Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Aug;15(8):4466–4478. doi: 10.1128/mcb.15.8.4466

Stereochemical selectivity of group II intron splicing, reverse splicing, and hydrolysis reactions.

M Podar 1, P S Perlman 1, R A Padgett 1
PMCID: PMC230686  PMID: 7542746

Abstract

We have previously shown, using phosphorothioate substitutions at splice site, that both transesterification steps of group II intron self-splicing proceed, by stereochemical inversion, with an Sp but not an Rp phosphorothioate. Under alternative reaction conditions or with various intron fragments, group II introns can splice following hydrolysis at the 5' splice site and can also hydrolyze the bond between spliced exons (the spliced-exon reopening reaction). In this study, we have determined the stereochemical specificities of all of the major model hydrolytic reactions carried out by the aI5 gamma intron from Saccharomyces cerevisiae mitochondria. For all substrates containing exon 1 and most of the intron, the stereospecificity of hydrolysis is the same as for the step 1 transesterification reaction. In contrast, the spliced-exon reopening reaction proceeds with an Rp but not an Sp phosphorothioate at the scissile bond, as does true reverse splicing. Thus, by stereochemistry, this reaction appears to be related to the reverse of step 2 of self-splicing. Finally, a substrate RNA that contains the first exon and nine nucleotides of the intron, when reacted with the intron ribozyme, releases the first exon regardless of the configuration of the phosphorothioate at the 5' splice site, suggesting that this substrate can be cleaved by either the step 1 or the step 2 reaction site. Our findings clarify the relationships of these model reactions to the transesterification reactions of the intact self-splicing system and permit new studies to be interpreted more rigorously.

Full Text

The Full Text of this article is available as a PDF (689.5 KB).

Selected References

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

  1. Altura R., Rymond B., Seraphin B., Rosbash M. Sequence requirements for branch formation in a group II self-splicing intron. Nucleic Acids Res. 1989 Jan 11;17(1):335–354. doi: 10.1093/nar/17.1.335. [DOI] [PMC free article] [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. Butow R. A., Perlman P. S. Introns in pieces. Curr Biol. 1991 Oct;1(5):331–333. doi: 10.1016/0960-9822(91)90103-4. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Celander D. W., Cech T. R. Visualizing the higher order folding of a catalytic RNA molecule. Science. 1991 Jan 25;251(4992):401–407. doi: 10.1126/science.1989074. [DOI] [PubMed] [Google Scholar]
  6. Chanfreau G., Jacquier A. Catalytic site components common to both splicing steps of a group II intron. Science. 1994 Nov 25;266(5189):1383–1387. doi: 10.1126/science.7973729. [DOI] [PubMed] [Google Scholar]
  7. Eckstein F. Nucleoside phosphorothioates. Annu Rev Biochem. 1985;54:367–402. doi: 10.1146/annurev.bi.54.070185.002055. [DOI] [PubMed] [Google Scholar]
  8. Franzen J. S., Zhang M., Peebles C. L. Kinetic analysis of the 5' splice junction hydrolysis of a group II intron promoted by domain 5. Nucleic Acids Res. 1993 Feb 11;21(3):627–634. doi: 10.1093/nar/21.3.627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Guthrie C. Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein. Science. 1991 Jul 12;253(5016):157–163. doi: 10.1126/science.1853200. [DOI] [PubMed] [Google Scholar]
  10. Herschlag D., Piccirilli J. A., Cech T. R. Ribozyme-catalyzed and nonenzymatic reactions of phosphate diesters: rate effects upon substitution of sulfur for a nonbridging phosphoryl oxygen atom. Biochemistry. 1991 May 21;30(20):4844–4854. doi: 10.1021/bi00234a003. [DOI] [PubMed] [Google Scholar]
  11. Jacquier A., Michel F. Base-pairing interactions involving the 5' and 3'-terminal nucleotides of group II self-splicing introns. J Mol Biol. 1990 Jun 5;213(3):437–447. doi: 10.1016/S0022-2836(05)80206-2. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Jacquier A., Rosbash M. Efficient trans-splicing of a yeast mitochondrial RNA group II intron implicates a strong 5' exon-intron interaction. Science. 1986 Nov 28;234(4780):1099–1104. doi: 10.1126/science.2430332. [DOI] [PubMed] [Google Scholar]
  14. Jarrell K. A., Dietrich R. C., Perlman P. S. Group II intron domain 5 facilitates a trans-splicing reaction. Mol Cell Biol. 1988 Jun;8(6):2361–2366. doi: 10.1128/mcb.8.6.2361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jarrell K. A., Peebles C. L., Dietrich R. C., Romiti S. L., Perlman P. S. Group II intron self-splicing. Alternative reaction conditions yield novel products. J Biol Chem. 1988 Mar 5;263(7):3432–3439. [PubMed] [Google Scholar]
  16. Kahle D., Küst B., Krupp G. Phosphorothioates in pre-tRNAs can change the specificities of RNAses P or reduce the cleavage efficiencies. Biochimie. 1993;75(11):955–962. doi: 10.1016/0300-9084(93)90145-i. [DOI] [PubMed] [Google Scholar]
  17. Maschhoff K. L., Padgett R. A. The stereochemical course of the first step of pre-mRNA splicing. Nucleic Acids Res. 1993 Nov 25;21(23):5456–5462. doi: 10.1093/nar/21.23.5456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Michels W. J., Jr, Pyle A. M. Conversion of a group II intron into a new multiple-turnover ribozyme that selectively cleaves oligonucleotides: elucidation of reaction mechanism and structure/function relationships. Biochemistry. 1995 Mar 7;34(9):2965–2977. doi: 10.1021/bi00009a028. [DOI] [PubMed] [Google Scholar]
  19. Moore M. J., Sharp P. A. Evidence for two active sites in the spliceosome provided by stereochemistry of pre-mRNA splicing. Nature. 1993 Sep 23;365(6444):364–368. doi: 10.1038/365364a0. [DOI] [PubMed] [Google Scholar]
  20. Moore M. J., Sharp P. A. Site-specific modification of pre-mRNA: the 2'-hydroxyl groups at the splice sites. Science. 1992 May 15;256(5059):992–997. doi: 10.1126/science.1589782. [DOI] [PubMed] [Google Scholar]
  21. Mörl M., Schmelzer C. Group II intron RNA-catalyzed recombination of RNA in vitro. Nucleic Acids Res. 1990 Nov 25;18(22):6545–6551. doi: 10.1093/nar/18.22.6545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Padgett R. A., Podar M., Boulanger S. C., Perlman P. S. The stereochemical course of group II intron self-splicing. Science. 1994 Dec 9;266(5191):1685–1688. doi: 10.1126/science.7527587. [DOI] [PubMed] [Google Scholar]
  23. Pecoraro V. L., Hermes J. D., Cleland W. W. Stability constants of Mg2+ and Cd2+ complexes of adenine nucleotides and thionucleotides and rate constants for formation and dissociation of MgATP and MgADP. Biochemistry. 1984 Oct 23;23(22):5262–5271. doi: 10.1021/bi00317a026. [DOI] [PubMed] [Google Scholar]
  24. Peebles C. L., Zhang M., Perlman P. S., Franzen J. S. Catalytically critical nucleotide in domain 5 of a group II intron. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4422–4426. doi: 10.1073/pnas.92.10.4422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sharp P. A. "Five easy pieces". Science. 1991 Nov 1;254(5032):663–663. doi: 10.1126/science.1948046. [DOI] [PubMed] [Google Scholar]
  26. Slim G., Gait M. J. Configurationally defined phosphorothioate-containing oligoribonucleotides in the study of the mechanism of cleavage of hammerhead ribozymes. Nucleic Acids Res. 1991 Mar 25;19(6):1183–1188. doi: 10.1093/nar/19.6.1183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Steitz T. A., Steitz J. A. A general two-metal-ion mechanism for catalytic RNA. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6498–6502. doi: 10.1073/pnas.90.14.6498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Suchy M., Schmelzer C. Restoration of the self-splicing activity of a defective group II intron by a small trans-acting RNA. J Mol Biol. 1991 Nov 20;222(2):179–187. doi: 10.1016/0022-2836(91)90204-j. [DOI] [PubMed] [Google Scholar]

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

RESOURCES