Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1998 Feb 1;26(3):787–795. doi: 10.1093/nar/26.3.787

Mutational analysis of the Tetrahymena telomerase RNA: identification of residues affecting telomerase activity in vitro.

C Autexier 1, C W Greider 1
PMCID: PMC147331  PMID: 9443971

Abstract

Telomere-specific repeat sequences are essential for chromosome end stability. Telomerase maintains telomere length by adding sequences de novo onto chromosome ends. The template domain of the telomerase RNA component dictates synthesis of species-specific telomeric repeats and other regions of the RNA have been suggested to be important for enzyme structure and/or catalysis. Using enzyme reconstituted in vitro with RNAs containing deletions or substitutions we identified nucleotides in the RNA component that are important for telomerase activity. Although many changes to conserved features in the RNA secondary structure did not abolish enzyme activity, levels of activity were often greatly reduced, suggesting that regions other than the template play a role in telomerase function. The template boundary was only altered by changes in stem II that affected the conserved region upstream of the template, not by changes in other regions, such as stems I, III and IV, consistent with a role of the conserved region in defining the 5' boundary of the template. Surprisingly, telomerase RNAs with substitutions or deletion of residues potentially abolishing the conserved pseudoknot structure had wild-type levels of telomerase activity. This suggests that this base pairing interaction may not be required for telomerase activity per se but may be conserved as a regulatory site for the enzyme in vivo.

Full Text

The Full Text of this article is available as a PDF (686.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Autexier C., Greider C. W. Boundary elements of the Tetrahymena telomerase RNA template and alignment domains. Genes Dev. 1995 Sep 15;9(18):2227–2239. doi: 10.1101/gad.9.18.2227. [DOI] [PubMed] [Google Scholar]
  2. Autexier C., Greider C. W. Functional reconstitution of wild-type and mutant Tetrahymena telomerase. Genes Dev. 1994 Mar 1;8(5):563–575. doi: 10.1101/gad.8.5.563. [DOI] [PubMed] [Google Scholar]
  3. Autexier C., Pruzan R., Funk W. D., Greider C. W. Reconstitution of human telomerase activity and identification of a minimal functional region of the human telomerase RNA. EMBO J. 1996 Nov 1;15(21):5928–5935. [PMC free article] [PubMed] [Google Scholar]
  4. Bhattacharyya A., Blackburn E. H. A functional telomerase RNA swap in vivo reveals the importance of nontemplate RNA domains. Proc Natl Acad Sci U S A. 1997 Apr 1;94(7):2823–2827. doi: 10.1073/pnas.94.7.2823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blasco M. A., Funk W., Villeponteau B., Greider C. W. Functional characterization and developmental regulation of mouse telomerase RNA. Science. 1995 Sep 1;269(5228):1267–1270. doi: 10.1126/science.7544492. [DOI] [PubMed] [Google Scholar]
  6. Collins K., Greider C. W. Tetrahymena telomerase catalyzes nucleolytic cleavage and nonprocessive elongation. Genes Dev. 1993 Jul;7(7B):1364–1376. doi: 10.1101/gad.7.7b.1364. [DOI] [PubMed] [Google Scholar]
  7. Collins K., Kobayashi R., Greider C. W. Purification of Tetrahymena telomerase and cloning of genes encoding the two protein components of the enzyme. Cell. 1995 Jun 2;81(5):677–686. doi: 10.1016/0092-8674(95)90529-4. [DOI] [PubMed] [Google Scholar]
  8. Feng J., Funk W. D., Wang S. S., Weinrich S. L., Avilion A. A., Chiu C. P., Adams R. R., Chang E., Allsopp R. C., Yu J. The RNA component of human telomerase. Science. 1995 Sep 1;269(5228):1236–1241. doi: 10.1126/science.7544491. [DOI] [PubMed] [Google Scholar]
  9. Gilley D., Blackburn E. H. Specific RNA residue interactions required for enzymatic functions of Tetrahymena telomerase. Mol Cell Biol. 1996 Jan;16(1):66–75. doi: 10.1128/mcb.16.1.66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Greider C. W., Blackburn E. H. A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature. 1989 Jan 26;337(6205):331–337. doi: 10.1038/337331a0. [DOI] [PubMed] [Google Scholar]
  11. Greider C. W., Blackburn E. H. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell. 1985 Dec;43(2 Pt 1):405–413. doi: 10.1016/0092-8674(85)90170-9. [DOI] [PubMed] [Google Scholar]
  12. Greider C. W. Telomerase is processive. Mol Cell Biol. 1991 Sep;11(9):4572–4580. doi: 10.1128/mcb.11.9.4572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  14. Lingner J., Hendrick L. L., Cech T. R. Telomerase RNAs of different ciliates have a common secondary structure and a permuted template. Genes Dev. 1994 Aug 15;8(16):1984–1998. doi: 10.1101/gad.8.16.1984. [DOI] [PubMed] [Google Scholar]
  15. McCormick-Graham M., Romero D. P. A single telomerase RNA is sufficient for the synthesis of variable telomeric DNA repeats in ciliates of the genus Paramecium. Mol Cell Biol. 1996 Apr;16(4):1871–1879. doi: 10.1128/mcb.16.4.1871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. McCormick-Graham M., Romero D. P. Ciliate telomerase RNA structural features. Nucleic Acids Res. 1995 Apr 11;23(7):1091–1097. doi: 10.1093/nar/23.7.1091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McEachern M. J., Blackburn E. H. Runaway telomere elongation caused by telomerase RNA gene mutations. Nature. 1995 Aug 3;376(6539):403–409. doi: 10.1038/376403a0. [DOI] [PubMed] [Google Scholar]
  18. Romero D. P., Blackburn E. H. A conserved secondary structure for telomerase RNA. Cell. 1991 Oct 18;67(2):343–353. doi: 10.1016/0092-8674(91)90186-3. [DOI] [PubMed] [Google Scholar]
  19. Shippen-Lentz D., Blackburn E. H. Functional evidence for an RNA template in telomerase. Science. 1990 Feb 2;247(4942):546–552. doi: 10.1126/science.1689074. [DOI] [PubMed] [Google Scholar]
  20. Singer M. S., Gottschling D. E. TLC1: template RNA component of Saccharomyces cerevisiae telomerase. Science. 1994 Oct 21;266(5184):404–409. doi: 10.1126/science.7545955. [DOI] [PubMed] [Google Scholar]
  21. Tuerk C., MacDougal S., Gold L. RNA pseudoknots that inhibit human immunodeficiency virus type 1 reverse transcriptase. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6988–6992. doi: 10.1073/pnas.89.15.6988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Yu G. L., Bradley J. D., Attardi L. D., Blackburn E. H. In vivo alteration of telomere sequences and senescence caused by mutated Tetrahymena telomerase RNAs. Nature. 1990 Mar 8;344(6262):126–132. doi: 10.1038/344126a0. [DOI] [PubMed] [Google Scholar]
  23. Zaug A. J., Cech T. R. Analysis of the structure of Tetrahymena nuclear RNAs in vivo: telomerase RNA, the self-splicing rRNA intron, and U2 snRNA. RNA. 1995 Jun;1(4):363–374. [PMC free article] [PubMed] [Google Scholar]
  24. ten Dam E., van Belkum A., Pleij K. A conserved pseudoknot in telomerase RNA. Nucleic Acids Res. 1991 Dec 25;19(24):6951–6951. doi: 10.1093/nar/19.24.6951. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES