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.

Structure of active dimeric human telomerase

Abstract

Telomerase contains a large RNA subunit, TER, and a protein catalytic subunit, TERT. Whether telomerase functions as a monomer or dimer has been a matter of debate. Here we report biochemical and labeling data that show that in vivo–assembled human telomerase contains two TERT subunits and binds two telomeric DNA substrates. Notably, catalytic activity requires both TERT active sites to be functional, which demonstrates that human telomerase functions as a dimer. We also present the three-dimensional structure of the active full-length human telomerase dimer, determined by single-particle EM in negative stain. Telomerase has a bilobal architecture with the two monomers linked by a flexible interface. The monomer reconstruction at 23-Å resolution and fitting of the atomic structure of the TERT subunit from beetle Tribolium castaneum into the EM density reveals the spatial relationship between RNA and protein subunits, providing insights into telomerase architecture.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Human telomerase is a dimer.
Figure 2: Active human telomerase functions as a dimer.
Figure 3: Analysis of the telomerase dimer by single-particle EM.
Figure 4: Independently refined monomers and composite dimers.
Figure 5: Assignment of the TERT and TER subunit within the 3D map of the open telomerase monomer.

Accession codes

Primary accessions

Electron Microscopy Data Bank

References

  1. 1

    de Lange, T. How telomeres solve the end-protection problem. Science 326, 948–952 (2009).

    CAS  Article  Google Scholar 

  2. 2

    Harley, C.B. Telomerase and cancer therapeutics. Nat. Rev. Cancer 8, 167–179 (2008).

    CAS  Article  Google Scholar 

  3. 3

    Greider, C.W. & Blackburn, E.H. A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature 337, 331–337 (1989).

    CAS  Article  Google Scholar 

  4. 4

    Yu, G.L., Bradley, J.D., Attardi, L.D. & Blackburn, E.H. In vivo alteration of telomere sequences and senescence caused by mutated Tetrahymena telomerase RNAs. Nature 344, 126–132 (1990).

    CAS  Article  Google Scholar 

  5. 5

    Cech, T.R. Beginning to understand the end of the chromosome. Cell 116, 273–279 (2004).

    CAS  Article  Google Scholar 

  6. 6

    Lingner, J. et al. Reverse transcriptase motifs in the catalytic subunit of telomerase. Science 276, 561–567 (1997).

    CAS  Article  Google Scholar 

  7. 7

    Blackburn, E.H. & Collins, K. Telomerase: an RNP enzyme synthesizes DNA. Cold Spring Harb. Perspect. Biol. 3, a003558 (2011).

    Article  Google Scholar 

  8. 8

    Weinrich, S.L. et al. Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. Nat. Genet. 17, 498–502 (1997).

    CAS  Article  Google Scholar 

  9. 9

    Autexier, C. & Lue, N. The structure and function of telomerase reverse transcriptase. Annu. Rev. Biochem. 75, 493–517 (2006).

    CAS  Article  Google Scholar 

  10. 10

    Cohen, S.B. et al. Protein composition of catalytically active human telomerase from immortal cells. Science 315, 1850–1853 (2007).

    CAS  Article  Google Scholar 

  11. 11

    Egan, E.D. & Collins, K. Specificity and stoichiometry of subunit interactions in the human telomerase holoenzyme assembled in vivo. Mol. Cell Biol. 30, 2775–2786 (2010).

    CAS  Article  Google Scholar 

  12. 12

    Venteicher, A.S. et al. A human telomerase holoenzyme protein required for Cajal body localization and telomere synthesis. Science 323, 644–648 (2009).

    CAS  Article  Google Scholar 

  13. 13

    Errington, T.M., Fu, D., Wong, J.M. & Collins, K. Disease-associated human telomerase RNA variants show loss of function for telomere synthesis without dominant-negative interference. Mol. Cell Biol. 28, 6510–6520 (2008).

    CAS  Article  Google Scholar 

  14. 14

    Wenz, C. et al. Human telomerase contains two cooperating telomerase RNA molecules. EMBO J. 20, 3526–3534 (2001).

    CAS  Article  Google Scholar 

  15. 15

    Arai, K. et al. Two independent regions of human telomerase reverse transcriptase are important for its oligomerization and telomerase activity. J. Biol. Chem. 277, 8538–8544 (2002).

    CAS  Article  Google Scholar 

  16. 16

    Beattie, T.L., Zhou, W., Robinson, M.O. & Harrington, L. Functional multimerization of the human telomerase reverse transcriptase. Mol. Cell Biol. 21, 6151–6160 (2001).

    CAS  Article  Google Scholar 

  17. 17

    Moriarty, T.J., Huard, S., Dupuis, S. & Autexier, C. Functional multimerization of human telomerase requires an RNA interaction domain in the N terminus of the catalytic subunit. Mol. Cell Biol. 22, 1253–1265 (2002).

    CAS  Article  Google Scholar 

  18. 18

    Gardano, L., Holland, L., Oulton, R., Le Bihan, T. & Harrington, L. Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin. Nucleic Acids Res. 40, e36 (2012).

    CAS  Article  Google Scholar 

  19. 19

    Sekaran, V.G., Soares, J. & Jarstfer, M. Structures of telomerase subunits provide functional insights. Biochim. Biophys. Acta 1804, 1190–1201 (2010).

    CAS  Article  Google Scholar 

  20. 20

    Wyatt, H., West, S. & Beattie, T. InTERTpreting telomerase structure and function. Nucleic Acids Res. 38, 5609–5622 (2010).

    CAS  Article  Google Scholar 

  21. 21

    Zhang, Q., Kim, N.K. & Feigon, J. Architecture of human telomerase RNA. Proc. Natl. Acad. Sci. USA 108, 20325–20332 (2011).

    CAS  Article  Google Scholar 

  22. 22

    Mitchell, M., Gillis, A., Futahashi, M., Fujiwara, H. & Skordalakes, E. Structural basis for telomerase catalytic subunit TERT binding to RNA template and telomeric DNA. Nat. Struct. Mol. Biol. 17, 513–518 (2010).

    CAS  Article  Google Scholar 

  23. 23

    Schuller, A.P., Harkisheimer, M.J. & Skordalakes, E. In vitro reconstitution of the active T. castaneum telomerase. J. Vis. Exp. e2799 (2011).

  24. 24

    Kohlstaedt, L.A., Wang, J., Friedman, J., Rice, P. & Steitz, T. Crystal structure at 3.5 Å resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science 256, 1783–1790 (1992).

    CAS  Article  Google Scholar 

  25. 25

    Gillis, A.J., Schuller, A. & Skordalakes, E. Structure of the Tribolium castaneum telomerase catalytic subunit TERT. Nature 455, 633–637 (2008).

    CAS  Article  Google Scholar 

  26. 26

    Jacobs, S.A., Podell, E. & Cech, T. Crystal structure of the essential N-terminal domain of telomerase reverse transcriptase. Nat. Struct. Mol. Biol. 13, 218–225 (2006).

    CAS  Article  Google Scholar 

  27. 27

    Theimer, C.A. & Feigon, J. Structure and function of telomerase RNA. Curr. Opin. Struct. Biol. 16, 307–318 (2006).

    CAS  Article  Google Scholar 

  28. 28

    Mitchell, J.R. & Collins, K. Human telomerase activation requires two independent interactions between telomerase RNA and telomerase reverse transcriptase. Mol. Cell 6, 361–371 (2000).

    CAS  Article  Google Scholar 

  29. 29

    Kelleher, C., Teixeira, M., Forstemann, K. & Lingner, J. Telomerase: biochemical considerations for enzyme and substrate. Trends Biochem. Sci. 27, 572–579 (2002).

    CAS  Article  Google Scholar 

  30. 30

    Cristofari, G. et al. Low- to high-throughput analysis of telomerase modulators with Telospot. Nat. Methods 4, 851–853 (2007).

    CAS  Article  Google Scholar 

  31. 31

    Cristofari, G. & Lingner, J. Telomere length homeostasis requires that telomerase levels are limiting. EMBO J. 25, 565–574 (2006).

    CAS  Article  Google Scholar 

  32. 32

    Mitchell, J.R., Wood, E. & Collins, K. A telomerase component is defective in the human disease dyskeratosis congenita. Nature 402, 551–555 (1999).

    CAS  Article  Google Scholar 

  33. 33

    Howarth, M. et al. A monovalent streptavidin with a single femtomolar biotin binding site. Nat. Methods 3, 267–273 (2006).

    CAS  Article  Google Scholar 

  34. 34

    Hahn, W.C. et al. Inhibition of telomerase limits the growth of human cancer cells. Nat. Med. 5, 1164–1170 (1999).

    CAS  Article  Google Scholar 

  35. 35

    Kastner, B. et al. GraFix: sample preparation for single-particle electron cryomicroscopy. Nat. Methods 5, 53–55 (2008).

    CAS  Article  Google Scholar 

  36. 36

    Scheres, S.H. et al. Modeling experimental image formation for likelihood-based classification of electron microscopy data. Structure 15, 1167–1177 (2007).

    CAS  Article  Google Scholar 

  37. 37

    Rosenthal, P.B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).

    CAS  Article  Google Scholar 

  38. 38

    Gardano, L., Holland, L., Oulton, R., Le Bihan, T. & Harrington, L. Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin. Nucleic Acids Res. 40, e36 (2012).

    CAS  Article  Google Scholar 

  39. 39

    Alves, D. et al. Single-molecule analysis of human telomerase monomer. Nat. Chem. Biol. 4, 287–289 (2008).

    CAS  Article  Google Scholar 

  40. 40

    Canudas, S. et al. A role for heterochromatin protein 1γ at human telomeres. Genes Dev. 25, 1807–1819 (2011).

    CAS  Article  Google Scholar 

  41. 41

    Fouché, N., Moon, I.K., Keppler, B.R., Griffith, J.D. & Jarstfer, M.B. Electron microscopic visualization of telomerase from Euplotes aediculatus bound to a model telomere DNA. Biochemistry 45, 9624–9631 (2006).

    Article  Google Scholar 

  42. 42

    Nunez-Ramirez, R. et al. Flexible tethering of primase and DNA Pol α in the eukaryotic primosome. Nucleic Acids Res. 39, 8187–8199 (2011).

    CAS  Article  Google Scholar 

  43. 43

    Ren, X. et al. Identification of a new RNA·RNA interaction site for human telomerase RNA (hTR): structural implications for hTR accumulation and a dyskeratosis congenita point mutation. Nucleic Acids Res. 31, 6509–6515 (2003).

    CAS  Article  Google Scholar 

  44. 44

    Graham, F.L., Smiley, J., Russell, W. & Nairn, R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol. 36, 59–74 (1977).

    CAS  Article  Google Scholar 

  45. 45

    Cristofari, G. et al. Human telomerase RNA accumulation in Cajal bodies facilitates telomerase recruitment to telomeres and telomere elongation. Mol. Cell 27, 882–889 (2007).

    CAS  Article  Google Scholar 

  46. 46

    Cohen, S.B. et al. Protein composition of catalytically active human telomerase from immortal cells. Science 315, 1850–1853 (2007).

    CAS  Article  Google Scholar 

  47. 47

    Kastner, B. et al. GraFix: sample preparation for single-particle electron cryomicroscopy. Nat. Methods 5, 53–55 (2008).

    CAS  Article  Google Scholar 

  48. 48

    Wenz, C. et al. Human telomerase contains two cooperating telomerase RNA molecules. EMBO J. 20, 3526–3534 (2001).

    CAS  Article  Google Scholar 

  49. 49

    Cristofari, G. et al. Low- to high-throughput analysis of telomerase modulators with Telospot. Nat. Methods 4, 851–853 (2007).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank P. Reichenbach, B. Zuber, S.J. Ludtke, R. Henderson, J. Short, J. Grimmett, S. Chen and A. Christen for help and advice, S. Thurnheer and D. Hacker for telomerase production, and P. Hunziker for protein identification. We thank the Human Frontier Science Program for funding through a grant (RGP0032/2005-C) awarded to J.L. and D.R. and a post-doctoral fellowship to A.S.; the European Molecular Biology Organization for post-doctorial fellowships to A.S. and S.S.; and the UK Medical Research Council for a career-development fellowship to S.S. Work in J.L.'s laboratory is supported by the Swiss National Science Foundation and a European Research Council advanced investigator grant (232812). Work in D.R.'s and S.H.W.S.'s laboratory is supported by the UK Medical Research Council (U10518433333 and MC_UP_A025_1013, respectively.).

Author information

Affiliations

Authors

Contributions

A.S. designed and carried out all the biochemical work and contributed to image processing. S.S. collected EM data and solved the structures. G.C. developed the super-telomerase cells. S.H.W.S designed and contributed to the structure refinement. J.L. and D.R. designed and supervised the project. All authors contributed to the writing of the paper.

Corresponding authors

Correspondence to Joachim Lingner or Daniela Rhodes.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Table 1 and Supplementary Note (PDF 1559 kb)

Supplementary Movie 1

Juxtaposition of the two 2D class average images suggests that telomerase has a flexible dimer interface. (MOV 238 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Sauerwald, A., Sandin, S., Cristofari, G. et al. Structure of active dimeric human telomerase. Nat Struct Mol Biol 20, 454–460 (2013). https://doi.org/10.1038/nsmb.2530

Download citation

Further reading

Search

Quick links