Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 May 1;25(9):1825–1829. doi: 10.1093/nar/25.9.1825

DNA polymerization catalysed by a group II intron RNA in vitro.

M Hetzer 1, R J Schweyen 1, M W Mueller 1
PMCID: PMC146660  PMID: 9108167

Abstract

The excised group II intron bI1 from Saccharomyces cerevisiae can act as a ribozyme catalysing various chemical reactions with different substrate RNAs in vitro . Recently, we have described an editing-like RNA polymerization reaction catalysed by the bI1 intron lariat that proceeds in the 3'-->5'direction. Here we show that the bI1 lariat RNA can also catalyse successive deoxyribonucleotide polymerization reactions on exogenous substrate molecules. The basic mechanism of the reaction involved interacting cycles between an alternative version of partial reverse splicing (lariat charging) and canonical forward splicing (lariat discharging by exon ligation). With an overall chain growth in the 3'-->5' direction, the 5' exon RNAs (IBS1dN) were elongated by successive insertion of deoxyribonucleotides derived from single deoxyribonucleotide substitutions (dA, dG, dC or dT). All four deoxyribonucleotides were used as substrates, although with different efficiencies. Our findings extend the catalytic repertoire of group II intron RNAs not only by a novel DNA polymerization activity, but also by a DNA-DNA ligation capacity, supporting the idea that ribozymes might have been part of the first primordial polymerization machinery for both RNA and DNA.

Full Text

The Full Text of this article is available as a PDF (140.9 KB).

Selected References

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

  1. Abramovitz D. L., Friedman R. A., Pyle A. M. Catalytic role of 2'-hydroxyl groups within a group II intron active site. Science. 1996 Mar 8;271(5254):1410–1413. doi: 10.1126/science.271.5254.1410. [DOI] [PubMed] [Google Scholar]
  2. Augustin S., Müller M. W., Schweyen R. J. Reverse self-splicing of group II intron RNAs in vitro. Nature. 1990 Jan 25;343(6256):383–386. doi: 10.1038/343383a0. [DOI] [PubMed] [Google Scholar]
  3. Bartel D. P., Doudna J. A., Usman N., Szostak J. W. Template-directed primer extension catalyzed by the Tetrahymena ribozyme. Mol Cell Biol. 1991 Jun;11(6):3390–3394. doi: 10.1128/mcb.11.6.3390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Bratty J., Chartrand P., Ferbeyre G., Cedergren R. The hammerhead RNA domain, a model ribozyme. Biochim Biophys Acta. 1993 Dec 14;1216(3):345–359. doi: 10.1016/0167-4781(93)90001-t. [DOI] [PubMed] [Google Scholar]
  6. Bussière F., Perreault J. P. On the road to a DNA-protein world. RNA. 1995 Jul;1(5):451–452. [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Cech T. R., Herschlag D., Piccirilli J. A., Pyle A. M. RNA catalysis by a group I ribozyme. Developing a model for transition state stabilization. J Biol Chem. 1992 Sep 5;267(25):17479–17482. [PubMed] [Google Scholar]
  9. 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]
  10. Chowrira B. M., Berzal-Herranz A., Burke J. M. Novel RNA polymerization reaction catalyzed by a group I ribozyme. EMBO J. 1993 Sep;12(9):3599–3605. doi: 10.1002/j.1460-2075.1993.tb06033.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cuenoud B., Szostak J. W. A DNA metalloenzyme with DNA ligase activity. Nature. 1995 Jun 15;375(6532):611–614. doi: 10.1038/375611a0. [DOI] [PubMed] [Google Scholar]
  12. Dai X., De Mesmaeker A., Joyce G. F. Cleavage of an amide bond by a ribozyme. Science. 1995 Jan 13;267(5195):237–240. doi: 10.1126/science.7809628. [DOI] [PubMed] [Google Scholar]
  13. Darnell J. E., Doolittle W. F. Speculations on the early course of evolution. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1271–1275. doi: 10.1073/pnas.83.5.1271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Guerrier-Takada C., Gardiner K., Marsh T., Pace N., Altman S. The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell. 1983 Dec;35(3 Pt 2):849–857. doi: 10.1016/0092-8674(83)90117-4. [DOI] [PubMed] [Google Scholar]
  16. Herschlag D., Cech T. R. DNA cleavage catalysed by the ribozyme from Tetrahymena. Nature. 1990 Mar 29;344(6265):405–409. doi: 10.1038/344405a0. [DOI] [PubMed] [Google Scholar]
  17. Hetzer M., Mueller M. W. PCR mediated analysis of RNA sequences. Nucleic Acids Res. 1993 Nov 25;21(23):5526–5527. doi: 10.1093/nar/21.23.5526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jacquier A., Michel F. Multiple exon-binding sites in class II self-splicing introns. Cell. 1987 Jul 3;50(1):17–29. doi: 10.1016/0092-8674(87)90658-1. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Kruger K., Grabowski P. J., Zaug A. J., Sands J., Gottschling D. E., Cech T. R. Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena. Cell. 1982 Nov;31(1):147–157. doi: 10.1016/0092-8674(82)90414-7. [DOI] [PubMed] [Google Scholar]
  21. Mueller M. W., Allmaier M., Eskes R., Schweyen R. J. Transposition of group II intron aI1 in yeast and invasion of mitochondrial genes at new locations. Nature. 1993 Nov 11;366(6451):174–176. doi: 10.1038/366174a0. [DOI] [PubMed] [Google Scholar]
  22. Mueller M. W., Hetzer M., Schweyen R. J. Group II intron RNA catalysis of progressive nucleotide insertion: a model for RNA editing. Science. 1993 Aug 20;261(5124):1035–1038. doi: 10.1126/science.8351516. [DOI] [PubMed] [Google Scholar]
  23. Mörl M., Niemer I., Schmelzer C. New reactions catalyzed by a group II intron ribozyme with RNA and DNA substrates. Cell. 1992 Sep 4;70(5):803–810. doi: 10.1016/0092-8674(92)90313-2. [DOI] [PubMed] [Google Scholar]
  24. Müller M. W., Schweyen R. J., Schmelzer C. Selection of cryptic 5' splice sites by group II intron RNAs in vitro. Nucleic Acids Res. 1988 Aug 11;16(15):7383–7395. doi: 10.1093/nar/16.15.7383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Müller M. W., Stocker P., Hetzer M., Schweyen R. J. Fate of the junction phosphate in alternating forward and reverse self-splicing reactions of group II intron RNA. J Mol Biol. 1991 Nov 20;222(2):145–154. doi: 10.1016/0022-2836(91)90201-g. [DOI] [PubMed] [Google Scholar]
  26. Peebles C. L., Perlman P. S., Mecklenburg K. L., Petrillo M. L., Tabor J. H., Jarrell K. A., Cheng H. L. A self-splicing RNA excises an intron lariat. Cell. 1986 Jan 31;44(2):213–223. doi: 10.1016/0092-8674(86)90755-5. [DOI] [PubMed] [Google Scholar]
  27. Piccirilli J. A., McConnell T. S., Zaug A. J., Noller H. F., Cech T. R. Aminoacyl esterase activity of the Tetrahymena ribozyme. Science. 1992 Jun 5;256(5062):1420–1424. doi: 10.1126/science.1604316. [DOI] [PubMed] [Google Scholar]
  28. Pyle A. M., Green J. B. Building a kinetic framework for group II intron ribozyme activity: quantitation of interdomain binding and reaction rate. Biochemistry. 1994 Mar 8;33(9):2716–2725. doi: 10.1021/bi00175a047. [DOI] [PubMed] [Google Scholar]
  29. Schmelzer C., Müller M. W. Self-splicing of group II introns in vitro: lariat formation and 3' splice site selection in mutant RNAs. Cell. 1987 Dec 4;51(5):753–762. doi: 10.1016/0092-8674(87)90098-5. [DOI] [PubMed] [Google Scholar]
  30. Schmelzer C., Schweyen R. J. Self-splicing of group II introns in vitro: mapping of the branch point and mutational inhibition of lariat formation. Cell. 1986 Aug 15;46(4):557–565. doi: 10.1016/0092-8674(86)90881-0. [DOI] [PubMed] [Google Scholar]
  31. Schmidt W. M., Mueller M. W. Controlled ribonucleotide tailing of cDNA ends (CRTC) by terminal deoxynucleotidyl transferase: a new approach in PCR-mediated analysis of mRNA sequences. Nucleic Acids Res. 1996 May 1;24(9):1789–1791. doi: 10.1093/nar/24.9.1789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Tasiouka K. I., Burke J. M. A modified group I intron can function as both a ribozyme and a 5' exon in a trans-exon ligation reaction. Gene. 1994 Jun 24;144(1):1–7. doi: 10.1016/0378-1119(94)90195-3. [DOI] [PubMed] [Google Scholar]
  34. Yang J., Zimmerly S., Perlman P. S., Lambowitz A. M. Efficient integration of an intron RNA into double-stranded DNA by reverse splicing. Nature. 1996 May 23;381(6580):332–335. doi: 10.1038/381332a0. [DOI] [PubMed] [Google Scholar]
  35. Zaug A. J., Cech T. R. The Tetrahymena intervening sequence ribonucleic acid enzyme is a phosphotransferase and an acid phosphatase. Biochemistry. 1986 Aug 12;25(16):4478–4482. doi: 10.1021/bi00364a002. [DOI] [PubMed] [Google Scholar]
  36. Zaug A. J., Cech T. R. The intervening sequence RNA of Tetrahymena is an enzyme. Science. 1986 Jan 31;231(4737):470–475. doi: 10.1126/science.3941911. [DOI] [PubMed] [Google Scholar]
  37. van der Veen R., Arnberg A. C., van der Horst G., Bonen L., Tabak H. F., Grivell L. A. Excised group II introns in yeast mitochondria are lariats and can be formed by self-splicing in vitro. Cell. 1986 Jan 31;44(2):225–234. doi: 10.1016/0092-8674(86)90756-7. [DOI] [PubMed] [Google Scholar]

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

RESOURCES