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Structures of telomerase at several steps of telomere repeat synthesis

Nature volume 593, pages 454–459 (2021)Cite this article

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Abstract

Telomerase is unique among the reverse transcriptases in containing a noncoding RNA (known as telomerase RNA (TER)) that includes a short template that is used for the processive synthesis of G-rich telomeric DNA repeats at the 3′ ends of most eukaryotic chromosomes1. Telomerase maintains genomic integrity, and its activity or dysregulation are critical determinants of human longevity, stem cell renewal and cancer progression2,3. Previous cryo-electron microscopy structures have established the general architecture, protein components and stoichiometries of Tetrahymena and human telomerase, but our understandings of the details of DNA–protein and RNA–protein interactions and of the mechanisms and recruitment involved remain limited4,5,6. Here we report cryo-electron microscopy structures of active Tetrahymena telomerase with telomeric DNA at different steps of nucleotide addition. Interactions between telomerase reverse transcriptase (TERT), TER and DNA reveal the structural basis of the determination of the 5′ and 3′ template boundaries, handling of the template–DNA duplex and separation of the product strand during nucleotide addition. The structure and binding interface between TERT and telomerase protein p50 (a homologue of human TPP17,8) define conserved interactions that are required for telomerase activation and recruitment to telomeres. Telomerase La-related protein p65 remodels several regions of TER, bridging the 5′ and 3′ ends and the conserved pseudoknot to facilitate assembly of the TERT–TER catalytic core.

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Fig. 1: Structure of Tetrahymena telomerase with sstDNA.
Fig. 2: Structure of p50 OB and interactions with TERT.
Fig. 3: Interactions between TERT and template–DNA duplex.
Fig. 4: Structural details for determining template boundaries.
Fig. 5: A model for telomere repeat synthesis.

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Data availability

Cryo-EM density maps have been deposited in the EMDB under accession numbers EMD-23437 (telomerase T3D2), EMD-23438 (telomerase T4D4) and EMD-23439 (telomerase T5D5). The atomic models have been deposited in the PDB under accession codes 7LMA (telomerase T3D2) and 7LMB (telomerase T5D5). The atomic model and cryo-EM density map of telomerase T3D3 were retrieved from the PDB (accession code 6D6V) and EMDB (accession code EMD-7821). Other structures used in this study were retrieved from the PDB with accession codes 2I46 (TPP1 OB), 3KYL (Tribolium TERT-like protein) and 2M22 (TER stem-loop 2). 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.

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Acknowledgements

This work was supported by NIH R35GM131901 and NSF MCB2016540 grants to J.F. and NIH grant GM071940 to Z.H.Z. We acknowledge use of instruments at the Electron Imaging Center for Nanomachines supported by UCLA and by instrumentation grants from NIH (1S10RR23057, 1S10OD018111 and U24GM116792) and NSF (DBI-1338135 and DMR-1548924). Some preliminary data were collected at the Stanford-SLAC Cryo-EM Center (S2C2) supported by the NIH Common Fund Transformative High Resolution Cryo-Electron Microscopy programme (U24 GM129541) and National Center for CryoEM Access and Training (NCCAT) and the Simons Electron Microscopy Center located at the New York Structural Biology Center, supported by the NIH Common Fund Transformative High Resolution Cryo-Electron Microscopy programme (U24 GM129539), and by grants from the Simons Foundation (SF349247) and NY State Assembly. We thank D. Weisman for help with illustration of Fig. 5.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA, USA

    Yao He, Yaqiang Wang, Baocheng Liu, Christina Helmling, Lukas Sušac, Ryan Cheng & Juli Feigon

  2. Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA

    Yao He & Z. Hong Zhou

  3. California NanoSystems Institute, University of California, Los Angeles, Los Angeles, CA, USA

    Z. Hong Zhou

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  1. Yao He
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Contributions

J.F. and Z.H.Z. supervised the project; Y.H. purified telomerase samples, screened cryo-EM grids, and performed cryo-EM data collection and processing; Y.H. and Y.W. built atomic models; Y.H. and B.L. performed telomerase activity assays; C.H., L.S. and R.C. helped with telomerase sample preparation; Y.H. and J.F. wrote the manuscript. All authors contributed to the final version.

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Correspondence to Z. Hong Zhou or Juli Feigon.

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Extended data figures and tables

Extended Data Fig. 1 Biochemical and biophysical evaluation of endogenously purified Tetrahymena telomerase with sstDNA.

a, Silver-stained SDS–PAGE gel of the tandem-affinity-purified telomerase. Serial diluted BSA samples were loaded together to assist concentration estimation of the telomerase sample. Gel image is representative of independent biological replicates (n = 3). b, Direct telomeric DNA extension assays of the purified telomerase bound with different sstDNA primers. A standard telomere addition pattern is observed when using a (GTTGGG)5 or (GTTGGG)3 primer (P1 and P2). However, the translocation of product is inhibited when using (GTTGGG)2GTTGGLGLGLT primer (P3), resulting in a single dark band (red asterisk). GL denotes an LNA nucleotide instead of a DNA nucleotide. The LNA-containing product (red asterisk) migrates slightly slower through the gel as compared to nonmodified DNA. Gel image is representative of independent biological replicates (n = 3). c, Motion-corrected cryo-EM micrograph. d, Representative 2D class averages of telomerase particles. All results from sample purification (a), activity assays (b) and cryo-EM experiments (c) were successfully reproduced at least three times. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 2 Cryo-EM data processing workflow of telomerase with sstDNA (GTTGGG)5 (telomerase T3D2) and the evaluation of the reconstruction.

a, Data processing workflow (detailed in Methods). Soft masks used in data processing are coloured in orange. b, Euler angle distributions of telomerase particles used for the 3.3 Å-resolution reconstruction. c, Local resolution evaluation of the 3.3 Å resolution cryo-EM map shown in surface views (left) and a slice view of the core region (right). d, Superposition of reconstructions P1, P2 and P3 that illustrates the rotation of TEN–TRAP. The three maps were low-pass-filtered to 6 Å and aligned on the TERT ring. p50 (red) and TEB bind to and move together with TEN–TRAP. e, Plot of the FSC as a function of the spatial frequency, with resolution of the final reconstruction indicated. f, FSC coefficients as a function of spatial frequency between model and cryo-EM density maps. Red curve, refined model versus half map 1 used for refinement; green curve, refined model versus half map 2 not used for refinement; black curve, refined model versus the combined final map. The generally similar appearances between the red and green curves suggests no substantial over-fitting. g, Representative cryo-EM densities (grey and mesh) encasing the related atomic models (colour sticks and ribbons).

Extended Data Fig. 3 Detailed interactions between TERT and p65 with TER.

a, Close-up view of motif 3N (amino acids 550–560). Motif 3A helix is bent towards motif 2, and motif 3N in between forms a finger-shaped architecture. b, Ribbon diagram of the TERT–TER ‘interlock’ with TERT domains coloured as indicated. c, Schematic of stem-loop 2, TBE and TBEL nucleotides and their interactions with RBD of TERT. Arrows indicate sites of polar interactions. Bold line represents the stacking interaction between Phe242 and C39. d–f, Structure of TER loop 4 and its interactions with RBD and CTE of TERT and xRRM of p65 (green). g, Rainbow-coloured ribbon diagram of La motif of p65 with secondary structural elements labelled. Positively charged and aromatic residues located on the interface between the La motif of p65 and TER are shown as spheres. h, Electrostatic surface representation of the La motif of p65 and its interactions with TER stem 1, pseudoknot and the 3′ poly-U. The La motifs of p65 in g and h are in the same orientation. i, Schematic of pseudoknot with regions that interact with TERT and p65 indicated. j, Interactions between motif 3 and the template. End of motif 3B and start of motif 3C are in the minor groove of the duplex. k, The eight TER nucleotides that stack inside the catalytic cavity. Cryo-EM densities are shown as transparent meshes. Ideal A-form stacking of eight nucleotides (white) is shown for comparison. Backbone of the final three TER nucleotides in the stacking deviate from ideal A-form conformation.

Extended Data Fig. 4 Interactions between TEN–TRAP and telomerase activity assays.

a, Ribbon representation of TERT with its domains coloured as indicated. Unmodelled regions of TERT are shown as dashed lines, including the linker between TEN and RBD (amino acids 180–215), flexible linkers within RBD (amino acids 252–280), and TRAP (amino acids 664–686). b, Hydrophobic interactions between the distal region of TRAP and the C-terminal helix of TEN domain, which is further stabilized by Gln168 via two hydrogen bonds. c, The extended β-sheet across TEN and TRAP. V791Y (Val791 in human corresponds to Val731 in Tetrahymena) mutation in human TERT that disrupts telomere length maintenance and cell immortalization is located at the interface69. d, e, In vitro-reconstituted telomerase activity assays with TERT mutations on the TEN–TRAP interface. The top panels are SDS–PAGE gels showing the expression level of 35S-Met incorporated TERT mutants. Quantification of activity and RAP for each mutant are shown in bar graphs below. f, g, Quantification of activity and RAP for gel shown in Figs. 1j, 3g. Data are mean ± s.d. from three independent experiments.

Extended Data Fig. 5 Comparison between TERT from Tetrahymena and human, and the TERT-like protein from Tribolium castaneum.

a, Sequence alignment of Tetrahymena TERT (TtTERT) human TERT (hTERT). Secondary structures and conserved motifs of Tetrahymena TERT are shown on top, with unmodelled regions shown as dashed lines. The alignments of the TEN, RBD, reverse transcriptase and CTE domains and TRAP motif were conducted separately with NIH COBALT70 and then merged together. The alignment of CP2 and TFLY region was adjusted manually according to the previously reported alignment37. b, Structural comparison of the TERT-ring of Tetrahymena TERT (colour) and Tribolium TERT-like protein (grey) (PDB 3KYL). Tribolium TERT-like protein lacks TEN, TRAP and TER, and was crystallized with an artificial template–DNA duplex. c, d, Ribbon diagrams of template–DNA duplexes and surrounding structural elements of Tetrahymena TERT (c) and Tribolium TERT-like protein (d). The palm, fingers, primer grip, TH, TL, motif 3 and T are structurally conserved between Tetrahymena TERT and Tribolium TERT-like protein. The ‘bridge loop’ of Tribolium TERT-like protein is in a similar position to that in Tetrahymena TERT; however, the tip residues (Ser82 and Phe83) have no contact with the template–DNA duplex. CP2, which participates in template 5′ boundary definition and template nucleotide guidance in Tetrahymena TERT, appears to be absent in Tribolium TERT-like protein.

Extended Data Fig. 6 Details of p50 OB–TERT and Teb1C–sstDNA interactions.

a–d, p50 OB–TERT interactions. a, Rainbow-coloured ribbon diagram of p50 OB with secondary structure elements labelled. b, Comparison of p50 OB (red) and human TPP1 OB (grey) (PDB 2I46) structures. c, TEN loop (amino acids 121–126) passes through a hydrophobic cleft of p50 OB. This loop is a disordered loop in the TEN-domain crystal structure71.d, Structure-based sequence alignment of p50 OB and human TPP1 OB. The secondary structure elements of p50 OB (red) and TPP1 OB (grey) are shown above and below the sequence alignment, respectively. Residues located at the interface between p50 OB and TERT are highlighted in yellow. The NOB and TEL patch residues in human TPP1 OB are indicated and coloured in yellow. The phosphorylation site Ser111 of TPP1 OB is coloured in green. Scaffold residues of Lα2–β4 shown in Fig. 2a (bottom) are coloured in blue. e–h, Teb1C–sstDNA interactions. e, Path of sstDNA from active site to Teb1C. Low-pass-filtered cryo-EM density of sstDNA (transparent surface) is superimposed with the unfiltered DNA density (green) to better show its flexible region from T20 to G22. Cryo-EM densities corresponding to TERT domains, TER and Teb1C are coloured as in Fig. 1d. f, Sequence of the sstDNA used for the cryo-EM sample preparation with the template and Teb1C-interacting regions indicated. Nucleotides from G1 to G16 are invisible in the cryo-EM map. g, Interactions between sstDNA nucleotides and Teb1C as indicated in e. Intermolecular hydrogen bonds and stacking interactions are shown as dashed yellow lines and black arrows, respectively. h, Specific interactions between Teb1C residues Lys660 and Glu667 and sstDNA nucleotide G19 are shown together with their cryo-EM densities. Hydrogen bonds and their lengths are indicated.

Extended Data Fig. 7 Cryo-EM reconstructions of telomerase with different sstDNA bound.

a, List of sstDNA primers used for cryo-EM sample preparation and their sequences. DNA or LNA nucleotides that pair with the template are underlined. b, Resolution of reconstructions determined by gold-standard FSC at the 0.143 criterion. c, d, Cryo-EM data processing workflow of telomerase T4D4 and T5D5, and evaluations of the final reconstructions. Initial particle screening processes are analogous to those described in the data processing workflow of telomerase T3D2 (Methods) and are omitted for brevity. Focused 3D classifications were performed to separate DNA-free and DNA-bound particles. Short duplexes were observed in both of T4D4 and T5D5 reconstructions. For telomerase T5D5, there is a subset of particles with a longer duplex that we attribute to the greater stability conferred on the duplex by LNA nucleotides at the thermodynamically most stable duplex (dGGGGT·rACCCC) formed in the previous step.

Extended Data Fig. 8 Template–DNA duplexes in telomerase structures at different steps of telomeric DNA synthesis.

Top, sequences of sstDNA primers. TLor GL denotes LNA nucleotide. DNA or LNA nucleotides that pair with the template are underlined. Middle, ribbon diagrams of the duplex, template-adjacent nucleotides, bridge loop, TH and TL superimposed with cryo-EM densities (transparent surfaces). Bottom, schematics of the duplexes. The active site (red star), bridge loop residues (Arg413 and Phe414), and catalytic cavity (grey shade) in different structures are aligned to show the relative positions of the duplex. TER and DNA nucleotides are colour-coded as in Fig. 4.

Extended Data Fig. 9 Structural details of template boundary determination (TBE, TBEL, TREL and TRE) in telomerase T5D5.

a, Telomerase catalytic cavity in telomerase T5D5 with TER (grey) and DNA (green) shown as ribbon and TERT shown as surface (coloured). TBE, TBEL, template, TREL and TRE nucleotides are highlighted as indicated. b–e, Detailed interactions between TERT and TER in regions as indicated in a. Intermolecular hydrogen bonds and stacking interactions are shown as dashed yellow lines and black arrows, respectively. The electrostatic surface of the TRAP–TH channel is shown in d. f, Schematic showing specific interactions between TERT and TREL–TRE as shown in c, e. Nucleotides from A54 to A58 are unmodelled and indicated as dashed orange lines. g, Predicted TRE and TREL conformation when the template is at the +1 position (template nucleotide C48 at the active site). TREL nucleotides C56, U57 and A58 would be fully stretched (about 5–6 Å phosphate-to-phosphate distance for each nucleotide) to span the distance from the neck of the TRAP–TH channel to the anchored TRE.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
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Supplementary information

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Reporting Summary

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Supplementary Video 1

Overall structure of Tetrahymena telomerase

Supplementary Video 2

Architecture of the TERT–TER interlock.

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He, Y., Wang, Y., Liu, B. et al. Structures of telomerase at several steps of telomere repeat synthesis. Nature 593, 454–459 (2021). https://doi.org/10.1038/s41586-021-03529-9

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