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.

  • Letter
  • Published:

Coenzyme recognition and gene regulation by a flavin mononucleotide riboswitch

Nature volume 458, pages 233–237 (2009)Cite this article

Abstract

The biosynthesis of several protein cofactors is subject to feedback regulation by riboswitches1,2,3. Flavin mononucleotide (FMN)-specific riboswitches4,5, also known as RFN elements6, direct expression of bacterial genes involved in the biosynthesis and transport of riboflavin (vitamin B2) and related compounds. Here we present the crystal structures of the Fusobacterium nucleatum riboswitch bound to FMN, riboflavin and antibiotic roseoflavin7. The FMN riboswitch structure, centred on an FMN-bound six-stem junction, does not fold by collinear stacking of adjacent helices, typical for folding of large RNAs. Rather, it adopts a butterfly-like scaffold, stapled together by opposingly directed but nearly identically folded peripheral domains. FMN is positioned asymmetrically within the junctional site and is specifically bound to RNA through interactions with the isoalloxazine ring chromophore and direct and Mg2+-mediated contacts with the phosphate moiety. Our structural data, complemented by binding and footprinting experiments, imply a largely pre-folded tertiary RNA architecture and FMN recognition mediated by conformational transitions within the junctional binding pocket. The inherent plasticity of the FMN-binding pocket and the availability of large openings make the riboswitch an attractive target for structure-based design of FMN-like antimicrobial compounds. Our studies also explain the effects of spontaneous and antibiotic-induced deregulatory mutations and provided molecular insights into FMN-based control of gene expression in normal and riboflavin-overproducing bacterial strains.

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: Overall structure and tertiary interactions of the FMN-bound F. nucleatum riboswitch.
Figure 2: Recognition of FMN by its riboswitch.
Figure 3: Interactions of FMN analogues with the riboswitch.
Figure 4: Probing the FMN riboswitch tertiary structure.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The coordinates of the X-ray structures of the FMN riboswitch are deposited in the RCSB Protein Data Bank under the following accession numbers: bound to FMN, transcribed RNA, 3F2Q; bound to FMN, two-strand composition, 3F4E; bound to riboflavin, 3F4G; bound to roseoflavin, 3F4H; [Ir(NH3)6]3+-soaked, 3F2T; Cs+-soaked 3F2X; Ba2+-soaked, 3F2W; Mn2+-soaked, 3F2Y; [Co(NH3)6]3+-soaked, 3F30.

References

  1. Nudler, E. & Mironov, A. S. The riboswitch control of bacterial metabolism. Trends Biochem. Sci. 29, 11–17 (2004)

    Article  CAS  Google Scholar 

  2. Serganov, A. & Patel, D. J. Ribozymes, riboswitches and beyond: regulation of gene expression without proteins. Nature Rev. Genet. 8, 776–790 (2007)

    Article  CAS  Google Scholar 

  3. Winkler, W. C. & Breaker, R. R. Regulation of bacterial gene expression by riboswitches. Annu. Rev. Microbiol. 59, 487–517 (2005)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Winkler, W. C., Cohen-Chalamish, S. & Breaker, R. R. An mRNA structure that controls gene expression by binding FMN. Proc. Natl Acad. Sci. USA 99, 15908–15913 (2002)

    Article  CAS  ADS  Google Scholar 

  6. 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  Google Scholar 

  7. Blount, K. F. & Breaker, R. R. Riboswitches as antibacterial drug targets. Nature Biotechnol. 24, 1558–1564 (2006)

    Article  CAS  Google Scholar 

  8. Barrick, J. E. & Breaker, R. R. The distributions, mechanisms, and structures of metabolite-binding riboswitches. Genome Biol. 8, R239 (2007)

    Article  Google Scholar 

  9. Serganov, A., Polonskaia, A., Phan, A. T., Breaker, R. R. & Patel, D. J. Structural basis for gene regulation by a thiamine pyrophosphate-sensing riboswitch. Nature 441, 1167–1171 (2006)

    Article  CAS  ADS  Google Scholar 

  10. Thore, S., Leibundgut, M. & Ban, N. Structure of the eukaryotic thiamine pyrophosphate riboswitch with its regulatory ligand. Science 312, 1208–1211 (2006)

    Article  CAS  ADS  Google Scholar 

  11. Edwards, T. E. & Ferre-D’Amare, A. R. Crystal structures of the thi-box riboswitch bound to thiamine pyrophosphate analogs reveal adaptive RNA-small molecule recognition. Structure 14, 1459–1468 (2006)

    Article  CAS  Google Scholar 

  12. Gilbert, S. D., Rambo, R. P., Van Tyne, D. & Batey, R. T. Structure of the SAM-II riboswitch bound to S-adenosylmethionine. Nature Struct. Mol. Biol. 15, 177–182 (2008)

    Article  CAS  Google Scholar 

  13. Montange, R. K. & Batey, R. T. Structure of the S-adenosylmethionine riboswitch regulatory mRNA element. Nature 441, 1172–1175 (2006)

    Article  CAS  ADS  Google Scholar 

  14. Nahvi, A. et al. Genetic control by a metabolite binding mRNA. Chem. Biol. 9, 1043–1049 (2002)

    Article  CAS  Google Scholar 

  15. Burgess, C. M., Smid, E. J., Rutten, G. & van Sinderen, D. A general method for selection of riboflavin-overproducing food grade micro-organisms. Microb. Cell Fact. 5, 24 (2006)

    Article  Google Scholar 

  16. Kapatral, V. et al. Genome sequence and analysis of the oral bacterium Fusobacterium nucleatum strain ATCC 25586. J. Bacteriol. 184, 2005–2018 (2002)

    Article  CAS  Google Scholar 

  17. Vitreschak, A. G., Rodionov, D. A., Mironov, A. A. & Gelfand, M. S. Regulation of riboflavin biosynthesis and transport genes in bacteria by transcriptional and translational attenuation. Nucleic Acids Res. 30, 3141–3151 (2002)

    Article  CAS  Google Scholar 

  18. Agmon, I., Bashan, A., Zarivach, R. & Yonath, A. Symmetry at the active site of the ribosome: structural and functional implications. Biol. Chem. 386, 833–844 (2005)

    Article  CAS  Google Scholar 

  19. Jaeger, L., Verzemnieks, E. J. & Geary, C. The UA_handle: a versatile submotif in stable RNA architectures. Nucleic Acids Res 10.1093/nar/gkn911 (28 November 2008)

  20. Lescoute, A. & Westhof, E. The interaction networks of structured RNAs. Nucleic Acids Res. 34, 6587–6604 (2006)

    Article  CAS  Google Scholar 

  21. Krasilnikov, A. S. & Mondragon, A. On the occurrence of the T-loop RNA folding motif in large RNA molecules. RNA 9, 640–643 (2008)

    Article  Google Scholar 

  22. Nagaswamy, U. & Fox, G. E. Frequent occurrence of the T-loop RNA folding motif in ribosomal RNAs. RNA 8, 1112–1119 (2002)

    Article  CAS  Google Scholar 

  23. Selmer, M. et al. Structure of the 70S ribosome complexed with mRNA and tRNA. Science 313, 1935–1942 (2006)

    Article  CAS  ADS  Google Scholar 

  24. Batey, R. T., Gilbert, S. D. & Montange, R. K. Structure of a natural guanine-responsive riboswitch complexed with the metabolite hypoxanthine. Nature 432, 411–415 (2004)

    Article  CAS  ADS  Google Scholar 

  25. Serganov, A. et al. Structural basis for discriminative regulation of gene expression by adenine- and guanine-sensing mRNAs. Chem. Biol. 11, 1729–1741 (2004)

    Article  CAS  Google Scholar 

  26. Garst, A. D., Heroux, A., Rambo, R. P. & Batey, R. T. Crystal structure of the lysine riboswitch regulatory mRNA element. J. Biol. Chem. 283, 22347–22351 (2008)

    Article  CAS  Google Scholar 

  27. Serganov, A., Huang, L. & Patel, D. J. Structural insights into amino acid binding and gene control by a lysine riboswitch. Nature 455, 1263–1267 (2008)

    Article  CAS  ADS  Google Scholar 

  28. Fan, P., Suri, A. K., Fiala, R., Live, D. & Patel, D. J. Molecular recognition in the FMN-RNA aptamer complex. J. Mol. Biol. 258, 480–500 (1996)

    Article  CAS  Google Scholar 

  29. Burgess, C., O’Connell-Motherway, M., Sybesma, W., Hugenholtz, J. & van Sinderen, D. Riboflavin production in Lactococcus lactis: potential for in situ production of vitamin-enriched foods. Appl. Environ. Microbiol. 70, 5769–5777 (2004)

    Article  CAS  Google Scholar 

  30. Wickiser, J. K., Winkler, W. C., Breaker, R. & Crothers, D. M. The speed of RNA transcription and metabolite binding kinetics operate an FMN riboswitch. Mol. Cell 18, 49–60 (2005)

    Article  CAS  Google Scholar 

  31. Serganov, A. et al. Ribosomal protein S15 from Thermus thermophilus: cloning, sequencing, overexpression of the gene and RNA-binding properties of the protein. Eur. J. Biochem. 246, 291–300 (1997)

    Article  CAS  Google Scholar 

  32. de La Fortelle, E. & Bricogne, G. in Methods in Enzymology 472–494 (Academic Press, 1997)

    Google Scholar 

  33. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    Article  CAS  Google Scholar 

  34. Feig, A. L. & Uhlenbeck, O. C. in The RNA World 2nd edn (eds Gesteland, R. F., Cech, T. R. & Atkins, J. F.) 287–319 (Cold Spring Harbor Laboratory Press, 1999)

    Google Scholar 

  35. Serganov, A., Polonskaia, A., Ehresmann, B., Ehresmann, C. & Patel, D. J. Ribosomal protein S15 represses its own translation via adaptation of an rRNA-like fold within its mRNA. EMBO J. 8, 1898–1908 (2003)

    Article  Google Scholar 

Download references

Acknowledgements

We thank the personnel of beamline X29 at the Brookhaven National Laboratory and beamline 24-ID-C at the Advanced Photon Source, Argonne National Laboratory, funded by the US Department of Energy. We thank L. Jaeger (University of California, Santa Barbara) for discussions and O. Ouerfelli (Memorial Sloan-Kettering Cancer Center, New York) for the synthesis of iridium hexamine. D.J.P. was supported by funds from the National Institutes of Health.

Author Contributions L.H. crystallized the F. nucleatum FMN riboswitch and performed binding experiments, A.S. determined the structures and performed footprinting experiments, and A.S. and D.J.P. wrote the manuscript with the assistance of L.H. All authors discussed the results and commented on the manuscript.

Author information

Author notes

  1. Alexander Serganov and Lili Huang: These authors contributed equally to this work.

Authors and Affiliations

  1. Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA,

    Alexander Serganov, Lili Huang & Dinshaw J. Patel

Authors
  1. Alexander Serganov
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lili Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dinshaw J. Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Alexander Serganov or Dinshaw J. Patel.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-19 with Legends, Supplementary Tables 1-2 and Supplementary References. This file was replaced on February 4th, 2009 to correct 2 chemical formulae. (PDF 9097 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Serganov, A., Huang, L. & Patel, D. Coenzyme recognition and gene regulation by a flavin mononucleotide riboswitch. Nature 458, 233–237 (2009). https://doi.org/10.1038/nature07642

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature07642

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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