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A miniature yeast telomerase RNA functions in vivo and reconstitutes activity in vitro

Nature Structural & Molecular Biology volume 12, pages 1072–1077 (2005)Cite this article

A Corrigendum to this article was published on 01 May 2006

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Abstract

The ribonucleoprotein enzyme telomerase synthesizes DNA at the ends of chromosomes. Although the telomerase catalytic protein subunit (TERT) is well conserved, the RNA component is rapidly evolving in both size and sequence. Here, we reduce the 1,157-nucleotide (nt) Saccharomyces cerevisiae TLC1 RNA to a size smaller than the 451-nt human RNA while retaining function in vivo. We conclude that long protein-binding arms are not essential for the RNA to serve its scaffolding function. Although viable, cells expressing Mini-T have shortened telomeres and reduced fitness as compared to wild-type cells, suggesting why the larger RNA has evolved. Previous attempts to reconstitute telomerase activity in vitro using TLC1 and yeast TERT (Est2p) have been unsuccessful. We find that substitution of Mini-T for wild-type TLC1 in a reconstituted system yields robust activity, allowing the contributions of individual yeast telomerase components to be directly assessed.

*Note: In the supplementary information initially published online to accompany this article, the secondary structure model for a 436-nt mini-T is shown in Supplementary Figure 1, but the legend describes a 500-nt mini-T model. The secondary structure model for the 500-nt mini-T has now been supplied by the authors. The error has been corrected online.

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Figure 1: Design of a smaller yeast telomerase RNA using the secondary structure model for the wild-type RNA.
Figure 2: Reduction of TLC1 to one-third of wild-type (WT) length with retention of function in vivo.
Figure 3: Competitive growth experiments show that the fitness of Mini-T–expressing cells is less than that of wild-type (WT) cells.
Figure 4: Mini-T allows reconstituted yeast telomerase activity in vitro.

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Acknowledgements

We thank A.J. Zaug (University of Colorado, Boulder, Colorado, USA) for plasmid T7-ProA-Est2p and A.G. Seto (present affiliation: Harvard Medical School, Cambridge, Massachusetts, USA) for telomerase immunopurified from yeast. Thanks also to A.J. Zaug for performing the telomerase activity assays that included human TERT. This research was supported in part by grant GM28039 from the US National Institutes of Health.

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  1. Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder, 80309-0215, Colorado, USA

    David C Zappulla, Karen Goodrich & Thomas R Cech

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  1. David C Zappulla
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  3. Thomas R Cech
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Correspondence to Thomas R Cech.

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Supplementary information

Supplementary Fig. 1

Mfold secondary structure model of Mini-T(500). (PDF 907 kb)

Supplementary Fig. 2

Mini-T(500) RNA accumulation when expressed from the chromosome. (PDF 355 kb)

Supplementary Fig. 3

Mini-T RNA levels during log phase cell growth. (PDF 734 kb)

Supplementary Fig. 4

Mini-T(64) and (67) do not yield reconstituted activity. (PDF 586 kb)

Supplementary Fig. 5

Reconstituted core mini-telomerase has activity very similar to wild-type telomerase from yeast. (PDF 1039 kb)

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Zappulla, D., Goodrich, K. & Cech, T. A miniature yeast telomerase RNA functions in vivo and reconstitutes activity in vitro. Nat Struct Mol Biol 12, 1072–1077 (2005). https://doi.org/10.1038/nsmb1019

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