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A telomerase holoenzyme protein enhances telomerase RNA assembly with telomerase reverse transcriptase

Nature Structural & Molecular Biology volume 12, pages 252–257 (2005)Cite this article

Abstract

Telomerase maintains the simple sequence repeats at chromosome ends, protecting cells from genomic rearrangement, proliferative senescence and death. The telomerase reverse transcriptase (TERT) and telomerase RNA (TER) alone can assemble into active enzyme in a heterologous cell extract, but the physiological process of telomerase biogenesis is more complex. The endogenous accumulation of Tetrahymena thermophila TERT and TER requires an additional telomerase holoenzyme protein, p65. Here, we reconstitute this cellular pathway for telomerase ribonucleoprotein biogenesis in vitro. We demonstrate that tandem RNA interaction domains in p65 recognize the sequence of the TER 3′ stem. Notably, the p65–TER complex recruits TERT much more efficiently than does TER alone. Using bacterially expressed p65 and TERT polypeptides, we show that p65 enhances TERT-TER interaction by a mechanism involving a conserved bulge in the protein-bridging TER molecule. These findings reveal a pathway for telomerase holoenzyme biogenesis that preassembles TER for TERT recruitment.

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Figure 1: Specific p65-TER interaction.
Figure 2: Direct interaction of p65 and TER.
Figure 3: Formation of ternary complex with p65, TER and TERT.
Figure 4: Protein association through a TER bridge.
Figure 5: Stimulation of TERT RNP assembly by p65.

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Acknowledgements

We thank Collins laboratory members and the RNA community for experimental discussion and comments on the manuscript. Funding was provided by a predoctoral fellowship from the US National Science Foundation (C.M.O.) and by US National Institutes of Health grant GM54198 (K.C.).

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Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, 94720, California, USA

    Ramadevi Prathapam, Keren L Witkin, Catherine M O'Connor & Kathleen Collins

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  1. Ramadevi Prathapam
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  2. Keren L Witkin
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  3. Catherine M O'Connor
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  4. Kathleen Collins
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Correspondence to Kathleen Collins.

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Supplementary information

Supplementary Fig. 1

TER stem I variants: EMSA competition. (PDF 245 kb)

Supplementary Fig. 2

No homomultimer. (PDF 437 kb)

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Prathapam, R., Witkin, K., O'Connor, C. et al. A telomerase holoenzyme protein enhances telomerase RNA assembly with telomerase reverse transcriptase. Nat Struct Mol Biol 12, 252–257 (2005). https://doi.org/10.1038/nsmb900

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