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Crystal structure of a hepatitis delta virus ribozyme

Nature volume 395, pages 567–574 (1998)Cite this article

Abstract

The self-cleaving ribozyme of the hepatitis delta virus (HDV) is the only catalytic RNA known to be required for the viability of a human pathogen. We obtained crystals of a 72-nucleotide, self-cleaved form of the genomic HDV ribozyme that diffract X-rays to 2.3 Å resolution by engineering the RNA to bind a small, basic protein without affecting ribozyme activity. The co-crystal structure shows that the compact catalytic core comprises five helical segments connected as an intricate nested double pseudoknot. The 5′-hydroxyl leaving group resulting from the self-scission reaction is buried deep within an active-site cleft produced by juxtaposition of the helices and five strand-crossovers, and is surrounded by biochemically important backbone and base functional groups in a manner reminiscent of protein enzymes.

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Figure 1: Pseudoknotted secondary-structure models of a, genomic and b, antigenomic HDV ribozymes proposed by Perrotta and Been7.
Figure 2: Complex of the HDV ribozyme and U1A-RBD.
Figure 3: Ribbon and stick representations of the crystal structure of the HDV ribozyme, colour-coded as in Fig. 2a.
Figure 4: Structural components of the active site.
Figure 5: Structural features of the active site.

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References

  1. Lai, M. M. The molecular biology of hepatitis delta virus. Annu. Rev. Biochem. 64, 259–286 (1995).

    Article  CAS  PubMed  Google Scholar 

  2. Been, M. D. & Wickham, G. S. Self-cleaving ribozymes of hepatitis delta virus RNA. Eur. J. Biochem. 247, 741–753 (1997).

    Article  CAS  PubMed  Google Scholar 

  3. Kuo, M. Y.-P., Sharmeen, L., Dinter-Gottlieb, G. & Taylor, J. Characterization of self-cleaving RNA sequences on the genome and antigenome of human hepatitis delta virus. J. Virol. 62, 4439–444 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Sharmeen, L., Kuo, M. Y.-P., Dinter-Gottlieb, G. & Taylor, J. Antigenomic RNA of human hepatitis delta virus can undergo self-cleavage. J. Virol. 62, 2674–2679 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Wu, H.-N. et al. Human hepatitis delta virus RNA subfragments contain an autocleavage activity. Proc. Natl Acad. Sci. USA 86, 1831–1835 (1989).

    Article  ADS  CAS  PubMed Central  PubMed  Google Scholar 

  6. Perrotta, A. T. & Been, M. D. The self-cleaving domain from the genomic RNA of hepatitis delta virus: sequence requirements and the effects of denaturant. Nucleic Acids Res. 18, 6821–6827 (1990).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Perrotta, A. T. & Been, M. D. Apseudoknot-like structure required for efficient self-cleavage of hepatitis delta virus RNA. Nature 350, 434–436 (1991).

    Article  ADS  CAS  PubMed  Google Scholar 

  8. Tanner, N. K. et al. Athree-dimensional model of hepatitis delta virus ribozyme based on biochemical and mutational analyses. Curr. Biol. 4, 488–498 (1994).

    Article  CAS  PubMed  Google Scholar 

  9. Thill, G., Vasseur, M. & Tanner, N. K. Structural and sequence elements required for the self-cleaving activity of the hepatitis delta virus ribozyme. Biochemistry 32, 4254–4262 (1993).

    Article  CAS  PubMed  Google Scholar 

  10. Pley, H. W., Flaherty, K. M. & McKay, D. B. Three-dimensional structure of a hammerhead ribozyme. Nature 372, 68–74 (1994).

    Article  ADS  CAS  PubMed  Google Scholar 

  11. Scott, W. G., Finch, J. T. & Klug, A. The crystal structure of an all-RNA hammerhead ribozyme: a proposed mechanism for RNA catalytic cleavage. Cell 81, 991–1002 (1995).

    Article  CAS  PubMed  Google Scholar 

  12. Richards, F. M. & Wyckoff, H. W. in The Enzymes (ed. Boyer, P. D.) 647–806 (Academic, New York, 1971).

    Google Scholar 

  13. Rosenstein, S. P. & Been, M. D. Self-cleavage of hepatitis delta virus genomic strand RNA is enhanced under partially denaturing conditions. Biochemistry 29, 8011–8016 (1990).

    Article  CAS  PubMed  Google Scholar 

  14. Duhamel, J. et al. Secondary structure content of the HDV ribozyme in 95% formamide. Nucleic Acids Res. 24, 3911–3917 (1996).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. 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  PubMed Central  PubMed  Google Scholar 

  16. Ferré-D'Amaré, A. R., Zhou, K. & Doudna, J. A. Ageneral module for RNA crystallization. J. Mol. Biol. 279, 621–631 (1998).

    Article  PubMed  Google Scholar 

  17. Oubridge, C., Ito, N. Evans, P. R., Teo, C.-H. & Nagai, K. Crystal structure at 1.92 Å resolution of the RNA-binding domain of the U1A spliceosomal protein complexed with an RNA hairpin. Nature 372, 432–438 (1994).

    Article  ADS  CAS  PubMed  Google Scholar 

  18. Kolk, M. H. et al. NMR structure of a classical pseudoknot: interplay of single- and double-stranded RNA. Science 280, 434–438 (1998).

    Article  ADS  CAS  PubMed  Google Scholar 

  19. Tang, C. K. & Draper, D. E. Unusual mRNA pseudoknot structure is recognized by a protein translational repressor. Cell 57, 531–536 (1989).

    Article  CAS  PubMed  Google Scholar 

  20. Gluick, T. C. & Draper, D. E. Thermodynamics of folding a pseudoknotted mRNA fragment. J. Mol. Biol. 241, 246–262 (1994).

    Article  CAS  PubMed  Google Scholar 

  21. Ekland, E. H., Szostak, J. W. & Bartel, D. P. Structurally complex and highly active RNA ligases derived from random RNA sequences. Science 269, 364–370 (1995).

    Article  ADS  CAS  PubMed  Google Scholar 

  22. Kolk, M. H., Heus, H. A. & Hilbers, C. W. The structure of the isolated, central hairpin of the HDV antigenomic ribozyme: novel structural features and similarity of the loop in the ribozyme and free in solution. EMBO J. 16, 3685–3692 (1997).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Lynch, S. R. & Tinoco, I. J The structure of the L3 loop from the hepatitis delta virus ribozyme: a syn cytidine. Nucleic Acids Res. 26, 980–987 (1998).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. Perrotta, A. T. & Been, M. D. Core sequences and a cleavage site wobble pair required for HDV antigenomic ribozyme self-cleavage. Nucleic Acids Res. 24, 1314–1321 (1996).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  25. Rosenstein, S. P. & Been, M. D. Hepatitis delta virus ribozymes fold to generate a solvent-inaccessible core with essential nucleotides near the cleavage site phosphate. Biochemistry 35, 11403–11413 (1996).

    Article  CAS  PubMed  Google Scholar 

  26. Nishikawa, F., Fauzi, H. & Nishikawa, S. Detailed analysis of base preferences at the cleavage site of a trans-acting HDV ribozyme: a mutation that changes cleavage site specificity. Nucleic Acids Res. 25, 1605–1610 (1997).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. Strobel, S. A. & Cech, T. R. Minor groove recognition of the conserved G-U pair at the Tetrahymena ribozyme reaction site. Science 267, 675–679 (1995).

    Article  ADS  CAS  PubMed  Google Scholar 

  28. Cate, J. H. et al. Crystal structure of a group I ribozyme domain: principles of RNA packing. Science 273, 1678–1685 (1996).

    Article  ADS  CAS  PubMed  Google Scholar 

  29. Pley, H. W., Flaherty, K. M. & McKay, D. B. Model for an RNA tertiary interaction from the structure of an intermolecular complex between a GAAA tetraloop and an RNA helix. Nature 372, 111–113 (1994).

    Article  ADS  CAS  PubMed  Google Scholar 

  30. Kumar, P. K. R. et al. Random mutations to evaluate the role of bases at two important single-stranded regions of the genomic HDV ribozyme. Nucleic Acids Res. 20, 3919–3924 (1992).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Kumar, P. K. R., Suh, Y.-A., Taira, K. & Nishikawa, S. Point and compensation mutations to evaluate essential stem structures of genomic HDV ribozyme. FASEB J. 7, 124–129 (1993).

    Article  CAS  PubMed  Google Scholar 

  32. Kumar, P. K. R., Taira, K. & Nishikawa, S. Chemical probing studies of variants of the genomic hepatitis delta virus ribozyme by primer extension analysis. Biochemistry 33, 583–592 (1994).

    Article  CAS  PubMed  Google Scholar 

  33. Belinsky, M. G., Britton, E. & Dinter-Gottlieb, G. Modification interference analysis of a self-cleaving RNA from hepatitis delta virus. FASEB J. 7, 130–136 (1993).

    Article  CAS  PubMed  Google Scholar 

  34. Wadkins, T. S. & Been, M. D. Core-associated non-duplex sequences distinguishing the genomic and antigenomic self-cleaving RNAs of hepatitis delta virus. Nucleic Acids Res. 25, 4085–4092 (1997).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Rosenstein, S. P. & Been, M. D. Evidence that genomic and antigenomic RNA self-cleaving elements from hepatitis delta virus have similar secondary structures. Nucleic Acids Res. 19, 5409–5416 (1991).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  36. Huang, Z.-S., Ping, Y.-H. & Wu, H.-N. An AU at the first base pair of helix 3 elevates the catalytic activity of hepatitis delta virus ribozymes. FEBS lett. 413, 299–303 (1997).

    Article  CAS  PubMed  Google Scholar 

  37. Jeoung, Y.-H., Kumar, P. K. R., Suh, Y.-A., Taira, K. & Nishikawa, S. Identification of phosphate oxygens that are important for self-cleavage activity of the HDV ribozyme by phosphorothioate substitution interference analysis. Nucleic Acids Res. 22, 3722–3727 (1994).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  38. Bravo, C., Lescure, F., Laugâa, P., Fourrey, J.-L. & Favre, A. Folding of the HDV antigenomic ribozyme pseudoknot structure deduced from long-range photocrosslinks. Nucleic Acids Res. 24, 1351–1359 (1996).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  39. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  PubMed  Google Scholar 

  40. Collaborative Computational Project Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–776 (1994).

  41. Terwilliger, T. C. & Kim, S.-H. Generalized method of determining heavy-atom positions using the difference Patterson function. Acta Crystallogr. A 43, 1–5 (1987).

    Article  Google Scholar 

  42. de la Fortelle, E. & Bricogne, G. Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472–494 (1997).

    Article  CAS  PubMed  Google Scholar 

  43. Abrahams, J. P. & Leslie, A. G. W. Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Crystallogr. D 54, 905–921 (1998).

    Google Scholar 

  44. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

    Article  PubMed  Google Scholar 

  45. Brünger, A. T. et al. Crystallography and NMR system: a new software system for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).

    Article  PubMed  Google Scholar 

  46. Brünger, A. T. Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. Nature 355, 472–475 (1992).

    Article  ADS  PubMed  Google Scholar 

  47. Laskowski, R. J., Macarthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: a program to check stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–290 (1993).

    Article  CAS  Google Scholar 

  48. Pleij, C. W. A., Rietveld, K. & Bosch, L. Anew principle of RNA folding based on pseudoknotting. Nucleic Acids Res. 13, 1717–1731 (1985).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  49. Nicholls, A., Sharp, K. A. & Honig, B. Protein folding and association: insight from the interfacial and thermodynamic properties of hydrocarbons. Proteins Struct. Funct. Genet. 11, 281–296 (1991).

    Article  CAS  PubMed  Google Scholar 

  50. Carson, M. Ribbons 2.0. J. Appl. Crystallogr. 24, 958–961 (1991).

    Article  Google Scholar 

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Acknowledgements

We thank K. Nagai for U1A-RBD expression plasmids; C. Ogata and D. Cook for help at beamline X4A of the National Synchrotron Light Source (Brookhaven National Laboratory); the staff of the Cornell High Energy Synchrotron Source for support at beamlines A-1 and F-1; R. Batey, D.Battle, J. Kieft, A. Luptak and R. Rambo for help with synchrotron data collection and useful comments; P. Adams, M. Been, S. Bellon, C. Correll, R. Gaudet, D. Herschlag, J. Ippolito, P. Moore, L.Silvian, T. Steitz, S. Strobel, O. Uhlenbeck and D. Wilson for helpful discussions; and the staff of the Yale Center for Structural Biology for crystallographic and computational support. A.R.F. was a Fellow of the Jane Coffin Childs Memorial Fund for Medical Research. This work was supported in part by grants from the Jane Coffin Childs Memorial Fund for Medical Research, the Searle Scholars program, the NIH, and a Beckman Young Investigator award. J.A.D. is an assistant investigator of the Howard Hughes Medical Institute, a Searle scholar, and a Fellow of the David and Lucile Packard Foundation.

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Authors and Affiliations

  1. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, 06520-8114, Connecticut, USA

    Adrian R. Ferré-D'Amaré & Jennifer A. Doudna

  2. Howard Hughes Medical Institute, Yale University, New Haven, 06520-8114, Connecticut, USA

    Kaihong Zhou & Jennifer A. Doudna

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Correspondence to Jennifer A. Doudna.

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Ferré-D'Amaré, A., Zhou, K. & Doudna, J. Crystal structure of a hepatitis delta virus ribozyme. Nature 395, 567–574 (1998). https://doi.org/10.1038/26912

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