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Structure of active dimeric human telomerase

Nature Structural & Molecular Biology volume 20, pages 454–460 (2013)Cite this article

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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.

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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.

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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.).

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Author notes

  1. Gaël Cristofari

    Present address: Present address: Institute for Research on Cancer and Aging, Faculty of Medicine, University of Nice–Sophia-Antipolis, Nice, France.,

  2. Anselm Sauerwald and Sara Sandin: These authors contributed equally to this work.

Authors and Affiliations

  1. Medical Research Council Laboratory of Molecular Biology, Cambridge, UK

    Anselm Sauerwald, Sara Sandin, Sjors H W Scheres & Daniela Rhodes

  2. Swiss Institute for Experimental Cancer Research, School of Life Sciences, Frontiers in Genetics National Center of Competence in Research, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

    Anselm Sauerwald, Gaël Cristofari & Joachim Lingner

  3. School of Biological Sciences, Nanyang Technological University, Singapore

    Sara Sandin & Daniela Rhodes

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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.

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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)

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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

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