Human telomerase is a RNA–protein complex that extends the 3′ end of linear chromosomes by synthesizing multiple copies of the telomeric repeat TTAGGG1. Its activity is a determinant of cancer progression, stem cell renewal and cellular aging2,3,4,5. Telomerase is recruited to telomeres and activated for telomere repeat synthesis by the telomere shelterin protein TPP16,7. Human telomerase has a bilobal structure with a catalytic core ribonuclear protein and a H and ACA box ribonuclear protein8,9. Here we report cryo-electron microscopy structures of human telomerase catalytic core of telomerase reverse transcriptase (TERT) and telomerase RNA (TER (also known as hTR)), and of telomerase with the shelterin protein TPP1. TPP1 forms a structured interface with the TERT-unique telomerase essential N-terminal domain (TEN) and the telomerase RAP motif (TRAP) that are unique to TERT, and conformational dynamics of TEN–TRAP are damped upon TPP1 binding, defining the requirements for recruitment and activation. The structures further reveal that the elements of TERT and TER that are involved in template and telomeric DNA handling—including the TEN domain and the TRAP–thumb helix channel—are largely structurally homologous to those in Tetrahymena telomerase10, and provide unique insights into the mechanism of telomerase activity. The binding site of the telomerase inhibitor BIBR153211,12 overlaps a critical interaction between the TER pseudoknot and the TERT thumb domain. Numerous mutations leading to telomeropathies13,14 are located at the TERT–TER and TEN–TRAP–TPP1 interfaces, highlighting the importance of TER–TERT and TPP1 interactions for telomerase activity, recruitment and as drug targets.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
The genetics of monogenic intestinal epithelial disorders
Human Genetics Open Access 23 November 2022
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Rent or buy this article
Get just this article for as long as you need it
Prices may be subject to local taxes which are calculated during checkout
Cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-26085 (H/ACA RNP), EMD-26086 (catalytic core combined), EMD-26087 (catalytic core with TPP1), EMD-26096 (catalytic core without TPP1), EMD-26088 (catalytic core with H2A–H2B), EMD-26090 (catalytic core with TPP1, P2a state 1-1), EMD-26091 (catalytic core with TPP1, P2a state 1-2), EMD-26092 (catalytic core with TPP1, P2a state 1-3), EMD-26093 (catalytic core with TPP1, P2a state 2), EMD-26094 (catalytic core without TPP1, P2a state 1) and EMD-26095 (catalytic core without TPP1, P2a state 2). The atomic models have been deposited in the Protein Data Bank under accession codes 7TRC (H/ACA RNP), 7TRD (catalytic core combined), 7TRE (catalytic core with TPP1) and 7TRF (catalytic core with H2A–H2B). Uncropped version of all the gels are included as Supplementary Fig. 1. Any other relevant data are available from the corresponding authors upon reasonable request.
Blackburn, E. H. & Collins, K. Telomerase: an RNP enzyme synthesizes DNA. Cold Spring Harb. Perspect. Biol. 3, a003558 (2011).
Chakravarti, D., LaBella, K. A. & DePinho, R. A. Telomeres: history, health, and hallmarks of aging. Cell 184, 306–322 (2021).
Trybek, T., Kowalik, A., Gozdz, S. & Kowalska, A. Telomeres and telomerase in oncogenesis. Oncol. Lett. 20, 1015–1027 (2020).
Roake, C. M. & Artandi, S. E. Regulation of human telomerase in homeostasis and disease. Nat. Rev. Mol. Cell Biol. 21, 384–397 (2020).
Shay, J. W. Role of telomeres and telomerase in aging and cancer. Cancer Discov. 6, 584–593 (2016).
Aramburu, T., Plucinsky, S. & Skordalakes, E. POT1–TPP1 telomere length regulation and disease. Comput. Struct. Biotechnol. J. 18, 1939–1946 (2020).
de Lange, T. Shelterin-mediated telomere protection. Annu. Rev. Genet. 52, 223–247 (2018).
Ghanim, G. E. et al. Structure of human telomerase holoenzyme with bound telomeric DNA. Nature 593, 449–453 (2021).
Nguyen, T. H. D. et al. Cryo-EM structure of substrate-bound human telomerase holoenzyme. Nature 557, 190–195 (2018).
He, Y. et al. Structures of telomerase at several steps of telomere repeat synthesis. Nature 593, 454–459 (2021).
Bryan, C. et al. Structural basis of telomerase inhibition by the highly specific BIBR1532. Structure 23, 1934–1942 (2015).
Pascolo, E. et al. Mechanism of human telomerase inhibition by BIBR1532, a synthetic, non-nucleosidic drug candidate. J. Biol. Chem. 277, 15566–15572 (2002).
Podlevsky, J. D., Bley, C. J., Omana, R. V., Qi, X. & Chen, J. J. The telomerase database. Nucleic Acids Res. 36, D339–D343 (2008).
Holohan, B., Wright, W. E. & Shay, J. W. Cell biology of disease: telomeropathies: an emerging spectrum disorder. J. Cell Biol. 205, 289–299 (2014).
Wu, R. A., Upton, H. E., Vogan, J. M. & Collins, K. Telomerase mechanism of telomere synthesis. Annu. Rev. Biochem. 86, 439–460 (2017).
Schmidt, J. C. & Cech, T. R. Human telomerase: biogenesis, trafficking, recruitment, and activation. Genes Dev. 29, 1095–1105 (2015).
Grill, S. & Nandakumar, J. Molecular mechanisms of telomere biology disorders. J. Biol. Chem. 296, 100064 (2021).
Schratz, K. E. et al. Cancer spectrum and outcomes in the Mendelian short telomere syndromes. Blood 135, 1946–1956 (2020).
Reilly, C. R. et al. The clinical and functional effects of TERT variants in myelodysplastic syndrome. Blood 138, 898–911 (2021).
Jiang, J. et al. Structure of telomerase with telomeric DNA. Cell 173, 1179–1190.e1113 (2018).
Jiang, J. et al. Structure of Tetrahymena telomerase reveals previously unknown subunits, functions, and interactions. Science 350, aab4070 (2015).
Lim, C. J. & Cech, T. R. Shaping human telomeres: from shelterin and CST complexes to telomeric chromatin organization. Nat. Rev. Mol. Cell Biol. 22, 283–298 (2021).
Lei, M., Podell, E. R. & Cech, T. R. Structure of human POT1 bound to telomeric single-stranded DNA provides a model for chromosome end-protection. Nat. Struct. Mol. Biol. 11, 1223–1229 (2004).
Nandakumar, J. et al. The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity. Nature 492, 285–289 (2012).
Zhong, F. L. et al. TPP1 OB-fold domain controls telomere maintenance by recruiting telomerase to chromosome ends. Cell 150, 481–494 (2012).
Sexton, A. N., Youmans, D. T. & Collins, K. Specificity requirements for human telomere protein interaction with telomerase holoenzyme. J. Biol. Chem. 287, 34455–34464 (2012).
Grill, S., Tesmer, V. M. & Nandakumar, J. The N terminus of the OB domain of telomere protein TPP1 is critical for telomerase action. Cell Rep. 22, 1132–1140 (2018).
Chen, C. et al. Structural insights into POT1–TPP1 interaction and POT1 C-terminal mutations in human cancer. Nat. Commun. 8, 14929 (2017).
Rice, C. et al. Structural and functional analysis of the human POT1–TPP1 telomeric complex. Nat. Commun. 8, 14928 (2017).
Schmidt, J. C., Zaug, A. J. & Cech, T. R. Live cell imaging reveals the dynamics of telomerase recruitment to telomeres. Cell 166, 1188–1197.e1189 (2016).
Mitchell, J. R., Cheng, J. & Collins, K. A box H/ACA small nucleolar RNA-like domain at the human telomerase RNA 3′ end. Mol. Cell. Biol. 19, 567–576 (1999).
Zhang, Q., Kim, N. K. & Feigon, J. Architecture of human telomerase RNA. Proc. Natl Acad. Sci. USA 108, 20325–20332 (2011).
Wang, F. et al. The POT1–TPP1 telomere complex is a telomerase processivity factor. Nature 445, 506–510 (2007).
Schmidt, J. C., Dalby, A. B. & Cech, T. R. Identification of human TERT elements necessary for telomerase recruitment to telomeres. eLife 3, e03563 (2014).
Tesmer, V. M., Smith, E. M., Danciu, O., Padmanaban, S. & Nandakumar, J. Combining conservation and species-specific differences to determine how human telomerase binds telomeres. Proc. Natl Acad. Sci. USA 116, 26505–26515 (2019).
Wang, Y., Gallagher-Jones, M., Susac, L., Song, H. & Feigon, J. A structurally conserved human and Tetrahymena telomerase catalytic core. Proc. Natl Acad. Sci. USA 117, 31078–31087 (2020).
Hong, K. et al. Tetrahymena telomerase holoenzyme assembly, activation, and inhibition by domains of the p50 central hub. Mol. Cell. Biol. 33, 3962–3971 (2013).
Forstemann, K. & Lingner, J. Telomerase limits the extent of base pairing between template RNA and telomeric DNA. EMBO Rep. 6, 361–366 (2005).
Jansson, L. I. et al. Structural basis of template-boundary definition in Tetrahymena telomerase. Nat. Struct. Mol. Biol. 22, 883–888 (2015).
Chen, J. L. & Greider, C. W. Template boundary definition in mammalian telomerase. Genes Dev. 17, 2747–2752 (2003).
Wan, F. et al. Zipper head mechanism of telomere synthesis by human telomerase. Cell Res. 31, 1275–1290 (2021).
Theimer, C. A., Blois, C. A. & Feigon, J. Structure of the human telomerase RNA pseudoknot reveals conserved tertiary interactions essential for function. Mol. Cell 17, 671–682 (2005).
Kim, N. K. et al. Solution structure and dynamics of the wild-type pseudoknot of human telomerase RNA. J. Mol. Biol. 384, 1249–1261 (2008).
Zhang, Q., Kim, N. K., Peterson, R. D., Wang, Z. & Feigon, J. Structurally conserved five nucleotide bulge determines the overall topology of the core domain of human telomerase RNA. Proc. Natl Acad. Sci. USA 107, 18761–18768 (2010).
Palka, C., Forino, N. M., Hentschel, J., Das, R. & Stone, M. D. Folding heterogeneity in the essential human telomerase RNA three-way junction. RNA 26, 1787–1800 (2020).
Huang, J. et al. Structural basis for protein–RNA recognition in telomerase. Nat. Struct. Mol. Biol. 21, 507–512 (2014).
Guterres, A. N. & Villanueva, J. Targeting telomerase for cancer therapy. Oncogene 39, 5811–5824 (2020).
Wong, J. M. & Collins, K. Telomerase RNA level limits telomere maintenance in X-linked dyskeratosis congenita. Genes Dev. 20, 2848–2858 (2006).
Gao, L., Frey, M. R. & Matera, A. G. Human genes encoding U3 snRNA associate with coiled bodies in interphase cells and are clustered on chromosome 17p11.2 in a complex inverted repeat structure. Nucleic Acids Res. 25, 4740–4747 (1997).
Fu, D. & Collins, K. Distinct biogenesis pathways for human telomerase RNA and H/ACA small nucleolar RNAs. Mol. Cell 11, 1361–1372 (2003).
Ferre-D’Amare, A. R., Zhou, K. & Doudna, J. A. Crystal structure of a hepatitis delta virus ribozyme. Nature 395, 567–574 (1998).
Chen, Y. Calcium phosphate transfection of eukaryotic cells. Bio Protoc. 2 (2012).
Kurth, I., Cristofari, G. & Lingner, J. An affinity oligonucleotide displacement strategy to purify ribonucleoprotein complexes applied to human telomerase. Methods Mol. Biol. 488, 9–22 (2008).
Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).
Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).
Bepler, T. et al. Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs. Nat. Methods 16, 1153–1160 (2019).
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).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).
Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).
Goddard, T. D. et al. UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).
DeLano, W. L. The PyMOL Molecular Graphics System (Delano Scientific, 2002).
Guillerez, J., Lopez, P. J., Proux, F., Launay, H. & Dreyfus, M. A mutation in T7 RNA polymerase that facilitates promoter clearance. Proc. Natl Acad. Sci. USA 102, 5958–5963 (2005).
Wang, Y., Yesselman, J. D., Zhang, Q., Kang, M. & Feigon, J. Structural conservation in the template/pseudoknot domain of vertebrate telomerase RNA from teleost fish to human. Proc. Natl Acad. Sci. USA 113, E5125–E5134 (2016).
Latrick, C. M. & Cech, T. R. POT1–TPP1 enhances telomerase processivity by slowing primer dissociation and aiding translocation. EMBO J. 29, 924–933 (2010).
Jansson, L. I. et al. Telomere DNA G-quadruplex folding within actively extending human telomerase. Proc. Natl Acad. Sci. USA 116, 9350–9359 (2019).
Leontis, N. B., Stombaugh, J. & Westhof, E. The non-Watson-Crick base pairs and their associated isostericity matrices. Nucleic Acids Res. 30, 3497–3531 (2002).
Robert, X. & Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320–W324 (2014).
Tummala, H. et al. Homozygous OB-fold variants in telomere protein TPP1 are associated with dyskeratosis congenita-like phenotypes. Blood 132, 1349–1353 (2018).
Hoffman, T. W. et al. Pulmonary fibrosis linked to variants in the ACD gene, encoding the telomere protein TPP1. Eur. Respir. J. 54, 1900809 (2019).
This work was supported by grants from NIH R35GM131901 and NSF MCB2016540 to J.F. and NIH R01GM071940 to Z.H.Z. We acknowledge use of instruments at the Electron Imaging Center for Nanomachines supported by UCLA and instrumentation grants from NIH (1S10RR23057, 1S10OD018111) and NSF (DBI-1338135 and DMR-1548924).
The authors declare no competing interests.
Peer review information
Nature thanks Thomas Cech and the other, anonymous, reviewers for their contribution to the peer review of this work. Peer review reports are available.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Cryo-EM data processing workflow of human telomerase (detailed in Methods).
a, Initial screening of sub-particles centered on the catalytic core and H/ACA RNP. b, Representative 2D class images of human telomerase as indicated in the red boxes in a. c, d, 3D classification and reconstruction processes for the H/ACA RNP (c) and catalytic core (d). Soft masks used in the data processing are shown in orange.
Extended Data Fig. 2 Evaluation of cryo-EM reconstructions.
a, Plot of the Fourier shell correlation (FSC) as a function of the spatial frequency of the final reconstructions. b, c, Local resolution evaluation of the H/ACA RNP (b) and catalytic core (c) reconstructions. d, e, Euler angle distribution of sub-particles used for the H/ACA RNP (d) and catalytic core (e) reconstructions. f, FSC coefficients as a function of spatial frequency between model and corresponding cryo-EM density maps. g–j, Representative cryo-EM densities encasing the corresponding atomic models of H/ACA proteins (g), TERT (h), TER (i) and the four base pairs in the template–DNA duplex (j).
Extended Data Fig. 3 Analysis of compositional and conformational heterogeneity of the catalytic core.
a, Cryo-EM 3D classification and reconstruction workflow with different masks. Insets show the FSC curves of final reconstructions. b, Superposition of H2A-H2B model and cryo-EM density. c, Local resolution evaluation of the 3.5 Å resolution reconstruction of catalytic core bond with TPP1. d, Euler angle distribution of sub-particles used for the reconstruction of catalytic core bound with TPP1. e, Model VS map FSC curve of the reconstruction with TPP1 or H2A-H2B. f, Superposition of TPP1 model and cryo-EM density.
Extended Data Fig. 4 Secondary structure of human TER and interactions with TERT.
a, Secondary structure observed in our telomerase structure (black), with unmodeled regions (gray) based on the telomerase database13. b, c, The previously predicted secondary structures of t/PK (b) and CR4/5 (c) based on phylogenetic analysis13. The dashed lines connecting residues in c indicate interactions forming the 3 bp P5.1 (highlighted) found in our structure, added for clarity. d, e, Structure-based schematics of TER P2b/P3-P1b (d) and CR4/5 (e) and interactions with TERT. Solid and dashed arrows from TERT residues to TER nucleotides indicate interactions with base and backbone, respectively. Base pair types are indicated using Leontis-Westhof symbols69. Red circles/ovals indicate residue positions with disease related mutations.
Extended Data Fig. 5 Comparison of human and Tetrahymena TERT structure.
a, Superposition of RBD–RT–CTE ring (TERT ring) structures of hTERT and Tetrahymena TERT (TtTERT, PDB: 7LMA). b, Overlay of TEN–IFD-TRAP structures in human (colored) and Tetrahymena (gray) telomerase. Inset shows the secondary structure of human TRAP and the extended β sheet between TEN and TRAP. c–f, Comparison of human and Tetrahymena TEN–TRAP position relative to the TERT ring. Ribbon diagrams of the TERT rings from hTERT (c–d) and TtTERT (e–f) are shown in the same orientations as in A. Human and Tetrahymena TER are shown as magenta surfaces in c and e, respectively. Transparent sticks in d and f indicate the orientations of TRAP domains in hTERT and TtTERT, respectively. Residues located at the distal end of TRAP domains are shown as spheres. The hinge of the “rotation movement” is at the proximal end of TRAP that is connected to the IFDa and IFDc helices.
Extended Data Fig. 6 Comparison of human TEN–TRAP–TPP1 and Tetrahymena TEN–TRAP–p50.
a, Comparison of telomerase-bound TPP1 and p50 (gray) (PDB: 7LMA). b, Superposition of human TEN–TRAP–TPP1 (colored as in Fig. 2) and Tetrahymena TEN–TRAP–p50 (gray). c, d, Overall comparison of human TEN–TRAP–TPP1 (c) and Tetrahymena TEN–TRAP–p50 (d) with the residues locate on the interfaces colored yellow. e, f, Open book views of the continuous interfaces between human TPP1–TERT (e) and Tetrahymena p50–TERT (f) shown as surface, respectively. g, h, A three-way junction shared by human TEN–TRAP–TPP1 (g) and Tetrahymena TEN–TRAP–p5010 (h). i, j, Density maps of TEN and TRAP without (i) and with (j) TPP1, highlighting the changes in density upon TPP1 binding. Density thresholds were adjusted for comparable density in the RT region. k, Structure-based sequence alignment (rendered with ESPript370) for human TPP1 (red) and Tetrahymena p50 (grey). Residues located on the interface are highlighted in yellow. Residues comprising TPP1 NOB and TEL patch are indicated on the top of alignment.
Extended Data Fig. 7 Disease related mutations and telomerase activity assays.
a-e Locations of disease related mutations. a, Back view of TEN–TRAP–TPP1 interface as in Extended Data Fig. 6. Residue positions with disease mutations in TEN, TRAP, and TPP1 (Supplementary Table 113,71,72) are shown as yellow (interface) and gray (non-interface) spheres. b, c, Zoomed-in views of intra-(b) and inter-(c) domain interactions from residues related to disease mutations that may disrupt TPP1–TERT interaction indirectly. d–i Telomerase activity assays for TERT variants with residue substitutions at the interface with TPP1. d, Telomerase activity assays corresponding to Fig. 2g. Gel is a representative from 3 independent experiments. The number of telomeric repeats added to primer are indicated at left, and number of nucleotides are indicated at right. RC, recovery control. e, f, Relative activity (e) and RAP (f) normalized to TERT WT without TPP1. Plotted values are mean ± s.d. from n = 3 biologically independent experiments. g, Telomerase activity assays without and with TPP1–POT1. Gel is a representative from 2 independent experiments. h, i, Relative activity (h) and RAP (i) normalized to TERT WT without TPP1-POT1. Open circles are values from n = 2 biologically independent experiments.
Extended Data Fig. 8 Structural details of the TBE, bridge loop and related activity assays.
a, Structure of the single-stranded RNA nucleotides at the 5′ side of the template in complex with TERT RBD (colored as in Fig. 3a). Cryo-EM densities of TER nucleotides are overlayed on the structure as transparent surfaces. Only the first two template-adjacent nucleotides (G44U45) and P1b have strong densities in the cryo-EM map, whereas the intervening nucleotides show only weak density indicative of positional dynamics. b, Comparison of the TBE-TBEL-RBD structures from human (color) and Tetrahymena (gray, PDB: 7LMA) telomerase. c, G44 and U45 in the kedge anchor pocket of TERT RBD. The linear distance from the phosphate group of U38 to the phosphate group of G44 is about 21 Å. d, Detailed interactions between G44-U45 and TERT RBD. Intermolecular hydrogen bonds are shown as dashed yellow lines. e, Schematic of TER TBE-TBEL-template conformation when the template is at the +3 position as in our structure. f, Predicted TBE-TBEL-template conformation when the template is at the +6 position. TBEL nucleotides U38–40 would be fully stretched to span the distance (21 Å) from the TBE anchor to the kedge anchor. g, Comparison of the template–DNA duplex and surrounding structural elements of human (color) and Tetrahymena (gray, PDB: 7LMA) telomerase. Residues located on the tip of the bridge loop are shown as sticks. h, Telomerase activity assays with substitutions of hTERT K499 and/or H500. Gel is a representative from 2 independent experiments. i, Detailed interactions surrounding the entrance of template nucleotides. Side chains of key residues are indicated with corresponding Tetrahymena TERT residues in parentheses. j, Ribbon diagram of the template-DNA duplex and the bridge loop superimposed with cryo-EM densities (transparent surface).
Extended Data Fig. 9 Electrostatic interactions and conformational dynamics of the pseudoknot on TERT.
a, Electrostatic surface of TERT shown in two different views. b–e, Cryo-EM density (upper) and model (lower) of telomerase catalytic core with P2 stem in State1-1 (b), State1-2 (c), State1–3 (d) and State2 (e). Through State1-1 to 1–3 (b–d), P2a.1 conducts an upward movement. From State1-1 (b) to State2 (e), P2a.1 and P2a move away from TEN domain of TERT with the J2a/b linker bulged out. The conformation of P2b and its location on TERT remain the same in the four structures.
This file contains Supplementary Figs. 1, 2, Table 1 and references.
Rights and permissions
About this article
Cite this article
Liu, B., He, Y., Wang, Y. et al. Structure of active human telomerase with telomere shelterin protein TPP1. Nature 604, 578–583 (2022). https://doi.org/10.1038/s41586-022-04582-8
This article is cited by
The genetics of monogenic intestinal epithelial disorders
Human Genetics (2023)
Structure of Tetrahymena telomerase-bound CST with polymerase α-primase
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.