Structure of active dimeric human telomerase

Abstract

Telomerase contains a large RNA subunit, TER, and a protein catalytic subunit, TERT. Whether telomerase functions as a monomer or dimer has been a matter of debate. Here we report biochemical and labeling data that show that in vivo-assembled human telomerase contains two TERT subunits and binds two telomeric DNA substrates. Notably, catalytic activity requires both TERT active sites to be functional, which demonstrates that human telomerase functions as a dimer. We also present the three-dimensional structure of the active full-length human telomerase dimer, determined by single-particle EM in negative stain. Telomerase has a bilobal architecture with the two monomers linked by a flexible interface. The monomer reconstruction at 23-Å resolution and fitting of the atomic structure of the TERT subunit from beetle Tribolium castaneum into the EM density reveals the spatial relationship between RNA and protein subunits, providing insights into telomerase architecture.

Figure 1. Human telomerase is a dimer

a, Schematic of hTERT domain arrangement: The essential N-terminal domain involved in DNA binding (TEN), the RNA binding domain (TRBD), the reverse transcriptase domain (RT) that contains the catalytic site and the C-terminal extension (CTE). b, Elution profile of the in vivo assembled human telomerase in complex with the G-overhang oligonucleotide 5′-[³²P]/biotin dT](TTAGGGT)₂-3′ fractionated on a sucrose gradient. c, Telomerase activity profile of a telomerase sample fractionated as in (b), but in the absence of G-overhang. G-overhang bound (b) and unbound (c) telomerase complexes migrate in the same position on the sucrose gradient. d, Electron micrograph of negatively stained telomerase bound to the G-overhang. Top views are indicated by black circles and side views by white circles. e, Example of reference-free 2D class averages. f, 3D structure obtained by subtomogram averaging. g and h, Electron micrographs of telomerase bound by 5 nm colloidal gold coated with streptavidin (g) and 5 nm colloidal gold coated with streptavidin in the absence of telomerase (h). Telomerase dimers in complex with one gold particle are circled in red and in complex with two gold particles circled in yellow. The arrows indicated colloidal gold particles that are not bound to telomerase. The scale bar in images (d), (g) and (h) is 50 nm and in images (e) and (f) is 5nm.

Figure 2. Active human telomerase functions as a dimer

Expression plasmids 13×Myc hTERT (WT) and hTER (WT) were transiently co-expressed in HEK293T cells together with either ZZ 3×Flag hTERT(WT), or the catalytically dead mutant ZZ 3×Flag hTERT(DN) at the ratios indicated at the top of the figure. a, Western blot analysis using an anti C-terminal hTERT antibody of TEV protease treated input whole cell lysates (left panel) and immunoaffinity purified and TEV protease released telomerase complexes (right panel). Whole cell lysate of untransfected HEK 293T cells was used as an expression control. For the left panel, the intensity of the TERT subunit is consistent with input expression plasmids. For the right hand panel showing the affinity purified and TEV released complexes, the intensities of the bands are consistent with the release of Flag_hTERT-Flag_hTERT dimers and Myc_hTERT-Flag_hTERT dimers, or Myc_hTERT-Flag_hTERT (DN) dimers b, Telospot telomerase activity assay of input whole cell lysate (left panel) and immunoaffinity purified and TEV released telomerase complexes (right panel). Telomerase activity in the transfected cells is shown as a multiple of the telomerase activity measured for untransfected HEK293T whole cell lysate. c, Direct telomerase activity assay of the immunoaffinity-purified and TEV released telomerase complexes. The presence of mutant hTERT (DN) has a dominant negative effect on telomerase activity.

Figure 3. Analysis of the telomerase dimer by single-particle EM

a, Field view of negatively stained telomerase dimers with individual particles circled in black (top and tilted views) and white (side views). b-d, Examples of reference-free 2D class averages with representative top views, tilted views and side views as indicated. f, Examples of 2D class averages (also shown in movie S1), in which the two monomers in the dimer exhibit considerable differences in their relative orientations. The double star (**) indicated the V-shaped opening in the top monomer and the single star (*) the closed view of the lower monomer. f, Four views of a refined dimer density map after MLF3D classification. The relative rotations are indicated g, Individual telomerase dimers in complex with biotinylated (TTAGGG)₂ bound to two streptavidin coated gold particles. The distance between the two gold particles in the dimer is 180-190Å. h, The locations of the two G-overhang binding site and the hinge region are shown on the structure of the dimer. The scale bar in image (a) 50nm and in images b-d,g) 10 nm.

Figure 4. Independently refined monomers and composite dimers

a, The left panels indicate the boxed out areas used for the independent refinement of both the open and closed monomer. b) Four views of the open monomer (upper panel) and closed monomer (lower panel), in which the side views are related by a 90° rotation around the horizontal axis. c, Comparison of three 2D class averages (top panel) from which three composite dimers were reconstructed (middle panel) and the corresponding projections (lower panel). d) Surface rendered refined monomer reconstructions (non-transparent) placed within the EM density of the telomerase dimer density (wire frame).

Figure 5. Assignment of the TERT and TER subunit within the 3D map of the open telomerase monomer

a-c, Three views of the three-dimensional map of the telomerase refined monomer into which the crystal structure of the beetle TERT subunit has been fitted. The upper panels show the EM density in transparent surface representation, together with the docked crystal structure. The lower panels show the EM density in non-transparent surface rendering. View (a) and (b) are related by a 90° rotation around the vertical axis. View (b) and (c) are related by a 90° rotation around the horizontal axis. Light blue depicts the RT domain; dark, blue the TRDB domain and red the DNA strand in the catalytic site. d, Electron micrographs of individual negatively stained telomerase containing His-tagged TERT subunit in complex with Ni-NTA-5nm colloidal gold. e, Distribution plot showing the distance between the Ni-NTA-gold and the dimer interface (y-axis), and the number of gold particles (x-axis). Although the large size of the gold particles complicates the analysis to some extent, a pronounced peak at 110 Å is in good agreement with the proposed location of TERT (blue) in the EM density map (a-c). f, Cartoon representation showing the interpretation of the EM density map. The colour coding for TERT is as in (a). A tentative location of the TEN domain is shown in green. Grey depicts the TER subunit, and the dotted line indicates the dimer interface.