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Crystal structure of the essential N-terminal domain of telomerase reverse transcriptase

Nature Structural & Molecular Biology volume 13, pages 218–225 (2006)Cite this article

An Addendum to this article was published on 01 October 2007

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Abstract

Telomerase, a ribonucleoprotein enzyme, adds telomeric DNA repeats to the ends of linear chromosomes. Here we report the first high-resolution structure of any portion of the telomerase reverse transcriptase, the telomerase essential N-terminal (TEN) domain from Tetrahymena thermophila. The structure, which seems to represent a novel protein fold, shows phylogenetically conserved amino acid residues in a groove on its surface. These residues are crucial for telomerase catalytic activity, and several of them are required for sequence-specific binding of a single-stranded telomeric DNA primer. The positively charged C terminus, which becomes ordered upon interaction with other macromolecules, is involved in binding RNA in a non–sequence-specific manner. The TEN domain's ability to bind both RNA and telomeric DNA, coupled with the notably strong effects on activity upon mutagenesis of single surface residues, suggest how this domain contributes to telomerase catalysis.

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Figure 1: Three-dimensional structure of the TEN domain.
Figure 2: Conservation of TEN domain structure.
Figure 3: In vitro telomerase activity of TERT mutants.
Figure 4: Interactions of TEN domain with telomeric DNA primers, as assessed by photo–cross-linking.
Figure 5: RNA-binding activity of the TEN domain.
Figure 6: Conceptual model of contributions of the TEN domain to the telomerase mechanism.

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  • 24 September 2007

    The recent paper by Romi et al. (Proc. Natl. Acad. Sci. USA 104, 8791–8796, 2007) was in general agreement with our study of the structure and function of the N-terminal domain of Tetrahymena telomerase reverse transcriptase. However, the two studies disagreed on the effect of a mutation of Trp187 on catalysis, with our study reporting a severe reduction in activity. Upon sequencing the entire gene encoding our W187A mutant, we found that it had a second distant mutation (R812W) in motif C of the reverse transcriptase domain, and we demonstrated that it was the mutation at position 812 that abolished catalytic activity. In addition, we confirmed that authentic W187A telomerase has catalytic activity similar to that of wild-type telomerase. Both studies concur that Trp187 is physically close to the primer-binding site, and in fact Romi et al. have mapped Trp187 as a site of photo-cross-linking to a telomeric DNA primer.

Notes

  1. *NOTE: The recent paper by Romi et al. (Proc. Natl. Acad. Sci. USA 104, 8791-8796, 2007) was in general agreement with our study of the structure and function of the N-terminal domain of Tetrahymena telomerase reverse transcriptase. However, the two studies disagreed on the effect of a mutation of Trp187 on catalysis, with our study reporting a severe reduction in activity. Upon sequencing the entire gene encoding our W187A mutant, we found that it had a second distant mutation (R812W) in motif C of the reverse transcriptase domain, and we demonstrated that it was the mutation at position 812 that abolished catalytic activity. In addition, we confirmed that authentic W187A telomerase has catalytic activity similar to that of wild-type telomerase. Both studies concur that Trp187 is physically close to the primer-binding site, and in fact Romi et al. have mapped Trp187 as a site of photo-cross-linking to a telomeric DNA primer. We gratefully acknowledge the work of Arthur J. Zaug (HHMI, University of Colorado-Boulder) in resolving this discrepancy.

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Acknowledgements

We thank A.J. Zaug and A. Gooding for help with X-ray diffraction data collection, the Advanced Light Source for beam time and helpful advice and L. Chen, D.S. Wuttke and M. Sousa for helpful discussions. S.A.J. was a Damon Runyon fellow supported by the Damon Runyon Cancer Research Foundation (grant DRG-#1821-04).

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

    Steven A Jacobs, Elaine R Podell & Thomas R Cech

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Correspondence to Thomas R Cech.

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Supplementary Fig. 1

SDS gel showing expression levels of the mutant TERT proteins (PDF 3554 kb)

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Jacobs, S., Podell, E. & Cech, T. Crystal structure of the essential N-terminal domain of telomerase reverse transcriptase. Nat Struct Mol Biol 13, 218–225 (2006). https://doi.org/10.1038/nsmb1054

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