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:

B12 cofactors directly stabilize an mRNA regulatory switch

Nature volume 492, pages 133–137 (2012)Cite this article

Subjects

Abstract

Structures of riboswitch receptor domains bound to their effector have shown how messenger RNAs recognize diverse small molecules, but mechanistic details linking the structures to the regulation of gene expression remain elusive1,2. To address this, here we solve crystal structures of two different classes of cobalamin (vitamin B12)-binding riboswitches that include the structural switch of the downstream regulatory domain. These classes share a common cobalamin-binding core, but use distinct peripheral extensions to recognize different B12 derivatives. In each case, recognition is accomplished through shape complementarity between the RNA and cobalamin, with relatively few hydrogen bonding interactions that typically govern RNA–small molecule recognition. We show that a composite cobalamin–RNA scaffold stabilizes an unusual long-range intramolecular kissing-loop interaction that controls mRNA expression. This is the first, to our knowledge, riboswitch crystal structure detailing how the receptor and regulatory domains communicate in a ligand-dependent fashion to regulate mRNA 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: Structures of cobalamins and cobalamin riboswitches.
Figure 2: Crystal structures of two distinct cobalamin riboswitches.
Figure 3: Cobalamin recognition by the receptor domain.
Figure 4: Formation of the kissing-loop interaction is the basis of cobalamin-dependent regulatory activity.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB) under accession numbers 4FRG (env8AqCbl(ΔJ1/13,P13)), 4FRN (env8AqCbl) and 4GMA (TteAdoCbl).

References

  1. Garst, A. D., Edwards, A. L. & Batey, R. T. Riboswitches: structures and mechanisms. Cold Spring Harb. Perspect. Biol. 3, a003533 (2011)

    Article  Google Scholar 

  2. Breaker, R. R. Riboswitches and the RNA world. Cold Spring Harb. Perspect. Biol. 4, a003566 (2012)

    Article  Google Scholar 

  3. Lundrigan, M. D., Koster, W. & Kadner, R. J. Transcribed sequences of the Escherichia coli btuB gene control its expression and regulation by vitamin B12 . Proc. Natl Acad. Sci. USA 88, 1479–1483 (1991)

    Article  ADS  CAS  Google Scholar 

  4. Franklund, C. V. & Kadner, R. J. Multiple transcribed elements control expression of the Escherichia coli btuB gene. J. Bacteriol. 179, 4039–4042 (1997)

    Article  CAS  Google Scholar 

  5. Nahvi, A., Barrick, J. E. & Breaker, R. R. Coenzyme B12 riboswitches are widespread genetic control elements in prokaryotes. Nucleic Acids Res. 32, 143–150 (2004)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  7. Rodionov, D. A., Vitreschak, A. G., Mironov, A. A. & Gelfand, M. S. Comparative genomics of the vitamin B12 metabolism and regulation in prokaryotes. J. Biol. Chem. 278, 41148–41159 (2003)

    Article  CAS  Google Scholar 

  8. Vitreschak, A. G., Rodionov, D. A., Mironov, A. A. & Gelfand, M. S. Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural element. RNA 9, 1084–1097 (2003)

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  10. Gardner, P. P. et al. Rfam: updates to the RNA families database. Nucleic Acids Res. 37, D136–D140 (2009)

    Article  CAS  Google Scholar 

  11. Fox, K. et al. Multiple posttranscriptional regulatory mechanisms partner to control ethanolamine utilization in Enterococcus faecalis. Proc. Natl Acad. Sci. USA 106, 4435–4440 (2009)

    Article  ADS  CAS  Google Scholar 

  12. Wilkinson, K. A., Merino, E. J. & Weeks, K. M. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution. Nature Protocols 1, 1610–1616 (2006)

    Article  CAS  Google Scholar 

  13. Lundrigan, M. D. & Kadner, R. J. Altered cobalamin metabolism in Escherichia coli btuR mutants affects btuB gene regulation. J. Bacteriol. 171, 154–161 (1989)

    Article  CAS  Google Scholar 

  14. Weinberg, Z. et al. Comparative genomics reveals 104 candidate structured RNAs from bacteria, archaea, and their metagenomes. Genome Biol. 11, R31 (2010)

    Article  Google Scholar 

  15. Taylor, R. T., Smucker, L., Hanna, M. L. & Gill, J. Aerobic photolysis of alkylcobalamins: quantum yields and light-action spectra. Arch. Biochem. Biophys. 156, 521–533 (1973)

    Article  CAS  Google Scholar 

  16. Adams, P. L., Stahley, M. R., Kosek, A. B., Wang, J. & Strobel, S. A. Crystal structure of a self-splicing group I intron with both exons. Nature 430, 45–50 (2004)

    Article  ADS  CAS  Google Scholar 

  17. Jaeger, L., Verzemnieks, E. J. & Geary, C. The UA_handle: a versatile submotif in stable RNA architectures. Nucleic Acids Res. 37, 215–230 (2009)

    Article  CAS  Google Scholar 

  18. 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 

  19. Krasilnikov, A. S. & Mondragon, A. On the occurrence of the T-loop RNA folding motif in large RNA molecules. RNA 9, 640–643 (2003)

    Article  CAS  Google Scholar 

  20. Serganov, A., Huang, L. & Patel, D. J. Coenzyme recognition and gene regulation by a flavin mononucleotide riboswitch. Nature 458, 233–237 (2009)

    Article  ADS  CAS  Google Scholar 

  21. Sussman, D., Nix, J. C. & Wilson, C. The structural basis for molecular recognition by the vitamin B12 RNA aptamer. Nature Struct. Biol. 7, 53–57 (2000)

    Article  CAS  Google Scholar 

  22. Ortiz-Guerrero, J. M., Polanco, M. C., Murillo, F. J., Padmanabhan, S. & Elias-Arnanz, M. Light-dependent gene regulation by a coenzyme B12-based photoreceptor. Proc. Natl Acad. Sci. USA 108, 7565–7570 (2011)

    Article  ADS  CAS  Google Scholar 

  23. Vicens, Q. & Cech, T. R. Atomic level architecture of group I introns revealed. Trends Biochem. Sci. 31, 41–51 (2006)

    Article  CAS  Google Scholar 

  24. Melnikov, S. et al. One core, two shells: bacterial and eukaryotic ribosomes. Nature Struct. Mol. Biol. 19, 560–567 (2012)

    Article  CAS  Google Scholar 

  25. Xin, Y., Laing, C., Leontis, N. B. & Schlick, T. Annotation of tertiary interactions in RNA structures reveals variations and correlations. RNA 14, 2465–2477 (2008)

    Article  CAS  Google Scholar 

  26. Butcher, S. E. & Pyle, A. M. The molecular interactions that stabilize RNA tertiary structure: RNA motifs, patterns, and networks. Acc. Chem. Res. 44, 1302–1311 (2011)

    Article  CAS  Google Scholar 

  27. Gregorian, R. S., Jr & Crothers, D. M. Determinants of RNA hairpin loop-loop complex stability. J. Mol. Biol. 248, 968–984 (1995)

    Article  CAS  Google Scholar 

  28. Pyle, A. M. Metal ions in the structure and function of RNA. J. Biol. Inorg. Chem. 7, 679–690 (2002)

    Article  CAS  Google Scholar 

  29. Reyes, F. E., Garst, A. D. & Batey, R. T. Strategies in RNA crystallography. Methods Enzymol. 469, 119–139 (2009)

    Article  CAS  Google Scholar 

  30. Edwards, A. L., Garst, A. D. & Batey, R. T. Determining structures of RNA aptamers and riboswitches by X-ray crystallography. Methods Mol. Biol. 535, 135–163 (2009)

    Article  CAS  Google Scholar 

  31. Milligan, J. F. & Uhlenbeck, O. C. Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol. 180, 51–62 (1989)

    Article  CAS  Google Scholar 

  32. Gilbert, S. D. & Batey, R. T. Monitoring RNA–ligand interactions using isothermal titration calorimetry. Methods Mol. Biol. 540, 97–114 (2009)

    Article  CAS  Google Scholar 

  33. Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006)

    Article  Google Scholar 

  34. Vonrhein, C. et al. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. D 67, 293–302 (2011)

    Article  CAS  Google Scholar 

  35. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

    Article  Google Scholar 

  36. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    Article  CAS  Google Scholar 

  37. Blanc, E. et al. Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. Acta Crystallogr. D 60, 2210–2221 (2004)

    Article  CAS  Google Scholar 

  38. Keating, K. S. & Pyle, A. M. Semiautomated model building for RNA crystallography using a directed rotameric approach. Proc. Natl Acad. Sci. USA 107, 8177–8182 (2010)

    Article  ADS  CAS  Google Scholar 

  39. Ferré-D’Amaré, A. R. Use of the spliceosomal protein U1A to facilitate crystallization and structure determination of complex RNAs. Methods 52, 159–167 (2010)

    Article  Google Scholar 

  40. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the National Institutes of Health (GM073850 and 1S10RR026516) to R.T.B. and by a Colorado Diversity Initiative Fellowship and NIH Ruth L. Kirschstein fellowship (F32GM095121) to J.E.J. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under contract no. DE-AC02-05CH11231.

Author information

Author notes

  1. Francis E. Reyes

    Present address: Present address: Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.,

  2. James E. Johnson Jr and Francis E. Reyes: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Colorado at Boulder, UCB 596, Boulder, Colorado 80309-0596, USA,

    James E. Johnson Jr, Francis E. Reyes, Jacob T. Polaski & Robert T. Batey

Authors
  1. James E. Johnson Jr
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Francis E. Reyes
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jacob T. Polaski
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Robert T. Batey
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

F.E.R. discovered the specificity of the AqCbl class and performed all aspects of the crystallography with assistance from J.T.P.; J.E.J. performed all biochemical experiments and fully characterized the specificities of the cobalamin family; J.T.P. obtained all in vivo data; and all authors contributed to the analysis of the data and the writing of this paper.

Corresponding authors

Correspondence to Francis E. Reyes or Robert T. Batey.

Ethics declarations

Competing interests

R.T.B. is a member of the Scientific Advisory Board of BioRelix, a company pursuing the development of antimicrobials that target riboswitches.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-12, Supplementary Tables 1-3 and additional references. (PDF 27942 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Johnson Jr, J., Reyes, F., Polaski, J. et al. B12 cofactors directly stabilize an mRNA regulatory switch. Nature 492, 133–137 (2012). https://doi.org/10.1038/nature11607

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature11607

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