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The architecture of Tetrahymena telomerase holoenzyme

A Corrigendum to this article was published on 12 February 2014

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Abstract

Telomerase adds telomeric repeats to chromosome ends using an internal RNA template and a specialized telomerase reverse transcriptase (TERT), thereby maintaining genome integrity. Little is known about the physical relationships among protein and RNA subunits within a biologically functional holoenzyme. Here we describe the architecture of Tetrahymena thermophila telomerase holoenzyme determined by electron microscopy. Six of the seven proteins and the TERT-binding regions of telomerase RNA (TER) have been localized by affinity labelling. Fitting with high-resolution structures reveals the organization of TERT, TER and p65 in the ribonucleoprotein (RNP) catalytic core. p50 has an unanticipated role as a hub between the RNP catalytic core, p75–p19–p45 subcomplex, and the DNA-binding Teb1. A complete in vitro holoenzyme reconstitution assigns function to these interactions in processive telomeric repeat synthesis. These studies provide the first view of the extensive network of subunit associations necessary for telomerase holoenzyme assembly and physiological function.

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Figure 1: Electron microscopy reconstruction of Tetrahymena telomerase holoenzyme and subunit localization.
Figure 2: Structure of the RNP catalytic core.
Figure 3: p50 anchors TERT, 7-1-4 and Teb1.
Figure 4: Contribution of Teb1 domains to holoenzyme structure and activity.
Figure 5: Positional dynamics of 7-1-4.

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Acknowledgements

This work was supported by grants from NSF MCB1022379 and NIH GM48123 to J.F., NIH GM54198 to K.C., GM071940 and AI069015 to Z.H.Z., Ruth L. Kirschstein NRSA postdoctoral fellowship GM101874 to E.J.M., and Ruth L. Kirschstein NRSA pre-doctoral training grant GM007185 fellowship for H.C. and D.D.C. We acknowledge the use of instruments at the Electron Imaging Center for NanoMachines supported by NIH (1S10RR23057 to ZHZ) and CNSI at UCLA.

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J.J. and E.J.M. purified and characterized electron microscopy samples, collected and analysed electron microscopy data, and wrote the paper; K.H., B.E. and B.M. designed and made strains, expression plasmids, initial purifications and reconstituted holoenzyme; H.C. purified telomerase; D.D.C. refined and modelled elements of TER; Z.H.Z., K.C. and J.F. supervised the research, analysed data and wrote the paper.

Corresponding authors

Correspondence to Z. Hong Zhou, Kathleen Collins or Juli Feigon.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-8 and a Supplementary Table outlining the strains and number of EM micrographs used in the EM studies. (PDF 7313 kb)

3D reconstruction of Teb1-f telomerase holoenzyme with fitting of high-resolution structures.

Rotation of Teb1-f telomerase holoenzyme 3D reconstruction followed by zoom in of Tetrahymena homology modeled TERT with RT (purple), TER template (magenta), CTE (light blue), and TRBD (blue) fit into the EM density. Zoom out and slight rotation showing fit of TEN domain (cyan), and stem-loop 2 (magenta), then La, RRM1, and xRRM2 of p65 (green), followed by incorporation of remaining TER components (black) stem 1, pseudoknot, and ssRNA regions, and Teb1C (orange). A final 360° rotation showing all high-resolution structures fit in and the locations of p75, p19, p45, and p50. (MOV 32810 kb)

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Jiang, J., Miracco, E., Hong, K. et al. The architecture of Tetrahymena telomerase holoenzyme. Nature 496, 187–192 (2013). https://doi.org/10.1038/nature12062

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