Skip to main content
NCBI home page
RNA logoLink to RNA
. 1998 Aug;4(8):890–900. doi: 10.1017/s1355838298971643

The two steps of group II intron self-splicing are mechanistically distinguishable.

M Podar 1, P S Perlman 1, R A Padgett 1
PMCID: PMC1369667  PMID: 9701281

Abstract

The two transesterification reactions catalyzed by self-splicing group II introns take place in either two active sites or two conformations of a single active site involving rearrangements of the positions of the reacting groups. We have investigated the effects on the rates of the chemical steps of the two reactions due to sulfur substitution of nonbridging oxygens at both the 5' and 3' splice sites as well as the deoxyribose substitution of the ribose 2' hydroxyl group at the 5' splice site. The data suggest that the two active sites differ in their interactions with several of these groups. Specifically, sulfur substitution of the pro-Sp nonbridging oxygen at the 5' splice site reduces the chemical rate of the step one branching reaction by at least 250-fold, whereas substitution of the pro-Sp oxygen at the 3' splice site has only a 4.5-fold effect on the chemical rate of step two. Previous work demonstrated that the Rp phosphorothioate substitutions at both the 5' and 3' splice sites reduced the rate of both steps of splicing to an undetectable level. These results suggest that either two distinct active sites catalyze the two steps or that more significant alterations must be made in a single bifunctional active site to accommodate the two different reactions.

Full Text

The Full Text of this article is available as a PDF (333.0 KB).

Selected References

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

  1. Bass B. L., Cech T. R. Specific interaction between the self-splicing RNA of Tetrahymena and its guanosine substrate: implications for biological catalysis by RNA. 1984 Apr 26-May 2Nature. 308(5962):820–826. doi: 10.1038/308820a0. [DOI] [PubMed] [Google Scholar]
  2. Chanfreau G., Jacquier A. An RNA conformational change between the two chemical steps of group II self-splicing. EMBO J. 1996 Jul 1;15(13):3466–3476. [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Chin K., Pyle A. M. Branch-point attack in group II introns is a highly reversible transesterification, providing a potential proofreading mechanism for 5'-splice site selection. RNA. 1995 Jun;1(4):391–406. [PMC free article] [PubMed] [Google Scholar]
  5. Dahm S. C., Uhlenbeck O. C. Role of divalent metal ions in the hammerhead RNA cleavage reaction. Biochemistry. 1991 Oct 1;30(39):9464–9469. doi: 10.1021/bi00103a011. [DOI] [PubMed] [Google Scholar]
  6. Daniels D. L., Michels W. J., Jr, Pyle A. M. Two competing pathways for self-splicing by group II introns: a quantitative analysis of in vitro reaction rates and products. J Mol Biol. 1996 Feb 16;256(1):31–49. doi: 10.1006/jmbi.1996.0066. [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. Griffin E. A., Jr, Qin Z., Michels W. J., Jr, Pyle A. M. Group II intron ribozymes that cleave DNA and RNA linkages with similar efficiency, and lack contacts with substrate 2'-hydroxyl groups. Chem Biol. 1995 Nov;2(11):761–770. doi: 10.1016/1074-5521(95)90104-3. [DOI] [PubMed] [Google Scholar]
  9. Herschlag D., Eckstein F., Cech T. R. Contributions of 2'-hydroxyl groups of the RNA substrate to binding and catalysis by the Tetrahymena ribozyme. An energetic picture of an active site composed of RNA. Biochemistry. 1993 Aug 17;32(32):8299–8311. doi: 10.1021/bi00083a034. [DOI] [PubMed] [Google Scholar]
  10. Herschlag D., Eckstein F., Cech T. R. The importance of being ribose at the cleavage site in the Tetrahymena ribozyme reaction. Biochemistry. 1993 Aug 17;32(32):8312–8321. doi: 10.1021/bi00083a035. [DOI] [PubMed] [Google Scholar]
  11. Herschlag D., Khosla M. Comparison of pH dependencies of the Tetrahymena ribozyme reactions with RNA 2'-substituted and phosphorothioate substrates reveals a rate-limiting conformational step. Biochemistry. 1994 May 3;33(17):5291–5297. doi: 10.1021/bi00183a036. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Jacquier A. Group II introns: elaborate ribozymes. Biochimie. 1996;78(6):474–487. doi: 10.1016/0300-9084(96)84754-7. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Kao T. H., Crothers D. M. A proton-coupled conformational switch of Escherichia coli 5S ribosomal RNA. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3360–3364. doi: 10.1073/pnas.77.6.3360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Knitt D. S., Herschlag D. pH dependencies of the Tetrahymena ribozyme reveal an unconventional origin of an apparent pKa. Biochemistry. 1996 Feb 6;35(5):1560–1570. doi: 10.1021/bi9521147. [DOI] [PubMed] [Google Scholar]
  17. Koizumi M., Ohtsuka E. Effects of phosphorothioate and 2-amino groups in hammerhead ribozymes on cleavage rates and Mg2+ binding. Biochemistry. 1991 May 28;30(21):5145–5150. doi: 10.1021/bi00235a005. [DOI] [PubMed] [Google Scholar]
  18. Konforti B. B., Abramovitz D. L., Duarte C. M., Karpeisky A., Beigelman L., Pyle A. M. Ribozyme catalysis from the major groove of group II intron domain 5. Mol Cell. 1998 Feb;1(3):433–441. doi: 10.1016/s1097-2765(00)80043-x. [DOI] [PubMed] [Google Scholar]
  19. Macaya R., Wang E., Schultze P., Sklenár V., Feigon J. Proton nuclear magnetic resonance assignments and structural characterization of an intramolecular DNA triplex. J Mol Biol. 1992 Jun 5;225(3):755–773. doi: 10.1016/0022-2836(92)90399-5. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Michel F., Ferat J. L. Structure and activities of group II introns. Annu Rev Biochem. 1995;64:435–461. doi: 10.1146/annurev.bi.64.070195.002251. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]
  26. Narlikar G. J., Herschlag D. Mechanistic aspects of enzymatic catalysis: lessons from comparison of RNA and protein enzymes. Annu Rev Biochem. 1997;66:19–59. doi: 10.1146/annurev.biochem.66.1.19. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Podar M., Perlman P. S., Padgett R. A. Stereochemical selectivity of group II intron splicing, reverse splicing, and hydrolysis reactions. Mol Cell Biol. 1995 Aug;15(8):4466–4478. doi: 10.1128/mcb.15.8.4466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Puglisi J. D., Wyatt J. R., Tinoco I., Jr Solution conformation of an RNA hairpin loop. Biochemistry. 1990 May 1;29(17):4215–4226. doi: 10.1021/bi00469a026. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Smith D., Pace N. R. Multiple magnesium ions in the ribonuclease P reaction mechanism. Biochemistry. 1993 May 25;32(20):5273–5281. doi: 10.1021/bi00071a001. [DOI] [PubMed] [Google Scholar]
  34. Sontheimer E. J., Sun S., Piccirilli J. A. Metal ion catalysis during splicing of premessenger RNA. Nature. 1997 Aug 21;388(6644):801–805. doi: 10.1038/42068. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Suh E., Waring R. B. A phosphorothioate at the 3' splice-site inhibits the second splicing step in a group I intron. Nucleic Acids Res. 1992 Dec 11;20(23):6303–6309. doi: 10.1093/nar/20.23.6303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zaug A. J., Dávila-Aponte J. A., Cech T. R. Catalysis of RNA cleavage by a ribozyme derived from the group I intron of Anabaena pre-tRNA(Leu). Biochemistry. 1994 Dec 13;33(49):14935–14947. doi: 10.1021/bi00253a033. [DOI] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES