Research Article

Structure of Tetrahymena telomerase reveals previously unknown subunits, functions, and interactions

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Science  15 Oct 2015:
DOI: 10.1126/science.aab4070


Telomerase helps maintain telomeres by processive synthesis of telomere repeat DNA at their 3′-ends using an integral telomerase RNA (TER) and telomerase reverse transcriptase (TERT). We report the cryoelectron microscopy structure of Tetrahymena telomerase at ~9 Å resolution. In addition to 7 known holoenzyme proteins, 2 new proteins are identified, which form a complex (TEB) with single-stranded telomere DNA-binding protein Teb1, paralogous to heterotrimeric Replication Protein A (RPA). The p75-p45-p19 subcomplex is identified as another RPA-related complex, CST. This study reveals the paths of TER in the TERT-TER-p65 catalytic core and ssDNA exit, extensive subunit interactions of TERT essential N-terminal domain, p50, and TEB, and new subunit identities and structures, including p19 and p45C crystal structures, providing unprecedented structural and mechanistic insights into telomerase holoenzyme function.

Telomerase is a ribonucleoprotein complex that extends the telomere DNA at the 3′ ends of linear chromosomes, thereby counteracting loss of DNA from replication and nucleolytic processing (1, 2). While telomerase is largely inactive in somatic cells, it is active in stem cells and highly active in most cancer cell lines, where its activity is necessary for their immortal phenotype (35). Thus it is an important regulator of aging, tumorigenesis, and stem cell renewal. Telomerase uses a template contained within the integral telomerase RNA (TER) and a unique telomerase reverse transcriptase (TERT) to synthesize multiple copies of the G-strand telomere repeat, TTGGGG in ciliates and TTAGGG in vertebrates. Telomerase recruitment to telomeres is cell cycle regulated, where its activity requires interplay between telomere end-protection and telomerase proteins (6). Telomere end maintenance also requires coordinated recruitment of telomerase and DNA polymerase α for synthesis of G- and C-strands, respectively (7, 8). In humans, telomerase is recruited to telomeres by components of shelterin (6). Specifically, the TERT essential N-terminal (TEN) domain interacts with TPP1 (911), which in complex with protection of telomeres POT1 is a processivity factor (6, 12, 13). Budding yeast telomerase holoenzyme subunit Est3 is a structural homolog of the oligosaccharide/oligonucleotide binding (OB)-fold of TPP1 (14) and like TPP1 interacts with the TEN domain (15). In addition to recruiting telomerase, the human TPP1-POT1 complex recruits the Replication Protein A (RPA)-like CST (CTC1-STN1-TEN1) complex. RPA binds sequence non-specifically to ssDNA and plays a central role in DNA replication and repair through protein recruitment (16); CST complexes have been proposed as telomere-specific RPAs (17). CST stimulates DNA polymerase α for C-strand synthesis and has apparently diverse other functions in different organisms (8, 1820). Mammalian CST acts as an inhibitor of telomerase action, determines telomeric 3′ overhang structure, and plays broader roles in telomere duplex replication and genome-wide replication restart (8, 18, 20, 21). Budding yeast CST (Cdc13-Stn1-Ten1) subunit Cdc13 recruits the telomerase holoenzyme to telomeres via interaction of Cdc13 with telomerase subunit Est1, associated to Est3 (2224).

Telomerase can synthesize telomere repeats in vitro with only TERT and TER, but physiological function requires a variety of other proteins (25, 26). Unlike yeast and mammalian telomerase, Tetrahymena telomerase is constitutively assembled (27, 28), making it possible to purify and study all holoenzyme components in a stable complex (29). In addition to TERT and TER, Tetrahymena telomerase holoenzyme contains six other known proteins, p65, p75, p45, p19, p50 and Teb1 (28). TER contains a template/pseudoknot (t/PK) domain enclosing a template with sequence complementarity to ~1.5 telomere repeats and a separate activating domain (30). While TER is essential for activity, the physical arrangement of TER on TERT has remained largely unknown. TERT comprises a telomerase RNA binding domain (TRBD), reverse transcriptase (RT), and C-terminal extension (CTE) that form a TERT “ring” (31) and a separate TEN domain (32) important for DNA handling and telomere repeat addition processivity (RAP) (6, 26). p65 binds the TER activating domain (stem-loop 4, SL4) inducing a large bend in the RNA that facilitates assembly of TERT with TER to form the RNP catalytic core (33, 34). Teb1 is a paralog of human RPA70, the large subunit of RPA (16, 28). Direct single-stranded telomere DNA binding by Teb1 is necessary for telomerase recruitment to telomeres (27), where it may compete for binding with the Tetrahymena telomere end-binding Pot1a (35). p75, p45, and p19 form a ternary complex whose structure and function remain largely unknown (28). The 25 Å resolution negative-stain electron microscopy (EM) structure and individual subunit affinity labeling revealed the overall architecture of Tetrahymena telomerase, where TERT occupies the center of the holoenzyme with p65 bound to SL4 below (29). p50, which forms a central hub linking the TERT-TER-p65 catalytic core, p75-p45-p19, and Teb1, can greatly increase RAP and with Teb1 dramatically enhance the rate and processivity of long-product synthesis (29, 36). A detailed mechanistic understanding of telomerase and its interaction at telomeres has been hampered by lack of structural models, due to difficulties in obtaining samples of sufficient quantity and quality, as well as subunit complexity and flexibility and low sequence identity among subunits from different organisms. Here we report 9.4 and 8.9-Å cryoelectron microscopy (cryoEM) structures of Tetrahymena telomerase holoenzyme. Combining the cryoEM structures with data from X-ray crystallography, NMR spectroscopy, negative-stain EM, and mass spectrometry, we discovered two RPA related complexes: an RPA paralog TEB, comprising Teb1 and previously undetected proteins Teb2 and Teb3, and a CST complex comprising the previously structurally uncharacterized p75-p45-p19. Both are tethered to p50, a potential structural and functional homolog of TPP1, which in turn binds TERT. A pseudoatomic model of the TERT-TER-p65 catalytic core reveals the path of TER on TERT and location of the TEN domain, and an exit path for the telomeric repeat DNA is proposed, providing insights into enzyme mechanism.

CryoEM reconstruction and overall structure

Telomerase holoenzyme endogenously assembled in Tetrahymena thermophila was affinity purified from a strain bearing a C-terminal 3×Flag (F) and tandem protein A (ZZ) tag on TERT (TERT-FZZ) (29). CryoEM specimens of Tetrahymena telomerase holoenzyme were prepared using holey carbon grids and imaged using a Gatan K2 Summit direct electron detection camera with drift correction (fig. S1). Particles showed various orientations, which are required for 3D reconstructions, besides the preferred “front view” (fig. S2 and methods). Using 40,754 particles, we obtained the intact structure of Tetrahymena telomerase holoenzyme at an overall resolution of 9.4 Å (Fig. 1, A to C, and fig. S1). The negative-stain EM study revealed that the p75-p45-p19 subcomplex is conformationally dynamic (29), which is also apparent in the cryoEM images (fig. S1, C and D). Therefore, to improve the resolution of the less flexible region, we used a soft mask to exclude the p75-p45-p19 subcomplex from the cryoEM structure refinement. The resulting reconstruction has an overall resolution of 8.9 Å with distinguishable secondary structure elements of proteins and RNA (Fig. 1, D to F, and figs. S1 and S3).

Fig. 1 CryoEM reconstructions of Tetrahymena telomerase holoenzyme.

(A) Front view of 9.4-Å cryoEM map with catalytic core, Teb1C-Teb2N-Teb3 (TEB), p75C-p45N-p19 (CST), and p50N colored blue, gold, copper, and red, respectively. (B) Front view of the 9.4-Å cryoEM map (gray surface) with pseudoatomic models of TERT-TER-p65 catalytic core, Teb1C-Teb2N-Teb3, and p75C-p45N-p19. (C) Side view of the cryoEM map and pseudoatomic models shown in (B). (D) Back view of 8.9-Å cryoEM map with catalytic core, Teb1C-Teb2N-Teb3 (TEB), p75C-p45N-p19 (CST), and p50N colored as in (A). (E) Back view of 8.9-Å cryoEM map with pseudoatomic models of TERT-TER-p65 and Teb1C-Teb2N-Teb3. (F) Close-up views of fitting of TER helical domains PK, SL2, S1, and SL4 and TEN domain into the 8.9-Å cryoEM map. TERT domains are TEN (cyan), TRBD (dark blue), RT (violet, with IFD labeled violet), and CTE (light blue). Other proteins and TER are colored individually.

Using the features of the secondary structure elements, we were able to rigid-body fit the available atomic-resolution and homology models of protein domains and RNA helical elements (table S1) unambiguously into the 8.9-Å cryoEM map except for p75-p45-p19 homologs that were fit into the 9.4-Å cryoEM map (Fig. 1 and fig. S3). All helical elements of TER, i.e., stem 1 (S1), stem-loop 2 (SL2), SL4, and the pseudoknot (PK), are clearly visible with distinct grooves (Fig. 1F), and the path of the single-stranded regions can be approximately traced although bases cannot be discerned. This allowed us to build a pseudoatomic model of the TERT-TER-p65 catalytic core and Teb1C. The locations of these subunit domains are the same as modeled in the negative-stain EM map within experimental resolution (29), with two exceptions: Teb1C [whose C terminus was correctly localized by Fab labeling (29)] is behind the TEN domain where the PK had been placed, while the PK is on the opposite side of TERT by the CTE. In addition, as discussed below, Teb1 forms a heterotrimer with two newly identified proteins, whose location was previously assigned to Teb1C. The p75-p45-p19 ternary complex, whose subunit boundaries could not be determined in the negative-stain EM map (29), is revealed to contain an RPA-like heterotrimer of OB-fold proteins with the domain structure of a CST complex. Overall the pseudoatomic models of the TERT-TER-p65 catalytic core, Teb1-Teb2-Teb3, and p75-p45-p19, which are all linked to p50 in the cryoEM maps, reveal an intricate network of interactions between the subunits.

The TER t/PK domain encircles the TERT ring

Tetrahymena TERT TRBD-RT-CTE forms a ring-shaped structure and was modeled using the crystal structure of a partial Tetrahymena TRBD (residues 259-265, 277-519) (37) and homology model based on the RT-CTE in Tribolium (flour beetle) TERT (Fig. 2A) (31, 38). Tribolium TERT lacks the TEN domain, and therefore the position of TEN relative to the TERT ring has remained speculative. Fitting of the crystal structure of the Tetrahymena TEN domain (32) into the cryoEM map (Fig. 1F) revealed it is on the active site side of the TERT ring, stacked over the CTE (Fig. 2A and fig. S4A). Residues 640-740 of the Insertion in Fingers Domain (IFD) in the RT, which are not modeled as the Tribolium TERT IFD is much shorter than in Tetrahymena and other organisms (39, 40), appear to be near the TEN domain (Figs. 1B and 2A). The evolutionarily non-conserved sequence (residues 178-258) that connects TEN to the TERT ring has no available atomic structure and appears undefined in the cryoEM maps. Consistent with this lack of a fixed conformation, the TEN domain can be assembled in trans with TER and the TERT ring to form an active catalytic core without linker residues 196-215 (41, 42).

Fig. 2 Structure of the TERT-TER-p65 catalytic core.

(A) Model structure of TERT with putative IFD (light violet), active site catalytic triad residues (red), linker (dotted blue) and TRE shown. (B) Secondary structure of TER. Locations of CTE and TRBD on TER are indicated with dashed lines. (C) Pseudoatomic model of TERT ring-TER showing TBE-Template-TRE and L4 on TERT, viewed from the active site side of TERT. (D) 8.9-Å cryoEM map with pseudoatomic model of TER, front view. (E) Pseudoatomic model TERT-TER-p65 catalytic core, front view. The putative location of IFD is shown as violet oval. (F) Region of 8.9-Å cryoEM map and pseudoatomic model showing L4 at the interface of TRBD (helix α8) and CTE (helix α22a, residues 975-983). The numbering of Tetrahymena TERT secondary structure elements follows that of Tribolium TERT structure.

Tetrahymena TER comprises a circular t/PK domain, closed by a short S1, that contains the template, 3′-flanking template recognition element (TRE), template boundary/TERT binding element (TBE), SL2, and PK, and an activating domain, SL4, which is connected to S1 by a short single-stranded linker (Fig. 2B) (30). TER was modeled starting with fitting previously determined (SL2, p65xRRM:S4, L4) and a new (PK; table S4) NMR structures into the cryoEM density (see methods). The core t/PK domain encircles the TERT ring approximately perpendicularly, such that the template extends across the bottom of the RT domain on one side of the TERT ring while the PK is on the other side next to the CTE (Fig. 2, C to E, and fig. S4). The bottom of S2 is adjacent to the TRBD CP-motif, and the 3′ TBE is also near the T-motif (Fig. 2C and fig. S4), as predicted (29, 37).

The PK, a conserved element of TER that has been proposed to contribute directly to catalysis (43), is on the opposite side of the TERT ring and far (~20 Å) from the active site (Fig. 2, C and E, and fig. S4A). The PKs of human and yeast TERs contain conserved interactions that stabilize the PK fold through formation of base triples between loops and stems that are important for activity (4345). The smaller Tetrahymena telomerase PK has two base triples in the NMR structure at 10°C, and is not stably folded at higher temperature in the absence of TERT (46). Based on the position of the PK on TERT, we conclude that rather than contributing directly to catalysis, the correct PK fold is important for proper positioning of TER on TERT. It is plausible that the PK acts like a watchband ratchet clasp during catalytic core assembly, partially or completely unfolding to allow the TER t/PK domain to fit around the TERT ring and then “locking” onto the TERT ring by folding of the PK. An indirect effect on assembly may explain why mutations that either stabilize or destabilize the PK, at least for human TER, affect telomerase activity in vitro (45).

Outside of the t/PK domain only L4 in S1/SL4 contacts TERT (Fig. 2, C, E, and F; and fig. S4A). The p65 La-RRM1 binds the 3′ polyU tail (33, 47). La-RRM1 has low resolution in the cryoEM map likely due to flexibility or partial disassociation (fig. S1), but appears to be associated with both the 3′ tail and the 5′ end of S1, thereby possibly linking the 3′ and 5′ ends of TER (Fig. 2E). p65 C-terminal xRRM2 (33) binds to and bends S4, inserting L4 between the end of TRBD helix α8 and a short helix from the CTE (α22a) that does not exist in Tribolium TERT but has well-defined density in the 8.9-Å cryoEM map (Fig. 2F). In complex with p65, SL4 stimulates activity, hierarchal assembly and holoenzyme stability (33, 34, 47, 48). L4 binds with high affinity to the TRBD, but its specificity for the CTE remains unknown. A hypothesis consistent with the above and location of L4 far from the active site and PK is that L4 stabilizes a closed conformation of the TERT ring via specific interactions with TRBD and CTE.

The TER single-stranded region containing the TRE–template–3′TBE spans the active site side of the TERT ring between the CTE and TEN domain, and across the RT and the TRBD T/CP pocket, respectively (Fig. 2C). The cryoEM structure is of telomerase in the apo state prior to binding telomeric ssDNA. The TRE nucleotides, on the 3′ side of the template, pass between the CTE and the TEN domain and could contact the TEN domain. There is clear cryoEM density for the 3′ half of the template only (fig. S3E), suggesting flexible positioning of the 5′ residues close to the TBE. Based on the distance from TER S2, the template appears to be positioned near where it would be at the end of synthesis of a telomere repeat, i.e., the 5′ end of the template is closest to the active site. It also appears to be ~7 Å displaced from its position at the active site in the crystal structure of Tribolium TERT in complex with an RNA-DNA hybrid helix (38), as proposed for the strand separation step (49). The position of S2/TBE on TERT relative to the 5′ end of the template suggests a structural mechanism for template boundary definition. The single stranded residues of the TBE, which flank S2, wrap on either side of the TERT ring (Fig. 2C and fig. S4B). At the end of a telomere repeat synthesis, the S2 and TBE-TRBD interactions could act as a stop to prevent residues beyond the template from being pulled into the active site (Fig. 2C and fig. S4B).

Teb1 forms an RPA-like complex with two newly identified proteins

Although Teb1 has four OB-folds, NABC (50), in the negative-stain EM class averages of Tetrahymena telomerase only the Teb1C domain and in some cases very weak density for Teb1B was apparently visible (29). Fitting of Teb1C into the cryoEM map revealed unaccounted-for density in the “knob” next to Teb1C (Fig. 3 and fig. S5A). An exhaustive analysis of the potential positions and fittings of other holoenzyme proteins revealed no known candidates for the “knob” density (fig. S5A). Since Teb1 is an RPA70 paralog we investigated whether this part of the cryoEM map might contain paralogs of the other two RPA subunits RPA32 and RPA14. We found that the crystal structure of the RPA heterotrimeric core complex (RPA70C-RPA32N-RPA14) (51) fit remarkably well into the cryoEM map, with RPA70C positioned in the cryoEM density for Teb1C and the RPA32N and RPA14 OB-folds in the “knob” (Fig. 3). RPA trimerization requires formation of a 3 α-helix bundle from the C-termini of RPA70, RPA14, and RPA32N. The predicted C-terminal α-helix of Teb1C is disordered in the crystal structure (50). However, in the cryoEM map there is clear density of an α-helix involved in the trimerization and near the C terminus of Teb1C, which we modeled in from the RPA70 structure (Fig. 3 and fig. S3H).

Fig. 3 Identification of two new holoenzyme proteins, Teb2 and Teb3.

Two views of “knob” and Teb1C region of 8.9-Å cryoEM map with model of Teb1C-Teb2N-Teb3 based on fitting of RPA70C-RPA32N-RPA14 into the cryoEM map followed by replacement of RPA70C with Teb1C except for the RPA70C C-terminal α-helix. The TEN domain is shown in cyan. The three helix bundle between the C-terminal residues of Teb1C, Teb2N, and Teb3 (modeled from RPA) is highlighted with dashed lines. Inset shows corresponding back view of the holoenzyme for reference.

To confirm the presence of putative Teb1 heterotrimer proteins in the holoenzyme, telomerase purified from the TERT-FZZ strain was subjected to analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). All 7 of the known Tetrahymena telomerase proteins plus two additional hypothetical proteins, TTHERM_001113129 and TTHERM_00439320 ( were detected with high confidence (table S2A). PSI-BLAST (52) searches of corrected cDNA sequences of these new 31 kDa and 14 kDa proteins showed homology with predicted RPA32 and RPA14 homologs, respectively. Secondary structure predictions by Jpred4 (53) are consistent with an OB-fold for the 14 kDa protein and an N-terminal OB-fold and C-terminal WH domain for the 31 kDa protein, and multiple sequence alignments of these domains with RPA32 and RPA14 orthologs show high similarity (fig. S6). We previously observed that telomerase purified from a Tetrahymena strain containing N-terminally ZZF-tagged p50 (F-p50 telomerase) lacks the “knob” (fig. S5B) and has low RAP, which was attributed to loss of Teb1 induced by the ZZF-tag on p50 (29). We analyzed F-p50 telomerase by LC-MS/MS and found that the two newly identified proteins and Teb1 are absent (table S2B), confirming that the two new proteins identified by mass spectrometry are indeed located at the “knob” in the cryoEM map and form a complex with Teb1. We conclude that there are two additional previously undetected proteins in Tetrahymena telomerase holoenzyme that form a heterotrimer with Teb1, here named TEB, paralogous to the ssDNA binding RPA except specific for telomeric G-strand DNA. In Tetrahymena, only the large subunit of RPA, Rfa1, has been identified (54). Transcript expression levels of the two new Teb1-binding proteins, here named Teb2 and Teb3, are much higher than other telomerase proteins and more similar to the level of Rfa1 as judged by expressed sequence tag abundance (55), suggesting the intriguing possibility that these two subunits may be shared between Tetrahymena RPA and TEB.

Multiple interactions between TEN, TEB, and p50 regulate telomerase activity

The TEN domain has been identified as a major determinant of telomerase activity (6, 42). In addition to its potential TRE interaction (Fig. 2), the cryoEM map reveals that TEN is neighbored by p50, Teb1C, Teb2N, and the IFD (Fig. 4, A and B). TEN, Teb1C, and p50 contact each other in a triangular arrangement. The Teb1C-TEN interaction is clearly defined in the pseudoatomic models (Fig. 4C). A Teb1 F590A/F648A double mutant was previously shown to ablate purification of telomerase by Teb1-FZZ expressed in cells (27). These two residues of Teb1 are on the Teb1C-TEN interface (Fig. 4C), accounting for the loss of function. In the cryoEM map, the density of TEN is also in contact with that of Teb2. TEN residues 77-87, which are missing in the crystal structure due to disorder (32), might form a structured interface with Teb2N in the holoenzyme (Fig. 4D). In in vitro telomerase reconstitution activity assays, which lacked Teb2 and Teb3, deletion of the Teb1C putative C-terminal α-helix (ΔCTαH) had little effect on activity (27, 54). However, purification of Teb1(ΔCTαH)-FZZ expressed in vivo did not recover any telomerase activity or holoenzyme subunits, indicating no in vivo telomerase assembly with Teb1(ΔCTαH) (27). A likely explanation for this is that Teb2 and Teb3, which interact with Teb1 C-terminal α-helix, stabilize association of Teb1 with telomerase via Teb2-TEN interaction.

Fig. 4 Subunit interactions between p50, TEN, IFD, Teb1C, Teb2N, and Teb3.

(A) Region of 8.9-Å cryoEM map showing density linking p50N, TEN, IFD, and TEB. Inset shows schematic of interactions. Dotted lines are inferred interactions from the cryoEM density and atomic models fitted to the cryoEM map. (B) Interactions between p50N (red, cryoEM density), TEN, Teb1C, and Teb2N. TEN residue sidechains K90 corresponding to human TEN domain residue K78 that interacts with TPP1, R137, and the β3-β4(112-120) hairpin are highlighted in violet. (C) Interactions between TEN and Teb1C. Teb1C F590/F648 residues (orange stick) that together abrogate Teb1C-TEN interaction and the TEN β3-β4 hairpin (violet) are indicated. TEN residues 122-127 disordered in the crystal structure are shown as dotted lines in (B) and (C). (D) Interactions between TEN and Teb2N. TEN residues 77-87 disordered in the crystal structure are shown as dotted lines. Pseudoatomic models of TEN, Teb1C, Teb2N interactions are based on crystal structures of TEN, Teb1C, and RPA32N, RPA14 fit into the 8.9-Å cryoEM map. (E and F) In vitro reconstitution telomerase activity assays for (E) effect of TEB proteins and (F) TEN domain mutations on catalytic core assembled with p50N30. TEN domain mutations were tested without (lanes 1-4) and with (lanes 5-8) TEB.

To provide biochemical evidence for these interactions, we investigated whether Teb2-Teb3 had any effect on telomerase activity by in vitro reconstitution of complexes with individual TEB subunits expressed in rabbit reticulocyte lysate (RRL) (Fig. 4E). Activity assays here were designed to sensitize for improved holoenzyme assembly of Teb1 by the use of a very low level of Teb1 expressed in RRL, rather than a saturating concentration of purified bacterially expressed protein, and by purification of RNP from unbound Teb1 prior to the activity assay. Addition of Teb1 to the catalytic core plus p50 greatly increases RAP, as expected from previous studies (29, 36). Co-expression of Teb2-Teb3 with Teb1 additionally increased overall activity, consistent with additional stabilization from synergistic interaction of Teb2 with the TEN domain and Teb1C. Addition of Teb2/Teb3 alone, without Teb1, provided no activity enhancement. The cryoEM structure, activity assays, and in vivo functional studies (27) discussed above together support a crucial physiological significance of the TEN-TEB protein interaction network.

The cryoEM density of p50N appears to contact the TEN domain, as well as Teb1C, the IFD, and p75-p45-p19 (Figs. 1, B and E, and 4A). p50, which has no known sequence or structural homology to other proteins, has a 30 kDa N-terminal domain (p50N) that is required for the high level of processive repeat synthesis conferred by p50 binding to the catalytic core (36). Only p50N is apparently visible in the class averages and 3D reconstructions of negative-stain EM images (36) and also in the cryoEM maps. Like TPP1, p50 contacts the TEN domain (Fig. 4, A and B) and like TPP1-POT1 (13), p50-Teb1 greatly increases RAP (36). These data suggest that p50 could be a structural and functional ortholog to vertebrate TPP1. Although TPP1 was initially identified as a telomere binding protein complex with POT1 bound to the G-strand overhang to block telomerase access, it has emerged as also being the direct mediator of telomerase recruitment, activation, and homeostasis set point regulation (6, 7, 56, 57). We fit the structure of TPP1 OB-fold into the 8.9-Å cryoEM map, but only the characteristic OB-fold β-barrel matches well to the cryoEM density (fig. S3, M to O). This is not surprising, due to the expected co-folding of p50 loops and helices with its interaction partners. While the fitting does not provide definitive structural homology to TPP1, it does provide evidence that p50N is an OB-fold.

The TEN domain has an extensive interface with p50 that apparently includes residues 122-127 missing in the crystal structure due to disorder (32) (Fig. 4B), which may co-fold with p50. We tested whether other TEN domain interactions with p50 inferred from the pseuodoatomic model and cryoEM map were important for RNP activity stimulation by p50, in a manner similar to TEN interactions with TPP1 and Est3. A cluster of residues on human TPP1 (and Est3), called the TEL patch, has been identified as the interaction surface with TEN (911, 14, 56, 57). This interaction is essential for human telomerase recruitment to telomeres. Human TEN residues whose substitution disrupts the interaction of telomerase with TPP1 without greatly affecting catalytic activity have been identified; among these a direct interaction between K78 and TPP1 has been demonstrated (57). The equivalent Tetrahymena TEN residue based on homology modeling of human TEN with the crystal structure of Tetrahymena TEN is K90, which is located near the density for p50 in the cryoEM map (Fig. 4B). We investigated whether K90A at the putative TEN-p50 TEL patch interface imposes a defect in p50 stimulation of telomerase activity using our in vitro telomerase reconstitution activity assay (Fig. 4F). K90A had a modest but significant effect on overall activity. The same results were obtained for TEN R137A, which is also at the TEN-p50 interface in the cryoEM map. These results support similar interactions between Tetrahymena TEN-p50 and human TEN-TPP1. TEN K90A and R137A did not abrogate TEB stimulation of high RAP (Fig. 4F), consistent with direct TEN domain interactions with TEB as well as p50-bridged TERT-TEB association. TEN residues 112-120 form a β-hairpin, unique to Tetrahymena TEN, that inserts at the interface between p50, Teb1C, and Teb2N (Fig. 4B). An adjacent block sequence substitution TEN(108-113NAAIRS) abolishes p50 binding (42). Here we replaced the β-hairpin with GSSG. This substitution abolished p50 activity stimulation even with added TEB, consistent with disruption of both p50 and TEB interactions, but did not affect catalytic core activity. Taken together, these results validate the network of TEN-p50-Teb1C-Teb2N interactions proposed based on the cryoEM structure (Fig. 4, A to D) and provide supporting evidence that p50 is a structural and functional paralog of TPP1.

CryoEM density that we attribute to the IFD appears to independently contact p50 and the TEN domain N terminus which is disordered in the crystal structure (32) (Figs. 1B and 4A). The IFD is unique to TERTs and is important for the translocation step required for RAP (39, 40). We suggest that the human TERT IFD may have parallel, as yet undetected, interactions with the TPP1 OB-fold and TEN domains.

p75-p45-p19 is a CST complex

We determined a 2.3-Å X-ray crystal structure of p19 (table S3), which revealed an OB-fold most structurally homologous to human Ten1 (Fig. 5A and fig. S7), suggesting the possibility that p75-p45-p19 might be a CST- or second RPA-like complex. We found that human RPA70C-RPA32N-RPA14, which fit the cryoEM density of TEB as described above, also fit equally well in the density at the “tip” of the p75-p45-p19 subcomplex in the 9.4-Å cryoEM map (Fig. 5B). For our model, RPA14 was then replaced with p19 crystal structure, except for its unstructured C terminus, which forms an α-helix in a three-helix bundle with the other two proteins in the RPA complex (Fig. 5B). p19 α2 and α3, which are lacking in RPA14, fit well into the cryoEM density at the very end of the “tip.” Significantly, the positions of the C-terminal α-helices of RPA70C and RPA14 correspond to the locations of the C-termini of p75 and p19 determined by Fab labeling in negative-stain EM (Fig. 5B) (29). We determined by co-purification and limited proteolysis that p45 is comprised of an N-terminal domain (p45N) which binds p19, and an independently folded C-terminal domain (p45C) (see methods); these results are consistent with the hypothesis that p45 is a Stn1 or RPA32 homolog. Previous attempts to locate the position of p45 in the holoenzyme by Fab labeling of its C terminus were unsuccessful, as Fab was apparently not visible in negative-stain EM class averages of telomerase holoenzyme (29). Based on the above we hypothesized that p45C might be connected to p45N in the p75-p45-p19 subcomplex via a flexible linker, and thus Fab-bound p45C was not visible in holoenzyme class averages due to positional flexibility relative to the holoenzyme. We screened >2000 negative-stain EM particles of Fab-labeled p45-F telomerase. The majority had a cluster of 3 Fab (the 3×FLAG tag can bind up to 3 Fab) located in various positions ~100 Å distant from the location of p45N (Fig. 5D and fig. S8). The C-terminal domains of RPA32 and Stn1 are comprised of winged-helix (WH) and tandem winged helix (WH1-WH2) domains, respectively (17, 19, 58, 59). We determined a 2.4-Å crystal structure of p45C (table S3) and found that it comprises WH1-WH2 domains (Fig. 5C). The OB-fold followed by 2 WH domains suggests that p45 is a Stn1 homolog (17, 19, 58, 59). Structural alignments (fig. S7), sequence alignments (fig. S6), and phylogenetic cluster analysis (fig. S9) of p19 and p45 domains also support their identification as Ten1 and Stn1 orthologs, despite low sequence homology of CST proteins between kingdoms. In the 9.4-Å cryoEM map, the remaining unmodeled density in the p75-p45-p19 complex, which appears to contain at least two additional OB-folds and contacts p50, can only be attributed to p75 (Fig. 5B). Consistent with this, among p75-p45-p19, p75 is necessary and sufficient to bind p50 in vitro (36). There are distinct differences between p75 and Teb1 in p50 interaction and effect on telomerase activity. The cryoEM maps show that p75 contacts p50 with a domain near its N terminus; in contrast, Teb1 binds p50 and the TEN domain with its C-terminal OB-fold. How the potential p75 ortholog CTC1 interacts with telomerase is unknown, but it is interesting to note that the functionally divergent Cdc13 binds yeast telomerase holoenzyme protein Est1 through a recruitment domain adjacent to its N-terminal OB-fold (8, 17, 24). In in vitro reconstitution assays, addition of p75 (or p75-p45-p19) to the catalytic core plus p50 stimulates activity slightly but without increase in RAP; in contrast, Teb1-p50 interaction stimulates high RAP (29, 36). We conclude that p75-p45-p19 is structurally and functionally distinct from TEB, and most similar to a CST complex. CST has been identified in yeasts, plants, and mammals (8, 19); this is the first evidence of a CST in ciliates.

Fig. 5 Identification of p75-p45-p19 as a TtCST complex.

(A) Crystal structure of p19, an OB-fold. (B) Model of p75C-p45N-p19 based on fitting of RPA70C-RPA32N-RPA14 into the 9.4-Å cryoEM map followed by replacement of RPA14 by p19 except for the RPA14 C-terminal helix. p19 α2 and α3 account for the density at the end of the “tip.” Gold and orange arrows point to the locations of p19 and p75 C-termini, respectively, previously determined by Fab labeling in negative-stain EM (29). The three helix bundle between the C-terminal residues of p75, p45N, and p19 (modeled from RPA) is highlighted with dashed lines. (C) Crystal structure of p45C, a WH-WH domain. β5 is domain swapped from a neighboring protein in the crystal lattice. (D) Negative-stain EM images of 4 typical p45-Fab labeled telomerase holoenzyme particles, showing a cluster of 3 Fab bound to the C terminus of p45 at various locations near the holoenzyme. Corresponding outlines of telomerase and Fabs are shown in black and blue, respectively, below, with red dots indicating the p45C (attached to 3 Fab) and p45N (on telomerase) domains and dotted line the linker. The side length of each image box in (D) is 44 nm.

The telomeric DNA exits the template toward Teb1C

To obtain information on the path of telomere DNA on telomerase we prepared holoenzyme bound to a short telomeric DNA, biotin-5′-d(GTTGGG)2GTLTLGLGLG, where TL and GL are locked nucleic acid (LNA) nucleotides (60). For this DNA, 6 nts should bind the template and 13 nts should extend out from its 3′ end. We then bound biotin with streptavidin, linking two telomerase holoenzymes together via the biotin binding sites in each streptavidin tetramer. Visualization of these “dimers” of telomerase holoenzyme by negative-stain EM confirms that the telomere DNA is bound (Fig. 6, A to C). The class averages and 3D reconstructions show clearly visible density of streptavidin located near Teb1C-Teb2N and the putative location of Teb1B (29) on the “backside” of telomerase holoenzyme (Fig. 6, B to D). These results reveal that telomeric DNA exits the template from the “backside” of telomerase and toward Teb1C (Fig. 6D). Furthermore, within the resolution (~30 Å) of negative-stain EM, there is no large scale structural rearrangement of TERT between the apo structure without DNA and the structure with telomeric DNA bound to the template, consistent with crystal structure of Tribolium TERT without TER (31) versus with an RNA-DNA hairpin mimicking a template-DNA hybrid (38).

Fig. 6 Telomere ssDNA exits from the “backside” of Tetrahymena telomerase holoenzyme.

(A) Negative-stain EM class averages of telomerase holoenzyme dimerized by primer-biotin-streptavidin-biotin-primer. The side length of each image box is 42 nm. (B and C) Random conical tilt (RCT) reconstruction of “dimeric” telomerase holoenzyme. One of two telomerase holoenzymes in (C) shows uninterpretable features due to flexible positioning of the two holoenzymes relative to each other. (D) The 9.4-Å cryoEM map of telomerase holoenzyme (gray surface) and the position of primer-attached streptavidin (black dashed circle) as identified by negative-stain EM RCT reconstruction in (B) and (C). Teb1B (purple) and ssDNA (green) exiting through Teb1C are modeled by fitting the crystal structure of RPA:ssDNA complex (PDB ID: 4GNX).

Implications for telomerase catalytic activity and association with telomeres

The cryoEM structures, p45C and p19 crystal structures, PK NMR structure, domain modeling, and mass spectrometry data presented here reveal a complex RNP composed of 3 ternary complexes, tethered by p50: a TERT-TER-p65 catalytic core; the RPA paralog TEB comprising Teb1-Teb2-Teb3; and a Tetrahymena CST complex comprising p75-p45-p19. TER wraps around the TERT ring and interacts with all 4 domains of TERT. The TEN domain is close to TER, the TERT ring IFD, p50, Teb1, and Teb2, signaling its importance in the catalytic cycle of telomerase. In addition to the protein domains visible in the cryoEM map, there are several domains that are not observed due to flexible positioning or intrinsic disorder in the absence of binding partners; these are Teb1 N, A, and B, putative Teb2C, p45C, p50C, p65N, and possibly p75N (schematized in Fig. 7A). Teb1A and Teb1B bind telomere DNA (54), while the others (except for p65N) may recruit proteins involved in C-strand synthesis, nucleolytic end-processing, unwinding of G-quadruplexes, telomere end-binding, and DNA repair, thereby coordinating telomere maintenance.

Fig. 7 Schematics of the complete Tetrahymena telomerase holoenzyme and DNA exit path.

(A) Arrangement of the subunits and domains of Tetrahymena telomerase catalytic core, TEB, and CST complexes tethered to p50, shown as front view. Domains connected by flexible linkers and not seen in the cryoEM map are shown as oval outline. (B) Arrangement of the holoenzyme with proposed path of telomere ssDNA, shown as back view.

In the catalytic core, the TER interactions with TERT are exclusively from the t/PK domain and L4. Since a t/PK and a stem terminus element like L4 are almost universally found in other organisms (2, 30) and TERT is highly conserved, Tetrahymena telomerase provides general insights into TER-TERT interactions and assembly. Human TER has a much larger pseudoknot, but the region containing the tertiary stem-loop interactions is comparable in size and likely interacts with TERT at the same location near the CTE, and the human TER t/PK could similarly encircle the TERT ring. This would place the other end of the pseudoknot close to the TEN domain, as implicated biochemically (41). Vertebrate telomerase contains a conserved three-way junction, CR4-CR5 domain, that binds the TRBD (61) and has a stem-loop (P6.1) that is critical for activity (62). P6.1 loop likely inserts at the TRBD-CTE interface (61, 63) similarly to Tetrahymena TER L4.

In the cryoEM structure of apo telomerase presented here, the template is apparently positioned with the 5′ end near the active site. This places the 3′ end of the template close to Teb1C, where the telomeric DNA binds as it exits the holoenzyme. We suggest that this would facilitate (re)-binding of the primer to the template 3′ end prior to translocation. Based on homology with the crystal structures of RPA-ssDNA complex (51), a model of Teb1AB bound to ssDNA based on the Pot1AB-ssDNA complex (50), and the position of the 5′ end of telomeric DNA on telomerase revealed by streptavidin labeling, we can model a possible path of the telomere DNA on telomerase (Fig. 7B). While the DNA is shown going straight from the template to Teb1, it is possible that during the catalytic cycle the DNA strand may interact with TEN, TRBD and/or CTE. Within the catalytic core, the TEN domain has been implicated to play a role in single-stranded DNA handling and act as an anchor site (32, 64) and/or in active site use of the DNA:template RNA hybrid (41). The TEN domain is positioned so that the exiting end of the short DNA:template helix could contact its (unmodeled) N- and C-termini at the end of a complete telomere repeat synthesis, where this interaction could facilitate template-DNA strand separation and help direct the DNA toward Teb1 (fig. S10).

The remarkable identification of two RPA related complexes in the Tetrahymena telomerase holoenzyme and their constitutive rather than cell-cycle regulated association provide a unique opportunity to use studies of Tetrahymena telomerase holoenzyme to inform models for interaction and function of human TPP1-POT1 and CST. Like p50-TEN, the interaction between human TPP1 and TEN is essential for bridging telomerase to telomeres (9, 11, 56, 57). Although Teb1C is homologous to RPA70C, Teb1AB are more similar to POT1 (50), suggesting that TEB functions to temporarily prevent rebinding of telomere-bound Tpt1-Pot1a complex (65). Consistent with the conformational flexibility of p75-p45-p19 around p50 (29), this CST complex could bind the telomeric DNA as it exits Teb1 to recruit DNA polymerase α for coordinated C-strand synthesis. In summary, the structure of the Tetrahymena telomerase catalytic core and identification of telomerase holoenzyme subcomplexes homologous to those found at mammalian, plant, and yeast telomeres provide new mechanistic insights and suggest commonalities of telomerase interaction, action, and regulation at telomeres.

Materials and methods

EM specimen preparation and data collection

Tetrahymena telomerase holoenzyme was purified following the previously described protocol (29) with some modifications. Briefly, 12 L cell culture of TERT-FZZ strain was used for tandem affinity purification using the established procedures (29). The final Flag elution was effected by incubating the telomerase-bound anti-Flag M2 affinity gel with 1.5 mL elution buffer (20 mM HEPES•NaOH, pH 8.0, 50 mM NaCl, 1 mM MgCl2, 1 mM TCEP•HCl, 0.025% IGEPAL CA-630, and 200 ng/μL 3×Flag peptide) at 4°C. The eluate was incubated with 30 mg Bio-Beads SM-2 absorbents (Bio-Rad) at 4°C for 2 hours by end-over-end rotation. The supernatant was then concentrated to 30 μL using Microcon YM-10 centrifugal filter (Millipore).

For cryoEM, 2.5 μL of sample was applied to a glow-discharged Quantifoil R2/1 grid. The grid was blotted with filter paper to remove excess sample and flash-frozen in liquid ethane with FEI Vitrobot Mark IV. The frozen-hydrated grids were loaded into an FEI Titan Krios electron microscope operated at 300 kV for automated image acquisition with Leginon (66). Micrographs were acquired with a Gatan K2 Summit direct electron detection camera operated in the electron counting mode at a calibrated magnification of 36,764× (pixel size of 1.36 Å on the sample level) and defocus values ranging from –2.0 to –7.0 μm. A GIF Quantum LS Imaging Filter (Gatan) was installed between the electron microscope and the K2 camera, but the energy filter (slit) was not used. The dose rate on the camera was set to ~8 e/pixel/s and the total exposure time was 12 s fractionated into 48 frames of images with 0.25 s exposure time for each frame. Frame images were aligned and averaged for correction of beam-induced drift using the GPU-accelerated motion correction program (67). The average images from all frames were used for defocus determination and particle picking, and those from the first 28 frames (corresponding to ~30 e2 total dose on sample) were used for further data processing, including image classification and 3D structure refinement. A total of 4210 micrographs were used in the data processing.

Image processing

The defocus value of each cryoEM micrograph was determined by CTFFIND (68) and the micrographs were corrected for contrast transfer function (CTF) by phase-flipping with the corresponding defocus and astigmatism values using Bsoft (69). A total of 478,698 particles were automatically picked using DoGpicker (70) and windowed out in 256×256 pixels using batchboxer in EMAN (71). The particles were binned to 128×128 pixels (pixel size of 2.72 Å) before the following processing. The binned particles were subjected to 2D and 3D classifications by RELION (72) following the recommended procedure ( Particles were classified into 100 classes for 25 iterations for each of two consecutive rounds of 2D classifications. After each round of 2D classification, class averages were visually inspected and particles in the “bad” classes with fuzzy or uninterpretable features or fuzzy density of p75-p45-p19 subcomplex (fig. S1B) were removed. About 20% of the class averages were kept after each round of 2D classification and 47,251 particles were selected for the following 3D classification. The negative-stain EM reconstruction at 25-Å resolution obtained previously (29) was low-pass filtered to 60 Å to serve as the initial model for 3D classification. The 3D classification generated 5 classes (fig. S1D), 4 of which showed an intact holoenzyme structure and were combined into a data set of 40,754 particles for 3D auto-refinements by RELION as follows. First, a 3D auto-refinement was performed using a spherical mask. The resolution was estimated to be 9.4 Å by the relion_postprocess program using the “gold-standard” FSC at 0.143 criterion with a soft mask for which the masking effect was corrected by phase randomization. Second, the density corresponding to the flexible p75-p45-p19 subcomplex was removed from the 9.4-Å cryoEM map using the “Volume Erase” in UCSF Chimera (73) and the resulting map was used to generate a soft-edge mask by the relion_mask_create program. This soft-edge mask was then used in the other 3D auto-refinement in which the p75-p45-p19 subcomplex was excluded during the structure refinement and only included in the final reconstruction. The resolution of this “focused” refinement was 8.9 Å estimated as described above. The cryoEM maps were sharpened with B-factor and low-pass filtered to the stated resolution using the relion_postprocess program. The local resolution was calculated by ResMap (74) using two cryoEM maps independently refined from halves of data.

Tetrahymena telomerase holoenzyme particles adopted a preferred “front view” in negative-stain EM grids (29), or in cryoEM Quantifoil grids with holey carbon or coated with continuous carbon film, probably due to its irregular and flattened shape. We tried numerous sample and cryoEM grid conditions to overcome this preferred orientation problem. The current protocol using Bio-Beads improved the orientation distribution the most. However, ~60% of the particles were still in the “front view” in the cryoEM images. To assess the impact of preferred orientation on reconstruction resolution, the particles were divided into halves (“uniform” and “preferred”) that showed more uniform or more preferred orientation distribution, respectively (fig. S2). Briefly, the particle orientation file (“” file generated by RELION 3D auto-refinement) of 40,754 particles was analyzed to group particles at the same orientation (same AngleRot and AngleTilt) together. A threshold of particle count was set so that the particles above the threshold in each orientation group (where the particle count in that group was larger than the threshold) were sent to the “preferred half” and the rest of the particles were kept in the “uniform half.” Separation of particles within an orientation group was random. These two halves of the data set of 40,754 particles were then subjected to 3D auto-refinement using the same parameters as those for the 8.9-Å reconstruction. Comparisons of orientation distributions and 3D reconstructions show that the “uniform half” had sufficient orientations to obtain a high-quality 3D reconstruction at an overall resolution better than 9.3 Å, and addition of the “preferred half” only improved the resolution modestly (fig. S2).

Fitting of atomic models into the cryoEM maps

For the pseudoatomic model of TERT-TER-p65 and Teb1C, the X-ray crystal and NMR structures of Tetrahymena TERT TRBD (2R4G) (37), TERT TEN (2B2A) (32), Teb1C (3U50) (50), TER SL2 (2FRL) (29), the model of p65 xRRM2:SL4 (33), and the homology model of Tetrahymena TERT RT-CTE domains based on Tribolium TERT (3DU6 and 3KYL; TERT CTE residues 1058-1117 were not modeled due to low sequence homology to Tribolium TERT) (29, 31, 38), which were previously assembled together by fitting into the negative-stain EM 3D reconstruction (29), were as a whole placed in an approximate position in the cryoEM maps, followed by fitting the individual domain structures into the cryoEM map using the “Fit in Map” function in UCSF Chimera (73). These domain structures or models fit into the cryoEM map with subtle rotations and translocations except for Teb1C, which was moved to and matched the cryoEM density behind TEN and next to TRBD with high consistency. The PK structure (PDB ID 2N6Q) was manually placed into cryoEM density matching its size and shape identified by exhaustive visual inspection of the cryoEM map and then fit into it using UCSF Chimera. S1 was modeled as an ideal A-form helix and fit into cryoEM density that matched its shape near the PK and SL4. The template was located by fitting the crystal structure of Tribolium TERT in complex with an RNA-DNA hybrid helix (PDB ID: 3KYL) (38) into the cryoEM map and finding unmodeled density near the RNA strand. The density assigned for the template is displaced by ~7 Å relative to that in the fitted Tribolium TERT. The remaining single-stranded regions of TER connecting the above fitted structure elements were modeled into the cryoEM densities using Coot (75) as follows. First, ideal A-form single-stranded RNA fragments were approximately placed between the flanking high-resolution RNA structures. The bond angles of the RNA backbone were then manually adjusted until the backbone fitted into the cryoEM density that was assigned to the RNA after the protein subunits were fitted into the cryoEM map. Last, standard bond angles and lengths of the backbone of the single-stranded RNA fragments were achieved using the “Regularize Zone” tool in Coot. Since the RNA bases were not distinguishable in the 8.9-Å cryoEM map, only the backbone of the single-stranded regions was modeled. For the pseudoatomic model of Teb1-Teb2-Teb3, RPA70C:RPA32N:RPA14 from the crystal structure of RPA (PDB ID: 1L1O) (51) was fit into the cryoEM maps by the colores program of Situs, using the exhaustive 6D search algorithm (76). The structure of RPA trimer fit with high consistency into the cryoEM map in the density assigned for Teb1C and the adjacent “knob.” For the final model, RPA70C was replaced by Teb1C, which fits in the same density, except for the C-terminal helix that is disordered in the Teb1C crystal structure but is apparently well ordered in the cryoEM maps. RPA32N and RPA14 in the model are paralogs of Teb2 and Teb3, respectively. During the Situs fitting of RPA70C:RPA32N:RPA14 to the 9.4-Å cryoEM map, a second location was identified with high confidence, corresponding to the “tip” of the density assigned to the p75-p45-p19 subcomplex, suggesting it was also an RPA homolog. For the pseudoatomic model of p75C-p45N-p19, RPA14 was manually replaced by p19 (PDB ID 5DFM), except for its C-terminal helix that is disordered in the p19 crystal structure. The crystal structure of TPP1 OB-fold (PDB ID: 2I46) (13) was placed in the cryoEM density assigned to p50N and then fit into it using UCSF Chimera. The cryoEM maps and the modeled protein and RNA structures were visualized with UCSF Chimera and PyMOL (The PyMOL Molecular Graphics System, Version 1.7.4 Schrödinger, LLC).

Structure determination of p19, p45C, and TER PK

Full length p19 (28) was fused via a triple alanine linker to the wild-type MBP vector as described (77), expressed in E. coli, and purified by amylose affinity and size exclusion chromatography. To obtain diffracting native crystals, a 2:1 mixture of 12 mg/ml MBP-p19 and the reservoir solution (0.1 M Sodium Acetate pH 4.6, 2.0 M Ammonium Sulfate) was set up in a 24 well hanging drop format. Full-length p45 (78) was fused to His-tagged MBP via a tobacco etch virus protease (TEV) cleavage site, expressed in E. coli, and co-purified with cells expressing 6×His-tagged p19 using Ni-NTA affinity and size exclusion chromatography. P45C (residues 162-373) was identified via limited chymotrypsin proteolysis of full-length p45 bound to p19. p45C was fused to His-tagged MBP via TEV cleavage site, expressed in E. coli, and purified by Ni-NTA affinity and size exclusion chromatography. To obtain diffracting native crystals, a 1:1 mixture of 14 mg/ml p45C and the reservoir solution (0.1M Na-HEPES pH 7.5, 5% MPD-v/v, 10%-w/v PEG 6000) was set up in a 24 well hanging drop format. All data was collected at 100 K at APS-NECAT beamline 24-ID-C on a DECTRIS-PILATUS 6 M pixel detector. Data were indexed, integrated, and scaled using XDS/XSCALE (79). Structures were solved using the molecular replacement program PHASER (80) using initial models from SeMet datasets. Final models were iteratively built and refined in Coot (75) and PHENIX (81). In the crystal structure of p45C, β5 in WH2 is domain-swapped from a neighboring protein in the crystal lattice. This domain-swapped β5 was used to build a “biological unit” model of p45C (Fig. 5C). NMR assignments were obtained and solution structure of the TER pseudoknot (nt 68-100) was determined following established protocols for human and yeast TER pseudoknots (44, 45, 82). Sample conditions were ~1 mM RNA in 10 mM NaPO4 pH 6.3, 50 mM KCl at 10°C. The structures were calculated with a total of 414 NOE, 171 dihedral angle, 82 H-bond, and 79 RDC restraints using standard protocols in Xplor-NIH 2.9.8. Pairwise RMSD for the 10 lowest energy structures (of 100) was 0.83 Å for all heavy atoms (table S4).

Mass spectrometry

Telomerase was purified from Tetrahymena TERT-FZZ and ZZF-p50 strains as described (29) up to the final anti-FLAG resin wash step before elution. Telomerase was batch washed on the resin 3 times by incubating in 1 ml IGEPAL-free wash buffer at 4°C rotating end-over-end for 10 min, and eluted twice by incubating at room temperature with 50 μl of 0.1% tri-fluoroacetic acid for 30 min each time. For enzymatic digestion of the samples, 70 μL of deoxycholic acid (0.1% w/v) in 1 M NH4HCO3 was added to the elution. Cysteines were alkylated by adding iodoacetamide to 4 mM and incubating at room temperature. After 30 min, excess dithiothreitol was added to quench residual iodoacetamide, and trypsin (300 ng) was added to initiate digestion. The solution was incubated at 37°C overnight, after which it was acidified to pH 2 with trifluoroacetic acid, to precipitate the deoxycholic acid. The deoxycholic acid was extracted from the aqueous layer with three 200 μL aliquots of water-saturated ethyl acetate (83). The digested peptides were dried and resuspended in 1% formic acid. The resulting tryptic peptides were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an EASY-nLC 1000 (Thermo Scientific, Waltham, MA) coupled to a Q-Exactive Orbitrap mass spectrometer (Thermo Scientific) and an EASY-Spray nano-ESI source. Peptides were injected onto a 75 μm × 15 cm, 3μ, 100Å PepMap C18 reversed-phase LC column and separated using a linear gradient from 5% solvent B (0.1% formic acid in acetonitrile), 95% solvent A (0.1% formic acid in water) to 50% solvent B in 2 hours at a constant flow of 300 nl/min. Eluted peptides were analyzed with a top-10 data-dependent acquisition method and identified using Proteome Discoverer (Version 1.4; Thermo Scientific) coupled to MASCOT (Version 2.4.1; Matrix Science, London, UK). Orbitrap MS resolving power was set to 70,000 at m/z 200 for MS1 and 17,500 at m/z 200 for MS2. Tryptic peptides with up to 2 missed cleavages were searched against a Tetrahymena thermophila protein database (2014) ( supplemented with common protein contaminant and enzyme sequences (27,046 sequences) with dynamic modifications for carbamidomethyl (C), oxidation (M), deamidation (N, Q), and peptide N-terminal cyclization to eliminate NH3 (Q, carbamidomethyl-cys) or H2O (E). Precursor and product ion mass tolerances were set to 10 ppm (or less) and 0.01 Da, respectively.

Telomerase reconstitution and activity assays

Telomerase subcomplexes were assembled separately and then combined. The RNP catalytic core was assembled by TERT expression in RRL in the presence of purified bacterially expressed p65 and in vitro transcribed TER. Additional RRL synthesis reactions generated p50N30, Teb1, or coexpressed TEB complex subunits. One subunit was tagged with 3×FLAG (p50N30-F for TEB subunit function studies or TERT-F for TERT TEN domain mutational analysis). Subcomplexes were combined, bound to FLAG antibody resin, and assayed by primer extension as described previously (29) using radiolabeled dGTP and primer (GTTGGG)3 at 200 nM final concentration. Elongation time was 10 min unless indicated otherwise. Products were resolved by denaturing gel electrophoresis, including an end-labeled oligonucleotide added before product precipitation as a recovery control.

Preparation of DNA-LNA bound telomerase linked by streptavidin and negative-stain EM

3-repeat biotinylated LNA/DNA primer was purchased from Exiqon as biotin-5′-GTTGGGGTTGGGGTLTLGLGLG, where TL and GL are LNA nucleotides. Telomerase was purified as described (29) from TERT-FZZ strain up to the TEV cleavage step, during which 5 μM 3-repeat primer and 50 μM dGTP were added to the IgG resin. Immediately before the 10 min wash steps of the telomerase bound flag resin, the resin was split, and one half was incubated in 50 μl of wash buffer while the other was incubated in 50 μl of wash buffer with 5 μM streptavidin, rotating end over end at 4°C for 30 min. Three wash steps were then performed as usual, after which the two resins were re-combined and batch eluted with 3×FLAG peptide according to the original protocol. Negative-stain EM samples were prepared, imaged, and processed as previously described (29).

Supplementary Materials

Figs. S1 to S10

Tables S1 to S4

References (8487)

References and Notes

  1. Acknowledgments: This work was supported in part by grants from the National Institutes of Health (NIH) (GM048123) and National Science Foundation (NSF) (MCB1022379) to J.F., NIH GM071940 to Z.H.Z., NIH GM103479 to J.A.L., NIH R01GM054198 to K.C., American Heart Association postdoctoral fellowship 14POST18870059 to J.J., NIH Ruth L. Kirschstein NRSA postdoctoral fellowship GM101874 to E.J.M., NIH Ruth L. Kirschstein NRSA pre-doctoral training grant fellowship under GM007185 to H.C. and R.O.J. and NSF Graduate Research Fellowship under DGE-1106400 to H.E.U. We acknowledge support for the NMR core, crystallization core, and X-ray core facilities by Department of Energy grant DE-FC0302ER63421 and the Electron Imaging Center for NanoMachines by NIH grants S10RR23057 and S10OD018111 and NSF DBI-133813. The Advanced Photon Source (APS) of Argonne National Laboratory is supported by NIH grants P41 RR015301 and P41 GM103403. Use of the APS is supported by DOE under Contract DE-AC02-06CH11357. The authors thank M. Sawaya and M. Collazo for help with crystallography, M. Capel, K. Rajashankar, N. Sukumar, F. Murphy, and I. Kourinov of NECAT. Additional support for electron microscopy time was provided by the National Center for Advancing Translational Sciences UCLA CTSI grant UL1TR000124 to J.F. The 9.4-Å and 8.9-Å cryoEM density maps of Tetrahymena telomerase holoenzyme have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-6442 and EMD-6443, respectively. Coordinates and structure factors for the X-ray crystal structures of p19 and p45C have been deposited in the PDB, accession codes 5DFM and 5DFN, respectively. Coordinates and restraints for the solution NMR structure of the TER PK and chemical shifts have been deposited in the PDB (accession code 2N6Q) and BMRB (accession code 25777), respectively. Nucleotide sequence data reported are available in the Third Party Annotation Section of the DDBJ/EMBL/GenBank databases under the accession numbers TPA: BK009378-BK009379.
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