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. 1989 Oct;86(19):7402–7406. doi: 10.1073/pnas.86.19.7402

RNA structure, not sequence, determines the 5' splice-site specificity of a group I intron.

J A Doudna 1, B P Cormack 1, J W Szostak 1
PMCID: PMC298070  PMID: 2678103

Abstract

The group I self-splicing introns act at exon-intron junctions without recognizing a particular sequence. In order to understand splice-site selection, we have developed an assay system based on the Tetrahymena ribozyme to allow the study of numerous 5'-splice-site variants. Cleavage at the correct site requires formation of the correct secondary structure and occurs most efficiently within a 3-base-pair window centered on base pair 5 from the bottom of the P1 stem. Within this window the ribozyme recognizes and cleaves at a "wobble" base pair; the base pair above the cleavage site also influences splicing efficiency. The recognition of RNA structure rather than sequence explains the ability of these transposable introns to splice out of a variety of sequence contexts.

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Selected References

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

  1. Been M. D., Barfod E. T., Burke J. M., Price J. V., Tanner N. K., Zaug A. J., Cech T. R. Structures involved in Tetrahymena rRNA self-splicing and RNA enzyme activity. Cold Spring Harb Symp Quant Biol. 1987;52:147–157. doi: 10.1101/sqb.1987.052.01.019. [DOI] [PubMed] [Google Scholar]
  2. Been M. D., Cech T. R. One binding site determines sequence specificity of Tetrahymena pre-rRNA self-splicing, trans-splicing, and RNA enzyme activity. Cell. 1986 Oct 24;47(2):207–216. doi: 10.1016/0092-8674(86)90443-5. [DOI] [PubMed] [Google Scholar]
  3. Burke J. M., Belfort M., Cech T. R., Davies R. W., Schweyen R. J., Shub D. A., Szostak J. W., Tabak H. F. Structural conventions for group I introns. Nucleic Acids Res. 1987 Sep 25;15(18):7217–7221. doi: 10.1093/nar/15.18.7217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Davies R. W., Waring R. B., Ray J. A., Brown T. A., Scazzocchio C. Making ends meet: a model for RNA splicing in fungal mitochondria. Nature. 1982 Dec 23;300(5894):719–724. doi: 10.1038/300719a0. [DOI] [PubMed] [Google Scholar]
  7. Doudna J. A., Gerber A. S., Cherry J. M., Szostak J. W. Genetic dissection of an RNA enzyme. Cold Spring Harb Symp Quant Biol. 1987;52:173–180. doi: 10.1101/sqb.1987.052.01.022. [DOI] [PubMed] [Google Scholar]
  8. McSwiggen J. A., Cech T. R. Stereochemistry of RNA cleavage by the Tetrahymena ribozyme and evidence that the chemical step is not rate-limiting. Science. 1989 May 12;244(4905):679–683. doi: 10.1126/science.2470150. [DOI] [PubMed] [Google Scholar]
  9. Michel F., Dujon B. Conservation of RNA secondary structures in two intron families including mitochondrial-, chloroplast- and nuclear-encoded members. EMBO J. 1983;2(1):33–38. doi: 10.1002/j.1460-2075.1983.tb01376.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Rajagopal J., Doudna J. A., Szostak J. W. Stereochemical course of catalysis by the Tetrahymena ribozyme. Science. 1989 May 12;244(4905):692–694. doi: 10.1126/science.2470151. [DOI] [PubMed] [Google Scholar]
  12. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Szostak J. W. Enzymatic activity of the conserved core of a group I self-splicing intron. Nature. 1986 Jul 3;322(6074):83–86. doi: 10.1038/322083a0. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Woodson S. A., Cech T. R. Reverse self-splicing of the tetrahymena group I intron: implication for the directionality of splicing and for intron transposition. Cell. 1989 Apr 21;57(2):335–345. doi: 10.1016/0092-8674(89)90971-9. [DOI] [PubMed] [Google Scholar]
  16. Zaug A. J., Grabowski P. J., Cech T. R. Autocatalytic cyclization of an excised intervening sequence RNA is a cleavage-ligation reaction. Nature. 1983 Feb 17;301(5901):578–583. doi: 10.1038/301578a0. [DOI] [PubMed] [Google Scholar]

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