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.
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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.
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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.
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Extended data figures and tables
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.
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.
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.
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.
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.
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.
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.
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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
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