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.

Structural basis for substrate binding, cleavage and allostery in the tRNA maturase RNase Z

Abstract

Transfer RNAs (tRNAs) are synthesized as part of longer primary transcripts that require processing of both their 3′ and 5′ extremities in every living organism known. The 5′ side is processed (matured) by the ubiquitously conserved endonucleolytic ribozyme, RNase P1, whereas removal of the 3′ tails can be either exonucleolytic2,3 or endonucleolytic4. The endonucleolytic pathway is catalysed by an enzyme known as RNase Z, or 3′ tRNase5,6. RNase Z cleaves precursor tRNAs immediately after the discriminator base (the unpaired nucleotide 3′ to the last base pair of the acceptor stem, used as an identity determinant by many aminoacyl-tRNA synthetases) in most cases6,7,8, yielding a tRNA primed for addition of the CCA motif by nucleotidyl transferase. Here we report the crystal structure of Bacillus subtilis RNase Z at 2.1 Å resolution, and propose a mechanism for tRNA recognition and cleavage. The structure explains the allosteric properties of the enzyme, and also sheds light on the mechanisms of inhibition by the CCA motif and long 5′ extensions. Finally, it highlights the extraordinary adaptability of the metallo-hydrolase domain of the β-lactamase family for the hydrolysis of covalent bonds.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Ribbon representation of RNase Z at 2.1 Å resolution.
Figure 2: Structural comparison of members of the metallo-β-lactamase family.
Figure 3: Model of RNase Z–tRNA complex and amino acids important for interaction.
Figure 4: Active sites of RNase Z subunits and the proposed cleavage mechanism.

References

  1. Frank, D. N. & Pace, N. R. Ribonuclease P: unity and diversity in a tRNA processing ribozyme. Annu. Rev. Biochem. 67, 153–180 (1998)

    CAS  Article  Google Scholar 

  2. Garber, R. L. & Altman, S. In vitro processing of B. mori transfer RNA precursor molecules. Cell 17, 389–397 (1979)

    CAS  Article  Google Scholar 

  3. Li, Z. & Deutscher, M. P. Maturation pathways for E. coli tRNA precursors: a random multienzyme process in vivo. Cell 86, 503–512 (1996)

    CAS  Article  Google Scholar 

  4. Garber, R. L. & Gage, L. P. Transcription of a cloned Bombyx mori tRNA2Ala gene: nucleotide sequence of the tRNA precursor and its processing in vitro. Cell 18, 817–828 (1979)

    CAS  Article  Google Scholar 

  5. Schiffer, S., Rosch, S. & Marchfelder, A. Assigning a function to a conserved group of proteins: the tRNA 3′-processing enzymes. EMBO J. 21, 2769–2777 (2002)

    CAS  Article  Google Scholar 

  6. Nashimoto, M. Distribution of both lengths and 5′ terminal nucleotides of mammalian pre-tRNA 3′ trailers reflects properties of 3′ processing endoribonuclease. Nucleic Acids Res. 25, 1148–1154 (1997)

    CAS  Article  Google Scholar 

  7. Kunzmann, A., Brennicke, A. & Marchfelder, A. 5′ end maturation and RNA editing have to precede tRNA 3′ processing in plant mitochondria. Proc. Natl Acad. Sci. USA 95, 108–113 (1998)

    ADS  CAS  Article  Google Scholar 

  8. Schierling, K., Rosch, S., Rupprecht, R., Schiffer, S. & Marchfelder, A. tRNA 3′ end maturation in archaea has eukaryotic features: the RNase Z from Haloferax volcanii. J. Mol. Biol. 316, 895–902 (2002)

    CAS  Article  Google Scholar 

  9. Pellegrini, O., Nezzar, J., Marchfelder, A., Putzer, H. & Condon, C. Endonucleolytic processing of CCA-less tRNA precursors by RNase Z in Bacillus subtilis. EMBO J. 22, 4534–4543 (2003)

    CAS  Article  Google Scholar 

  10. Tavtigian, S. V. et al. A candidate prostate cancer susceptibility gene at chromosome 17p. Nature Genet. 27, 172–180 (2001)

    CAS  Article  Google Scholar 

  11. Minagawa, A., Takaku, H., Takagi, M. & Nashimoto, M. A novel endonucleolytic mechanism to generate the CCA 3′ termini of tRNA molecules in Thermotoga maritima. J. Biol. Chem. 279, 15688–15697 (2004)

    CAS  Article  Google Scholar 

  12. Mohan, A., Whyte, S., Wang, X., Nashimoto, M. & Levinger, L. The 3′ end CCA of mature tRNA is an antideterminant for eukaryotic 3′-tRNase. RNA 5, 245–256 (1999)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  14. Schiffer, S., Helm, M., Theobald-Dietrich, A., Giege, R. & Marchfelder, A. The plant tRNA 3′ processing enzyme has a broad substrate spectrum. Biochemistry 40, 8264–8272 (2001)

    CAS  Article  Google Scholar 

  15. Cameron, A. D., Ridderstrom, M., Olin, B. & Mannervik, B. Crystal structure of human glyoxalase II and its complex with a glutathione thiolester substrate analogue. Struct. Fold. Des. 7, 1067–1078 (1999)

    CAS  Article  Google Scholar 

  16. Nashimoto, M., Wesemann, D. R., Geary, S., Tamura, M. & Kaspar, R. L. Long 5′ leaders inhibit removal of a 3′ trailer from a precursor tRNA by mammalian tRNA 3′ processing endoribonuclease. Nucleic Acids Res. 27, 2770–2776 (1999)

    CAS  Article  Google Scholar 

  17. Vogel, A., Schilling, O., Niecke, M., Bettmer, J. & Meyer-Klaucke, W. ElaC encodes a novel binuclear zinc phosphodiesterase. J. Biol. Chem. 277, 29078–29085 (2002)

    CAS  Article  Google Scholar 

  18. Daiyasu, H., Osaka, K., Ishino, Y. & Toh, H. Expansion of the zinc metallo-hydrolase family of the β-lactamase fold. FEBS Lett. 503, 1–6 (2001)

    ADS  CAS  Article  Google Scholar 

  19. Carfi, A. et al. The 3-D structure of a zinc metallo-beta-lactamase from Bacillus cereus reveals a new type of protein fold. EMBO J. 14, 4914–4921 (1995)

    CAS  Article  Google Scholar 

  20. Frazao, C. et al. Structure of a dioxygen reduction enzyme from Desulfovibrio gigas. Nature Struct. Biol. 7, 1041–1045 (2000)

    CAS  Article  Google Scholar 

  21. Matthews, B. W. Solvent content of protein crystals. J. Mol. Biol. 33, 491–497 (1968)

    CAS  Article  Google Scholar 

  22. CCP4. The CCP4 Suite: Programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

    Article  Google Scholar 

  23. Otwinowski, Z. & Minor, W. in Methods in Enzymology Vol. 276, Macromolecular Crystallography Part A (eds Carter, C. W. Jr & Sweet, R. M.) 307–326 (Academic, New York, 1997)

    Book  Google Scholar 

  24. Uson, I. & Sheldrick, G. M. Advances in direct methods for protein crystallography. Curr. Opin. Struct. Biol. 9, 643–648 (1999)

    CAS  Article  Google Scholar 

  25. Schneider, T. R. & Sheldrick, G. M. Substructure solution with SHELXD. Acta Crystallogr. D 58, 1772–1779 (2002)

    Article  Google Scholar 

  26. Lu, G. FINDNCS: A program to detect non-crystallographic symmetries in protein crystals from heavy atom sites. J. Appl. Crystallogr. 32, 365–368 (1999)

    CAS  Article  Google Scholar 

  27. Perrakis, A., Morris, R. & Lamzin, V. S. Automated protein model building combined with iterative structure refinement. Nature Struct. Biol. 6, 458–463 (1999)

    CAS  Article  Google Scholar 

  28. Kleywegt, G. J. & Jones, T. A. in From First Map to Final Model (eds Bailey, S. R. H. & Waller, D.) 59–66 (SERC Daresbury Laboratory, Daresbury, UK, 1994)

    Google Scholar 

  29. Berman, H. M. et al. The Protein Data Bank. Nucleic Acids Res. 28, 235–242 (2000)

    ADS  CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank M. Springer, D. Picot, R. Giégé, F. Allemand, V. Arluison and F. Dardel for discussions, S. Fieulaine and M. Pirocchi for help with the beam-line BM30A at the European Synchrotron Radiation Facility, and J.L. Popot for use of crystallography facilities and X-ray generator at the IBPC. This work was supported by the CNRS (UPR 9073), Université Paris VII-Denis Diderot, PRFMMIP 2001/2003, and ACI Jeunes Chercheurs from the Ministère de l'Education Nationale.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Ciarán Condon.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Figure S1

Shows an alignment COGs representing different members of the metallo -lactamase family. (PPT 1058 kb)

Supplementary Figure S2

Shows both the secondary structure features of RNase Z superimposed on its amino acid sequence and the secondary structure topology of the protein. (PPT 52 kb)

Supplementary Figure S3

Shows the crystal packing of RNase Z. (PPT 122 kb)

Supplementary Methods

Describes the seleno-methionine labelling of RNase Z. (RTF 3 kb)

Supplementary Table

Shows data-collection, phasing and refinement statistics for resolution of RNase Z structure. (RTF 304 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

de la Sierra-Gallay, I., Pellegrini, O. & Condon, C. Structural basis for substrate binding, cleavage and allostery in the tRNA maturase RNase Z. Nature 433, 657–661 (2005). https://doi.org/10.1038/nature03284

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature03284

Further reading

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

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