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Crystal structure of a phage Twort group I ribozyme–product complex

Nature Structural & Molecular Biology volume 12, pages 8289 (2005) | Download Citation

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Abstract

Group I introns are catalytic RNAs capable of orchestrating two sequential phosphotransesterification reactions that result in self-splicing. To understand how the group I intron active site facilitates catalysis, we have solved the structure of an active ribozyme derived from the orf142-I2 intron from phage Twort bound to a four-nucleotide product RNA at a resolution of 3.6 Å. In addition to the three conserved domains characteristic of all group I introns, the Twort ribozyme has peripheral insertions characteristic of phage introns. These elements form a ring that completely envelops the active site, where a snug pocket for guanosine is formed by a series of stacked base triples. The structure of the active site reveals three potential binding sites for catalytic metals, and invokes a role for the 2′ hydroxyl of the guanosine substrate in organization of the active site for catalysis.

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References

  1. 1.

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

  2. 2.

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

  3. 3.

    , , & A preorganized active site in the crystal structure of the Tetrahymena ribozyme. Science 282, 259–264 (1998).

  4. 4.

    , , & Structural basis of the enhanced stability of a mutant ribozyme domain and a detailed view of RNA-solvent interactions. Structure 9, 221–231 (2001).

  5. 5.

    , , , & Crystal structure of a self-splicing group I intron with both exons. Nature 430, 45–50 (2004).

  6. 6.

    , & Structure of the Tetrahymena ribozyme: base triple sandwich and metal ion at the active site. Mol. Cell 16, 351–362 (2004).

  7. 7.

    , , , & The guanosine binding site of the Tetrahymena ribozyme. Nature 342, 391–395 (1989).

  8. 8.

    & A hydrogen-bonding triad stabilizes the chemical transition state of a group I ribozyme. Chem. Biol. 6, 153–165 (1999).

  9. 9.

    , , & Defining the catalytic metal ion interactions in the Tetrahymena ribozyme reaction. Biochemistry 40, 5161–5171 (2001).

  10. 10.

    , , & Identification of an active site ligand for a group I ribozyme catalytic metal ion. Biochemistry 41, 2516–2525 (2002).

  11. 11.

    Self-splicing of group I introns. Annu. Rev. Biochem. 59, 543–68 (1990).

  12. 12.

    , & The Tetrahymena ribozyme acts like an RNA restriction endonuclease. Nature 324, 429–433 (1986).

  13. 13.

    , & Sequence-specific endoribonuclease activity of the Tetrahymena ribozyme: enhanced cleavage of certain oligonucleotide substrates that form mismatched ribozyme-substrate complexes. Biochemistry 27, 8924–8931 (1988).

  14. 14.

    & Modelling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis. J. Mol. Biol. 216, 585–610 (1990).

  15. 15.

    , & Representation of the secondary and tertiary structure of group I introns. Nat. Struct. Biol. 1, 273–280 (1994).

  16. 16.

    & Group I intron self-splicing with adenosine: evidence for a single nucleoside-binding site. Science 252, 434–437 (1991).

  17. 17.

    & Unexpected abundance of self-splicing introns in the genome of bacteriophage Twort: introns in multiple genes, a single gene with three introns, and exon skipping by group I ribozymes. Proc. Natl. Acad. Sci. USA 96, 7005–7010 (1999).

  18. 18.

    , , & Two self-splicing group I introns in the ribonucleotide reductase large subunit gene of Staphylococcus aureus phage Twort. Nucleic Acids Res. 30, 1935–1943 (2002).

  19. 19.

    , & Catalysis of RNA cleavage by a ribozyme derived from the group I intron of Anabaena pre-tRNA(Leu). Biochemistry 33, 14935–14947 (1994).

  20. 20.

    Phage T4 introns: self-splicing and mobility. Annu. Rev. Genet. 24, 363–385 (1990).

  21. 21.

    , , & Architecture and folding mechanism of the Azoarcus group I pre-tRNA. J. Mol. Biol. 339, 41–51 (2004).

  22. 22.

    & A common motif organizes the structure of multi-helix loops in 16 S and 23 S ribosomal RNAs. J. Mol. Biol. 283, 571–583 (1998).

  23. 23.

    & Structure of Escherichia coli ribosomal protein L25 complexed with a 5S rRNA fragment at 1.8-Å resolution. Proc. Natl. Acad. Sci. USA 97, 2023–2028 (2000).

  24. 24.

    , , , & RNA tertiary interactions in the large ribosomal subunit: the A-minor motif. Proc. Natl. Acad. Sci. USA 98, 4899–4903 (2001).

  25. 25.

    , , , & The common and the distinctive features of the bulged-G motif based on a 1.04 Å resolution RNA structure. Nucleic Acids Res. 31, 6806–6818 (2003).

  26. 26.

    et al. Activation of the catalytic core of a group I intron by a remote 3′ splice junction. Genes Dev. 6, 1373–1385 (1992).

  27. 27.

    & Structural features that give rise to the unusual stability of RNA hairpins containing GNRA loops. Science 253, 191–194 (1991).

  28. 28.

    , , & New loop-loop tertiary interactions in self-splicing introns of subgroup IC and ID: a complete 3D model of the Tetrahymena thermophila ribozyme. Chem. Biol. 3, 993–1009 (1996).

  29. 29.

    & Ribozyme inhibitors: deoxyguanosine and dideoxyguanosine are competitive inhibitors of self-splicing of the Tetrahymena ribosomal ribonucleic acid precursor. Biochemistry 25, 4473–4478 (1986).

  30. 30.

    , , & The chemical basis of adenosine conservation throughout the Tetrahymena ribozyme. RNA 4, 498–519 (1998).

  31. 31.

    , & Protonated 2′-aminoguanosine as a probe of the electrostatic environment of the active site of the Tetrahymena group I ribozyme. Biochemistry 38, 10976–10988 (1999).

  32. 32.

    et al. RNA tertiary structure mediation by adenosine platforms. Science 273, 1696–1699 (1996).

  33. 33.

    , , & A second catalytic metal ion in group I ribozyme. Nature 388, 805–808 (1997).

  34. 34.

    , & Binding of guanosine and 3′ splice site analogues to a group I ribozyme: interactions with functional groups of guanosine and with additional nucleotides. Biochemistry 32, 5247–5256 (1993).

  35. 35.

    , , & Metal ion catalysis in the Tetrahymena ribozyme reaction. Nature 361, 85–88 (1993).

  36. 36.

    , , & Metal ion interaction with cosubstrate in self-splicing of group I introns. Nucleic Acids Res. 25, 648–653 (1997).

  37. 37.

    & A general two-metal-ion mechanism for catalytic RNA. Proc. Natl. Acad. Sci. USA 90, 6498–6502 (1993).

  38. 38.

    , , , & Three metal ions at the active site of the Tetrahymena group I ribozyme. Proc. Natl. Acad. Sci. USA 96, 12299–12304 (1999).

  39. 39.

    et al. The comparative RNA web (CRW) site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs. BMC Bioinformatics 3, 2 (2002).

  40. 40.

    , & Cooperative and anticooperative binding to a ribozyme. Proc. Natl. Acad. Sci. USA 90, 8357–8361 (1993).

  41. 41.

    , , & Guanosine binds to the Tetrahymena ribozyme in more than one step, and its 2′-OH and the nonbridging pro-Sp phosphoryl oxygen at the cleavage site are required for productive docking. Biochemistry 36, 12477–12485 (1997).

  42. 42.

    , & The importance of being ribose at the cleavage site in the Tetrahymena ribozyme reaction. Biochemistry 32, 8312–8321 (1993).

  43. 43.

    & Extraordinarily slow binding of guanosine to the Tetrahymena group I ribozyme: implications for RNA preorganization and function. Proc. Natl. Acad. Sci. USA 100, 2300–2305 (2003).

  44. 44.

    & Conformational switches involved in orchestrating the successive steps of group I RNA splicing. Biochemistry 35, 3754–3763 (1996).

  45. 45.

    , , & Crystals by design: a strategy for crystallization of a ribozyme derived from the Tetrahymena group I intron. J. Mol. Biol. 270, 711–723 (1997).

  46. 46.

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

  47. 47.

    et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).

  48. 48.

    Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

  49. 49.

    , , & . 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).

  50. 50.

    Ribbons. Methods Enzymol. 277, 493–505 (1997).

  51. 51.

    MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991).

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Acknowledgements

We are grateful to P. Bevilacqua, J. Bolin, C. Correll, J. Piccirilli, E. Westhof and members of the Golden laboratory for critical discussions, and to J. Hougland, D. Herschlag and J. Piccirilli for communication of results prior to publication. We thank the staff of BioCars, NE-CAT and SBC for assistance with data collection. This work was supported by NASA (NAG8-1833), the Pew Scholars Program in Biomedical Sciences and the Purdue University Cancer Center. This is journal paper number 2004-17476 from the Purdue University Agricultural Experiment Station.

Author information

Affiliations

  1. Department of Biochemistry, Purdue University, 175 South University Street, West Lafayette, Indiana 47907, USA.

    • Barbara L Golden
    • , Hajeong Kim
    •  & Elaine Chase

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Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Barbara L Golden.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    The Twort self-splicing reaction.

  2. 2.

    Supplementary Fig. 2

    The Twort ribozyme reaction.

  3. 3.

    Supplementary Fig. 3

    Ribozyme activity in manganese.

  4. 4.

    Supplementary Fig. 4

    A manganese-binding site.

  5. 5.

    Supplementary Fig. 5

  6. 6.

    Supplementary Table 1

    Michaelis-Menten parameters for the Twort intron.

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DOI

https://doi.org/10.1038/nsmb868

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