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Backbone and nucleobase contacts to glucosamine-6-phosphate in the glmS ribozyme

Nature Structural & Molecular Biology volume 13, pages 517–523 (2006)Cite this article

Abstract

The glmS ribozyme resides in the 5′ untranslated region of glmS mRNA and functions as a catalytic riboswitch that regulates amino sugar metabolism in certain Gram-positive bacteria. The ribozyme catalyzes self-cleavage of the mRNA and ultimately inhibits gene expression in response to binding of glucosamine-6-phosphate (GlcN6P), the metabolic product of the GlmS protein. We have used nucleotide analog interference mapping (NAIM) and suppression (NAIS) to investigate backbone and nucleobase functional groups essential for ligand-dependent ribozyme function. NAIM using GlcN6P as ligand identified requisite structural features and potential sites of ligand and/or metal ion interaction, whereas NAIS using glucosamine as ligand analog revealed those sites that orchestrate recognition of ligand phosphate. These studies demonstrate that the ligand-binding site lies in close proximity to the cleavage site in an emerging model of ribozyme structure that supports a role for ligand within the catalytic core.

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Figure 1: B. cereus glmS ribozyme and ligand structures.
Figure 2: Magnesium dependence of P1–P3 and P1–P4 B. cereus glmS ribozymes.
Figure 3: Phosphorothioate NAIM of B. cereus glmS ribozymes.
Figure 4: Summary of backbone analog interference and rescue effects in the P1–P3 and P1–P4 B. cereus glmS ribozymes.
Figure 5: GlcN-dependent phosphorothioate-interference suppression in the P1–P4 B. cereus glmS ribozyme.
Figure 6: Summary of nucleobase interference and interference suppression effects in the P1–P4 B. cereus glmS ribozyme.
Figure 7: Ligand phosphate recognition in the B. cereus glmS ribozyme.

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References

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Winkler, W.C., 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  Google Scholar 

  4. Barrick, J.E. et al. New RNA motifs suggest an expanded scope for riboswitches in bacterial genetic control. Proc. Natl. Acad. Sci. USA 101, 6421–6426 (2004).

    Article  CAS  Google Scholar 

  5. Soukup, G.A. Core requirements for glmS ribozyme self-cleavage reveal a putative pseudoknot structure. Nucleic Acids Res. 34, 968–975 (2006).

    Article  CAS  Google Scholar 

  6. Wilkinson, S.R. & Been, M.D. A pseudoknot in the 3′ non-core region of the glmS ribozyme enhances self-cleavage activity. RNA 11, 1788–1794 (2005).

    Article  CAS  Google Scholar 

  7. Doherty, E.A. & Doudna, J.A. Ribozyme structures and mechanisms. Annu. Rev. Biochem. 69, 597–615 (2000).

    Article  CAS  Google Scholar 

  8. Fedor, M.J. & Williamson, J.R. The catalytic diversity of RNAs. Nat. Rev. Mol. Cell Biol. 6, 399–412 (2005).

    Article  CAS  Google Scholar 

  9. Roth, A., Nahvi, A., Lee, M., Jona, I. & Breaker, R.R. Characteristics of the glmS ribozyme suggest only structural roles for divalent metal ions. RNA 12, 607–619 (2006).

    Article  CAS  Google Scholar 

  10. Chowrira, B.M., Berzal-Herranz, A. & Burke, J.M. Ionic requirements for RNA binding, cleavage, and ligation by the hairpin ribozyme. Biochemistry 32, 1088–1095 (1993).

    Article  CAS  Google Scholar 

  11. Collins, R.A. & Olive, J.E. Reaction conditions and kinetics of self-cleavage of a ribozyme derived from Neurospora VS RNA. Biochemistry 32, 2795–2799 (1993).

    Article  CAS  Google Scholar 

  12. Suh, Y.A., Kumar, P.K.R., Taira, K. & Nishikawa, S. Self-cleavage activity of the genomic HDV ribozyme in the presence of various divalent metal ions. Nucleic Acids Res. 21, 3277–3280 (1993).

    Article  CAS  Google Scholar 

  13. Dahm, S.C. & Uhlenbeck, O.C. Role of divalent metal ions in the hammerhead RNA cleavage reaction. Biochemistry 30, 9464–9469 (1991).

    Article  CAS  Google Scholar 

  14. McCarthy, T.J. et al. Ligand requirements for glmS ribozyme self-cleavage. Chem. Biol. 12, 1221–1226 (2005).

    Article  CAS  Google Scholar 

  15. Ryder, S. & Strobel, S. Nucleotide analog interference mapping. Methods 18, 38–50 (1999).

    Article  CAS  Google Scholar 

  16. Ryder, S., Ortoleva-Donnelly, L., Kosek, A. & Strobel, S. Chemical probing of RNA by nucleotide analog interference mapping. Methods Enzymol. 317, 92–109 (2000).

    Article  CAS  Google Scholar 

  17. Waring, R.B. Identification of phosphate groups important to self-splicing of the Tetrahymena rRNA intron as determined by phosphorothioate substitution. Nucleic Acids Res. 17, 10281–10293 (1989).

    Article  CAS  Google Scholar 

  18. Ruffner, D.E. & Uhlenbeck, O.C. Thiophosphate interference experiments locate phosphates important for the hammerhead RNA self-cleavage reaction. Nucleic Acids Res. 18, 6025–6029 (1990).

    Article  CAS  Google Scholar 

  19. Basu, S. & Strobel, S. Thiophilic metal ion rescue of phosphorothioate interference within the Tetrahymena ribozyme P4–P6 domain. RNA 5, 1399–1407 (1999).

    Article  CAS  Google Scholar 

  20. Basu, S. & Strobel, S. Biochemical detection of monovalent metal ion binding sites within RNA. Methods 23, 264–275 (2001).

    Article  CAS  Google Scholar 

  21. Strobel, S., Ortoleva-Donnelly, L., Ryder, S., Cate, J. & Moncoeur, E. Complementary sets of noncanonical base pairs mediate RNA helix packing in the group I intron active site. Nat. Struct. Biol. 5, 60–66 (1998).

    Article  CAS  Google Scholar 

  22. Szewczak, A., Ortoleva-Donnelly, L., Ryder, S., Moncouer, E. & Strobel, S. A minor groove triple helix in the active site of the Tetrahymena group I intron. Nat. Struct. Biol. 5, 1037–1042 (1998).

    Article  CAS  Google Scholar 

  23. Soukup, J., Minakawa, N., Matsuda, A. & Strobel, S. Identification of A-minor tertiary interactions within a bacterial group I intron active site by 3-deazaadenosine interference mapping. Biochemistry 41, 10426–10438 (2002).

    Article  CAS  Google Scholar 

  24. Brautigam, C.A. & Steitz, T.A. Structural principles for the inhibition of the 3′-5′ exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates. J. Mol. Biol. 277, 363–377 (1998).

    Article  CAS  Google Scholar 

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Acknowledgements

N-methylguanosine was provided by S.A. Strobel (Yale University). J.K.S. is supported by the Clare Boothe Luce Program of the Henry Luce Foundation and US National Institutes of Health (NIH) grant P20 RR016469, from the Institutional Development Award Program for Networks of Biomedical Research Excellence of the National Center for Research Resources (NCRR). G.A.S. is supported by the Health Future Foundation and NIH grant P20 RR018788, from the Centers of Biomedical Research Excellence Program of the NCRR. This investigation was in part conducted in a facility constructed with support from NIH grant C06 RR017417, from the Research Facilities Improvement Program of the NCRR.

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Authors and Affiliations

  1. Department of Chemistry, Creighton University, 2500 California Plaza, Omaha, 68178, Nebraska, USA

    Joshua A Jansen, Tom J McCarthy & Juliane K Soukup

  2. Department of Biomedical Sciences, Creighton University School of Medicine, 2500 California Plaza, Omaha, 68178, Nebraska, USA

    Garrett A Soukup

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  1. Joshua A Jansen
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  2. Tom J McCarthy
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Contributions

J.A.J. and T.J.M. performed the experiments. G.A.S. and J.K.S. designed and performed experiments, analyzed data and cowrote the paper.

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Correspondence to Garrett A Soukup or Juliane K Soukup.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Summary of interference, interference suppression and manganese rescue data for the P1–P4 B. cereus glmS ribozyme (PDF 59 kb)

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Jansen, J., McCarthy, T., Soukup, G. et al. Backbone and nucleobase contacts to glucosamine-6-phosphate in the glmS ribozyme. Nat Struct Mol Biol 13, 517–523 (2006). https://doi.org/10.1038/nsmb1094

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