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. 1996 Jan;16(1):66–75. doi: 10.1128/mcb.16.1.66

Specific RNA residue interactions required for enzymatic functions of Tetrahymena telomerase.

D Gilley 1, E H Blackburn 1
PMCID: PMC230979  PMID: 8524330

Abstract

The ribonucleoprotein enzyme telomerase is a specialized reverse transcriptase that synthesizes telomeric DNA by copying a template sequence within the telomerase RNA. Here we analyze the actions of telomerase from Tetrahymena thermophila assembled in vivo with mutated or wild-type telomerase RNA to define further the roles of particular telomerase RNA residues involved in essential enzymatic functions: templating, substrate alignment, and promotion of polymerization. Position 49 of the telomerase RNA defined the 3' templating residue boundary, demonstrating that seven positions, residues 43 to 49, are capable of acting as templating residues. We demonstrate directly that positioning of the primer substrate involves Watson-Crick base pairing between the primer with telomerase RNA residues. Unexpectedly, formation of a Watson-Crick base pair specifically between the primer DNA and telomerase RNA residue 50 is critical in promoting primer elongation. In contrast, mutant telomerase with the cytosine at position 49 mutated to a G exhibited efficient 3' mispair extension. This work provides new evidence for specific primer-telomerase interactions, as well as base-specific interactions involving the telomerase RNA, playing roles in essential active-site functions of telomerase.

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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. 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]
  2. Bhattacharyya A., Blackburn E. H. Architecture of telomerase RNA. EMBO J. 1994 Dec 1;13(23):5721–5731. doi: 10.1002/j.1460-2075.1994.tb06910.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blackburn E. H. Telomerases. Annu Rev Biochem. 1992;61:113–129. doi: 10.1146/annurev.bi.61.070192.000553. [DOI] [PubMed] [Google Scholar]
  4. Blackburn E. H. Telomeres: no end in sight. Cell. 1994 Jun 3;77(5):621–623. doi: 10.1016/0092-8674(94)90046-9. [DOI] [PubMed] [Google Scholar]
  5. Cohn M., Blackburn E. H. Telomerase in yeast. Science. 1995 Jul 21;269(5222):396–400. doi: 10.1126/science.7618104. [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. Gaertig J., Gorovsky M. A. Efficient mass transformation of Tetrahymena thermophila by electroporation of conjugants. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9196–9200. doi: 10.1073/pnas.89.19.9196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gilley D., Lee M. S., Blackburn E. H. Altering specific telomerase RNA template residues affects active site function. Genes Dev. 1995 Sep 15;9(18):2214–2226. doi: 10.1101/gad.9.18.2214. [DOI] [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. The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity. Cell. 1987 Dec 24;51(6):887–898. doi: 10.1016/0092-8674(87)90576-9. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Kohlstaedt L. A., Wang J., Friedman J. M., Rice P. A., Steitz T. A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992 Jun 26;256(5065):1783–1790. doi: 10.1126/science.1377403. [DOI] [PubMed] [Google Scholar]
  14. Kunkel T. A. DNA replication fidelity. J Biol Chem. 1992 Sep 15;267(26):18251–18254. [PubMed] [Google Scholar]
  15. Lee M. S., Blackburn E. H. Sequence-specific DNA primer effects on telomerase polymerization activity. Mol Cell Biol. 1993 Oct;13(10):6586–6599. doi: 10.1128/mcb.13.10.6586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lee M. S., Gallagher R. C., Bradley J., Blackburn E. H. In vivo and in vitro studies of telomeres and telomerase. Cold Spring Harb Symp Quant Biol. 1993;58:707–718. doi: 10.1101/sqb.1993.058.01.078. [DOI] [PubMed] [Google Scholar]
  17. Lin J. J., Zakian V. A. An in vitro assay for Saccharomyces telomerase requires EST1. Cell. 1995 Jun 30;81(7):1127–1135. doi: 10.1016/s0092-8674(05)80017-0. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Mantell L. L., Greider C. W. Telomerase activity in germline and embryonic cells of Xenopus. EMBO J. 1994 Jul 1;13(13):3211–3217. doi: 10.1002/j.1460-2075.1994.tb06620.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Morin G. B. Recognition of a chromosome truncation site associated with alpha-thalassaemia by human telomerase. Nature. 1991 Oct 3;353(6343):454–456. doi: 10.1038/353454a0. [DOI] [PubMed] [Google Scholar]
  22. Ollis D. L., Brick P., Hamlin R., Xuong N. G., Steitz T. A. Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP. 1985 Feb 28-Mar 6Nature. 313(6005):762–766. doi: 10.1038/313762a0. [DOI] [PubMed] [Google Scholar]
  23. Prowse K. R., Avilion A. A., Greider C. W. Identification of a nonprocessive telomerase activity from mouse cells. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1493–1497. doi: 10.1073/pnas.90.4.1493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Romero D. P., Blackburn E. H. Circular rDNA replicons persist in Tetrahymena thermophila transformants synthesizing GGGGTC telomeric repeats. J Eukaryot Microbiol. 1995 Jan-Feb;42(1):32–43. doi: 10.1111/j.1550-7408.1995.tb01537.x. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Sousa R., Chung Y. J., Rose J. P., Wang B. C. Crystal structure of bacteriophage T7 RNA polymerase at 3.3 A resolution. Nature. 1993 Aug 12;364(6438):593–599. doi: 10.1038/364593a0. [DOI] [PubMed] [Google Scholar]
  28. Yu G. L., Blackburn E. H. Developmentally programmed healing of chromosomes by telomerase in Tetrahymena. Cell. 1991 Nov 15;67(4):823–832. doi: 10.1016/0092-8674(91)90077-c. [DOI] [PubMed] [Google Scholar]
  29. Yu G. L., Blackburn E. H. Transformation of Tetrahymena thermophila with a mutated circular ribosomal DNA plasmid vector. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8487–8491. doi: 10.1073/pnas.86.21.8487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. 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]

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