Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1991 Jun;11(6):3390–3394. doi: 10.1128/mcb.11.6.3390

Template-directed primer extension catalyzed by the Tetrahymena ribozyme.

D P Bartel 1, J A Doudna 1, N Usman 1, J W Szostak 1
PMCID: PMC360195  PMID: 2038341

Abstract

The Tetrahymena ribozyme has been shown to catalyze an RNA polymerase-like reaction in which an RNA primer is extended by the sequential addition of pN nucleotides derived from GpN dinucleotides, where N = A, C, or U. Here, we show that this reaction is influenced by the presence of a template; bases that can form Watson-Crick base pairs with a template add as much as 25-fold more efficiently than mismatched bases. A mutant enzyme with an altered guanosine binding site can catalyze template-directed primer extension with all four bases when supplied with dinucleotides of the form 2-aminopurine-pN.

Full text

PDF
3391

Images in this article

Selected References

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

  1. Been M. D., Cech T. R. RNA as an RNA polymerase: net elongation of an RNA primer catalyzed by the Tetrahymena ribozyme. Science. 1988 Mar 18;239(4846):1412–1416. doi: 10.1126/science.2450400. [DOI] [PubMed] [Google Scholar]
  2. Benner S. A., Ellington A. D., Tauer A. Modern metabolism as a palimpsest of the RNA world. Proc Natl Acad Sci U S A. 1989 Sep;86(18):7054–7058. doi: 10.1073/pnas.86.18.7054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boosalis M. S., Petruska J., Goodman M. F. DNA polymerase insertion fidelity. Gel assay for site-specific kinetics. J Biol Chem. 1987 Oct 25;262(30):14689–14696. [PubMed] [Google Scholar]
  4. Bridson P. K., Orgel L. E. Catalysis of accurate poly(C)-directed synthesis of 3'-5'-linked oligoguanylates by Zn2+. J Mol Biol. 1980 Dec 25;144(4):567–577. doi: 10.1016/0022-2836(80)90337-x. [DOI] [PubMed] [Google Scholar]
  5. Cech T. R. A model for the RNA-catalyzed replication of RNA. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4360–4363. doi: 10.1073/pnas.83.12.4360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cech T. R., Tanner N. K., Tinoco I., Jr, Weir B. R., Zuker M., Perlman P. S. Secondary structure of the Tetrahymena ribosomal RNA intervening sequence: structural homology with fungal mitochondrial intervening sequences. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3903–3907. doi: 10.1073/pnas.80.13.3903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cech T. R. The chemistry of self-splicing RNA and RNA enzymes. Science. 1987 Jun 19;236(4808):1532–1539. doi: 10.1126/science.2438771. [DOI] [PubMed] [Google Scholar]
  8. Doudna J. A., Szostak J. W. RNA-catalysed synthesis of complementary-strand RNA. Nature. 1989 Jun 15;339(6225):519–522. doi: 10.1038/339519a0. [DOI] [PubMed] [Google Scholar]
  9. Doudna J. A., Szostak J. W., Rich A., Usman N. Chemical synthesis of oligoribonucleotides containing 2-aminopurine: substrates for the investigation of ribozyme function. J Org Chem. 1990 Oct 12;55(21):5547–5549. doi: 10.1021/jo00308a003. [DOI] [PubMed] [Google Scholar]
  10. Freier S. M., Kierzek R., Caruthers M. H., Neilson T., Turner D. H. Free energy contributions of G.U and other terminal mismatches to helix stability. Biochemistry. 1986 Jun 3;25(11):3209–3213. doi: 10.1021/bi00359a019. [DOI] [PubMed] [Google Scholar]
  11. Inoue T., Joyce G. F., Grzeskowiak K., Orgel L. E., Brown J. M., Reese C. B. Template-directed synthesis on the pentanucleotide CpCpGpCpC. J Mol Biol. 1984 Sep 25;178(3):669–676. doi: 10.1016/0022-2836(84)90244-4. [DOI] [PubMed] [Google Scholar]
  12. Inoue T., Orgel L. E. A nonenzymatic RNA polymerase model. Science. 1983 Feb 18;219(4586):859–862. doi: 10.1126/science.6186026. [DOI] [PubMed] [Google Scholar]
  13. Inoue T., Orgel L. E. Oligomerization of (guanosine 5'-phosphor)-2-methylimidazolide on poly(C). An RNA polymerase model. J Mol Biol. 1982 Nov 25;162(1):201–217. doi: 10.1016/0022-2836(82)90169-3. [DOI] [PubMed] [Google Scholar]
  14. Joyce G. F. RNA evolution and the origins of life. Nature. 1989 Mar 16;338(6212):217–224. doi: 10.1038/338217a0. [DOI] [PubMed] [Google Scholar]
  15. Kay P. S., Inoue T. Catalysis of splicing-related reactions between dinucleotides by a ribozyme. 1987 May 28-Jun 3Nature. 327(6120):343–346. doi: 10.1038/327343a0. [DOI] [PubMed] [Google Scholar]
  16. Martin C. T., Muller D. K., Coleman J. E. Processivity in early stages of transcription by T7 RNA polymerase. Biochemistry. 1988 May 31;27(11):3966–3974. doi: 10.1021/bi00411a012. [DOI] [PubMed] [Google Scholar]
  17. Michel F., Hanna M., Green R., Bartel D. P., Szostak J. W. The guanosine binding site of the Tetrahymena ribozyme. Nature. 1989 Nov 23;342(6248):391–395. doi: 10.1038/342391a0. [DOI] [PubMed] [Google Scholar]
  18. Milligan J. F., Groebe D. R., Witherell G. W., Uhlenbeck O. C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 1987 Nov 11;15(21):8783–8798. doi: 10.1093/nar/15.21.8783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mullis K. B., Faloona F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. doi: 10.1016/0076-6879(87)55023-6. [DOI] [PubMed] [Google Scholar]
  20. NIYOGI S. K., STEVENS A. STUDIES OF THE RIBONUCLEIC ACID POLYMERASE FROM ESCHERICHIA COLI. IV. EFFECT OF OLIGONUCLEOTIDES ON THE RIBONUCLEIC ACID POLYMERASE REACTION WITH SYNTHETIC POLYRIBONUCLEOTIDES AS TEMPLATES. J Biol Chem. 1965 Jun;240:2593–2598. [PubMed] [Google Scholar]
  21. Sharp P. A. On the origin of RNA splicing and introns. Cell. 1985 Sep;42(2):397–400. doi: 10.1016/0092-8674(85)90092-3. [DOI] [PubMed] [Google Scholar]
  22. Turner D. H., Sugimoto N., Freier S. M. RNA structure prediction. Annu Rev Biophys Biophys Chem. 1988;17:167–192. doi: 10.1146/annurev.bb.17.060188.001123. [DOI] [PubMed] [Google Scholar]
  23. Waring R. B., Scazzocchio C., Brown T. A., Davies R. W. Close relationship between certain nuclear and mitochondrial introns. Implications for the mechanism of RNA splicing. J Mol Biol. 1983 Jul 5;167(3):595–605. doi: 10.1016/s0022-2836(83)80100-4. [DOI] [PubMed] [Google Scholar]
  24. el-Deiry W. S., So A. G., Downey K. M. Mechanisms of error discrimination by Escherichia coli DNA polymerase I. Biochemistry. 1988 Jan 26;27(2):546–553. doi: 10.1021/bi00402a007. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES