoire of natural ribozymes. Nature 418, 222–228 (2002).

    Article  CAS  PubMed  Google Scholar 

  7. Steward, P., Molin, S. & Nordstrom, K. RNAs involved in copy-number control and incompatibility of plasmid R1. Proc. Natl. Acad. Sci. USA 78, 6008–6012 (1981).

    Article  Google Scholar 

  8. Wagner, E.G.H. & Simons, R.W. Antisense RNA control in bacteria, phages, and plasmids. Annu. Rev. Microbiol. 48, 713–742 (1994).

    Article  CAS  PubMed  Google Scholar 

  9. Lee, R.C., Feinbaum, R.L. & Ambros, V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843–854 (1993).

    Article  CAS  PubMed  Google Scholar 

  10. Gottesman, S. Stealth regulation: biological circuits with small RNA switches. Genes Dev. 16, 2829–2842 (2002).

    Article  CAS  PubMed  Google Scholar 

  11. Gelfand, M.S., Mironov, A.A., Jomantas, J., Kozlov, Y.I. & Perumov, D.A. A conserved RNA structure element involved in the regulation of bacterial riboflavin synthesis genes. Trends Genet. 15, 439–442 (1999).

    Article  CAS  PubMed  Google Scholar 

  12. Winkler, W., Nahvi, A. & Breaker, R.R. Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419, 952–656 (2002).

    Article  CAS  PubMed  Google Scholar 

  13. Johansson, J. et al. An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes. Cell 110, 551–561 (2002).

    Article  PubMed  Google Scholar 

  14. Mironov, A. et al. Sensing small molecules by nascent RNA: a mechanism to control transcription in bacteria. Cell 111, 747–756 (2002).

    Article  CAS  PubMed  Google Scholar 

  15. Morita, M.T. et al. Translational induction of heat shock transcription factor sigma32: evidence for a built-in RNA thermosensor. Genes Dev. 13, 655–665 (2002).

    Article  Google Scholar 

  16. Mandal, M., Boese, B., Barrick, J.E., Winkler, W.C. & Breaker, R.R. Riboswitches control fundamental biochemical pathways in Bacillus subtilis and other bacteria. Cell 113, 577–586 (2003).

    Article  CAS  PubMed  Google Scholar 

  17. Winkler, W., Nahvi, A., Roth, A., Collins, J.A. & Breaker, R.R. Control of gene expression by a natural metabolite-responsive ribozyme. Nature 428, 281–286 (2004).

    Article  CAS  PubMed  Google Scholar 

  18. Lease, R.A. & Belfort, M. A trans-acting RNA as a control switch in Escherichia coli: DsrA modulates function by forming alternative structures. Proc. Natl. Acad. Sci. USA 97, 9919–9924 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Majdalani, N., Hernandez, D. & Gottesman, S. Regulation and mode of action of the second small RNA activator of RpoS translation, RprA. Mol. Microbiol. 46, 813–826 (2002).

    Article  CAS  PubMed  Google Scholar 

  20. Opalinska, J.B. & Gewirtz, A.M. Nucleic–acid therapeutics: basic principles and recent applications. Nat. Rev. Drug Discov. 1, 503–514 (2002).

    Article  CAS  PubMed  Google Scholar 

  21. Good, L. Translation repression by antisense sequences. Cell. Mol. LifeSci. 60, 854–861 (2003).

    Article  CAS  Google Scholar 

  22. Ji, Y. et al. Identification of critical Staphylococcal gene using conditional phenotypes generated by antisense RNA. Science 293, 2266–2269 (2001).

    Article  CAS  PubMed  Google Scholar 

  23. Dykxhoorn, D., Novina, C.D. & Sharp, P.A. Killing the messenger: Short RNAs that silence gene expression. Nat. Rev. Mol. Cell Biol. 4, 457–467 (2003).

    Article  CAS  PubMed  Google Scholar 

  24. Wagner, E.G.H. & Flardh, K. Antisense RNAs everywhere? Trends Genet. 18, 223–226 (2002).

    Article  CAS  PubMed  Google Scholar 

  25. Engdahl, H.M., Lindell, M. & Wagner, E.G.H. Introduction of an RNA stability element at the 5′–end of an antisense RNA cassette increases the inhibition of target RNA translation. Antisense Nucleic A. 11, 29–40 (2001).

    CAS  Google Scholar 

  26. Morfeldt, E., Taylor, D., von Gabain, A. & Arvidson, S. Activation of alpha-toxin translation in Staphylococcus aureus by the trans-encoded antisense RNA, RNAIII. EMBO J. 14, 4569–4577 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Franch, T., Petersen, M., Wagner, E.G.H., Jacobsen, J.P. & Gerdes, K. Antisense RNA regulation in prokaryotes: rapid RNA/RNA interaction facilitated by a general U-turn loop structure. J. Mol. Biol. 294, 1115–1125 (1999).

    Article  CAS  PubMed  Google Scholar 

  28. Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Lutz, R. & Bujard, H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraCI1-I2 regulatory elements. Nucleic Acids Res. 25, 1203–1210 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Cormack, B.P., Valdivia, R.C. & Falkow, S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38 (1996).

    Article  CAS  PubMed  Google Scholar 

  31. Hjalt, T.A.H. & Wagner, E.G.H. Bulged-out nucleotides protect an antisense RNA from RNase III cleavage. Nucleic Acids Res. 23, 571–579 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Court, D. RNA processing and degradation by RNase III. Control of Messenger RNA Stability, (eds. Belasco, J. & Brawerman, G.) 71–116. (Academic Press, New York, 1993).

    Chapter  Google Scholar 

  33. Rivas, E., Klein, R.J., Jones, T.A. & Eddy, S.R. Computational identification of noncoding RNAs in E. coli by comparative genomics. Curr. Biol. 11, 1369–1373 (2001).

    Article  CAS  PubMed  Google Scholar 

  34. Altuvia, S. & Wagner, E.G.H. Switching on and off with RNA. Proc. Natl. Acad. Sci. USA 97, 9824–9826 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Hasty, J., McMillen, D. & Collins, J.J. Engineered gene circuits. Nature 420, 224–230 (2002).

    Article  CAS  PubMed  Google Scholar 

  36. Gardner, T.S., di Bernardo, D., Lorenz, D. & Collins, J.J. Inferring genetic networks and identifying compound mode of action via expression profiling. Science 301, 102–105 (2003).

    Article  CAS  PubMed  Google Scholar 

  37. Bartel, D.P. & Szostak, J.W. Isolation of new ribozymes from a large pool of random sequences. Science 261, 1411–1418 (1993).

    Article  CAS  PubMed  Google Scholar 

  38. Wilson, D.S. & Szostak, J.W. In vitro selection of functional nucleic acids. Annu. Rev. Biochem. 68, 611–647 (1999).

    Article  CAS  PubMed  Google Scholar 

  39. Ellington, A.D. & Szostak, J.W. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822 (1990).

    Article  CAS  PubMed  Google Scholar 

  40. Tuerk, C. & Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505–510 (1990).

    Article  CAS  PubMed  Google Scholar 

  41. Joyce, G.F. Amplification, mutation and selection of catalytic RNA. Gene 82, 83–87 (1989).

    Article  CAS  PubMed  Google Scholar 

  42. Robertson, M.P. & Ellington, A.D. In vitro selection of an allosteric ribozyme that transduces analytes to amplicons. Nat. Biotechnol. 17, 62–66 (1999).

    Article  CAS  PubMed  Google Scholar 

  43. Faulhammer, D., Cukras, A.R., Lipton, R.J. & Landweber, L.F. Molecular computation: RNA solutions to chess problems. Proc. Natl. Acad. Sci. USA 97, 1385–1389 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Smith, H.O., Hutchison, C.A. III, Pfannkoch, C. & Venter, J.C. Generating a synthetic genome by whole genome assembly: φx174 bacteriophage from synthetic oligonucleotides. Proc. Natl. Acad. Sci. USA 100, 15440–15445 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Tabor, J.J. & Ellington, A.D. Playing to win at DNA computation. Nat. Biotechnol. 21, 1013–1015 (2003).

    Article  CAS  PubMed  Google Scholar 

  46. Stojanovic, M.N. & Stefanovic, D. A deoxyribozyme–based molecular automaton. Nat. Biotechnol. 21, 1069–1074 (2003).

    Article  CAS  PubMed  Google Scholar 

  47. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular Cloning: A Laboratory Manual, edn. 2 (Cold Spring Harbor Laboratory Press, Plainview, New York, 1989).

    Google Scholar 

  48. Andersen, J.B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240–2246 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Ding, C. & Cantor, C.R. A high-throughput gene expression analysis technique using competitive PCR and matrix-assisted laser desorption ionization time-of-flight MS. Proc. Natl. Acad. Sci. USA 100, 3059–3064 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Marky, L.A. & Breslauer, K.J. Calculating thermodynamic data for transitions of any molecularity from equilibrium melting curves. Biopolymers 26, 1601–1620 (1987).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank T. Yoshida for providing access to the UV spectrophotometer; E. Protozanova for discussions and advice with RNA melting experiments; I. Smolina for help and advice with reverse transcription experiments; W. Blake, J. Hasty, D.H. Lee, J. Graber and members of our lab for helpful discussions and advice in preparing the manuscript. This work was supported by the National Science Foundation and Defense Advanced Research Projects Agency.

Author information

Authors and Affiliations

  1. Center for BioDynamics, Boston University, 44 Cummington Street, Boston, 02215, Massachusetts, USA

    Farren J Isaacs, Daniel J Dwyer, Dmitri D Pervouchine & James J Collins

  2. Center for Advanced Biotechnology, Boston University, 44 Cummington Street, Boston, 02215, Massachusetts, USA

    Farren J Isaacs, Daniel J Dwyer, Chunming Ding, Dmitri D Pervouchine, Charles R Cantor & James J Collins

  3. Bioinformatics Program, Boston University, 44 Cummington Street, Boston, 02215, Massachusetts, USA

    Farren J Isaacs, Chunming Ding, Charles R Cantor & James J Collins

  4. Department of Biomedical Engineering, Boston University, 44 Cummington Street, Boston, 02215, Massachusetts, USA

    Farren J Isaacs, Charles R Cantor & James J Collins

  5. Department of Biology, Boston University, 44 Cummington Street, Boston, 02215, Massachusetts, USA

    Daniel J Dwyer

Authors
  1. Farren J Isaacs
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daniel J Dwyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chunming Ding
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dmitri D Pervouchine
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Charles R Cantor
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. James J Collins
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to James J Collins.

Ethics declarations

Competing interests

The authors declare a pending patent application whose value may be affected by publication of this paper.

Supplementary information

Supplementary Table 1

List of plasmids in this study. aThe pBADHisA vector was obtained from Invitrogen. (PDF 32 kb)

Supplementary Table 2

Sequences of cis-repressed RNA constructs, loop containing the YUNR (TTGG) recognition motif, ribosome binding site (RBS), and trans-activating RNA constructs used in this work. (PDF 80 kb)

Supplementary Table 3

Real-competitive PCR assay design. List of primers used to amplify RTPCR products obtained from RNA cell preparations. A terminator mix contains three different ddNTPs and one dNTP. For example, CGT mix for 16S rRNA is ddCTP/ddGTP/ddTTP/dATP. (PDF 9 kb)

Supplementary Notes

Rational attempts to increase dynamic range of taR12-crR12 (PDF 100 kb)

Supplementary Fig. 1

Set of plasmids used in the artificial riboregulator systems. (PDF 49 kb)

Supplementary Fig. 2

Reverse transcription profiles of taRNA-crRNA complexes. (PDF 140 kb)

Supplementary Fig. 3

Determination of equilibrium dissociation constants for the taR7-crR12 pair. (PDF 34 kb)

Supplementary Fig. 4

RNA Melting curves for crR7, crR10, and crR12. Absorbance measurements at 260 nm (OD260) were determined between 10–95°C for each construct. (PDF 47 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Isaacs, F., Dwyer, D., Ding, C. et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol 22, 841–847 (2004). https://doi.org/10.1038/nbt986

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt986

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing