Skip to main content
RNA logoLink to RNA
. 1998 Aug;4(8):901–914. doi: 10.1017/s1355838298980621

Destabilizing effect of an rRNA stem-loop on an attenuator hairpin in the 5' exon of the Tetrahymena pre-rRNA.

Y Cao 1, S A Woodson 1
PMCID: PMC1369668  PMID: 9701282

Abstract

Self-splicing of the Tetrahymena group I intron is attenuated by an rRNA stem-loop in the 5' exon, which competes with formation of the P1 splice site helix. The equilibrium between the P1 and P(-1) stem-loops is influenced by rRNA sequences upstream and downstream of the intron. To investigate the mechanism of this conformational switch, internal deletions and point mutations were introduced in the second rRNA stem-loop upstream of the 5' splice site. Nuclease protection, native gel electrophoresis, and self-splicing results show that this helix is important for maintaining self-splicing activity. Co-axial base stacking of adjacent helices in the 5' exon is proposed to enable exchange between inactive and active conformations of the pre-rRNA.

Full Text

The Full Text of this article is available as a PDF (623.8 KB).

Selected References

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

  1. Altuvia S., Kornitzer D., Teff D., Oppenheim A. B. Alternative mRNA structures of the cIII gene of bacteriophage lambda determine the rate of its translation initiation. J Mol Biol. 1989 Nov 20;210(2):265–280. doi: 10.1016/0022-2836(89)90329-x. [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. Cech T. R. Self-splicing of group I introns. Annu Rev Biochem. 1990;59:543–568. doi: 10.1146/annurev.bi.59.070190.002551. [DOI] [PubMed] [Google Scholar]
  4. Clark C. G., Tague B. W., Ware V. C., Gerbi S. A. Xenopus laevis 28S ribosomal RNA: a secondary structure model and its evolutionary and functional implications. Nucleic Acids Res. 1984 Aug 10;12(15):6197–6220. doi: 10.1093/nar/12.15.6197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dammel C. S., Noller H. F. A cold-sensitive mutation in 16S rRNA provides evidence for helical switching in ribosome assembly. Genes Dev. 1993 Apr;7(4):660–670. doi: 10.1101/gad.7.4.660. [DOI] [PubMed] [Google Scholar]
  6. Donis-Keller H. Phy M: an RNase activity specific for U and A residues useful in RNA sequence analysis. Nucleic Acids Res. 1980 Jul 25;8(14):3133–3142. doi: 10.1093/nar/8.14.3133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Donis-Keller H. Site specific enzymatic cleavage of RNA. Nucleic Acids Res. 1979 Sep 11;7(1):179–192. doi: 10.1093/nar/7.1.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ehresmann C., Baudin F., Mougel M., Romby P., Ebel J. P., Ehresmann B. Probing the structure of RNAs in solution. Nucleic Acids Res. 1987 Nov 25;15(22):9109–9128. doi: 10.1093/nar/15.22.9109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Emerick V. L., Pan J., Woodson S. A. Analysis of rate-determining conformational changes during self-splicing of the Tetrahymena intron. Biochemistry. 1996 Oct 15;35(41):13469–13477. doi: 10.1021/bi960865i. [DOI] [PubMed] [Google Scholar]
  10. Emerick V. L., Woodson S. A. Fingerprinting the folding of a group I precursor RNA. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9675–9679. doi: 10.1073/pnas.91.21.9675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Emerick V. L., Woodson S. A. Self-splicing of the Tetrahymena pre-rRNA is decreased by misfolding during transcription. Biochemistry. 1993 Dec 21;32(50):14062–14067. doi: 10.1021/bi00213a040. [DOI] [PubMed] [Google Scholar]
  12. Emerick V. L., Woodson S. A. Self-splicing of the Tetrahymena pre-rRNA is decreased by misfolding during transcription. Biochemistry. 1993 Dec 21;32(50):14062–14067. doi: 10.1021/bi00213a040. [DOI] [PubMed] [Google Scholar]
  13. Fedor M. J., Uhlenbeck O. C. Substrate sequence effects on "hammerhead" RNA catalytic efficiency. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1668–1672. doi: 10.1073/pnas.87.5.1668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fortner D. M., Troy R. G., Brow D. A. A stem/loop in U6 RNA defines a conformational switch required for pre-mRNA splicing. Genes Dev. 1994 Jan;8(2):221–233. doi: 10.1101/gad.8.2.221. [DOI] [PubMed] [Google Scholar]
  15. Franch T., Gultyaev A. P., Gerdes K. Programmed cell death by hok/sok of plasmid R1: processing at the hok mRNA 3'-end triggers structural rearrangements that allow translation and antisense RNA binding. J Mol Biol. 1997 Oct 17;273(1):38–51. doi: 10.1006/jmbi.1997.1294. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Jaeger J. A., Turner D. H., Zuker M. Predicting optimal and suboptimal secondary structure for RNA. Methods Enzymol. 1990;183:281–306. doi: 10.1016/0076-6879(90)83019-6. [DOI] [PubMed] [Google Scholar]
  18. Joseph S., Noller H. F. Mapping the rRNA neighborhood of the acceptor end of tRNA in the ribosome. EMBO J. 1996 Feb 15;15(4):910–916. [PMC free article] [PubMed] [Google Scholar]
  19. Kim J., Walter A. E., Turner D. H. Thermodynamics of coaxially stacked helixes with GA and CC mismatches. Biochemistry. 1996 Oct 29;35(43):13753–13761. doi: 10.1021/bi960913z. [DOI] [PubMed] [Google Scholar]
  20. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  21. Laing L. G., Draper D. E. Thermodynamics of RNA folding in a conserved ribosomal RNA domain. J Mol Biol. 1994 Apr 15;237(5):560–576. doi: 10.1006/jmbi.1994.1255. [DOI] [PubMed] [Google Scholar]
  22. LeCuyer K. A., Crothers D. M. The Leptomonas collosoma spliced leader RNA can switch between two alternate structural forms. Biochemistry. 1993 May 25;32(20):5301–5311. doi: 10.1021/bi00071a004. [DOI] [PubMed] [Google Scholar]
  23. Leviev I., Levieva S., Garrett R. A. Role for the highly conserved region of domain IV of 23S-like rRNA in subunit-subunit interactions at the peptidyl transferase centre. Nucleic Acids Res. 1995 May 11;23(9):1512–1517. doi: 10.1093/nar/23.9.1512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ma C. K., Kolesnikow T., Rayner J. C., Simons E. L., Yim H., Simons R. W. Control of translation by mRNA secondary structure: the importance of the kinetics of structure formation. Mol Microbiol. 1994 Dec;14(5):1033–1047. doi: 10.1111/j.1365-2958.1994.tb01337.x. [DOI] [PubMed] [Google Scholar]
  25. Michel F., Westhof E. Modelling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis. J Mol Biol. 1990 Dec 5;216(3):585–610. doi: 10.1016/0022-2836(90)90386-Z. [DOI] [PubMed] [Google Scholar]
  26. Milligan J. F., Uhlenbeck O. C. Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol. 1989;180:51–62. doi: 10.1016/0076-6879(89)80091-6. [DOI] [PubMed] [Google Scholar]
  27. Mitchell P., Osswald M., Brimacombe R. Identification of intermolecular RNA cross-links at the subunit interface of the Escherichia coli ribosome. Biochemistry. 1992 Mar 24;31(11):3004–3011. doi: 10.1021/bi00126a023. [DOI] [PubMed] [Google Scholar]
  28. Moazed D., Noller H. F. Interaction of tRNA with 23S rRNA in the ribosomal A, P, and E sites. Cell. 1989 May 19;57(4):585–597. doi: 10.1016/0092-8674(89)90128-1. [DOI] [PubMed] [Google Scholar]
  29. Narlikar G. J., Herschlag D. Isolation of a local tertiary folding transition in the context of a globally folded RNA. Nat Struct Biol. 1996 Aug;3(8):701–710. doi: 10.1038/nsb0896-701. [DOI] [PubMed] [Google Scholar]
  30. Noller H. F., Green R., Heilek G., Hoffarth V., Hüttenhofer A., Joseph S., Lee I., Lieberman K., Mankin A., Merryman C. Structure and function of ribosomal RNA. Biochem Cell Biol. 1995 Nov-Dec;73(11-12):997–1009. doi: 10.1139/o95-107. [DOI] [PubMed] [Google Scholar]
  31. Noller H. F., Kop J., Wheaton V., Brosius J., Gutell R. R., Kopylov A. M., Dohme F., Herr W., Stahl D. A., Gupta R. Secondary structure model for 23S ribosomal RNA. Nucleic Acids Res. 1981 Nov 25;9(22):6167–6189. doi: 10.1093/nar/9.22.6167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Panyutin I. G., Hsieh P. The kinetics of spontaneous DNA branch migration. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2021–2025. doi: 10.1073/pnas.91.6.2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Petersheim M., Turner D. H. Base-stacking and base-pairing contributions to helix stability: thermodynamics of double-helix formation with CCGG, CCGGp, CCGGAp, ACCGGp, CCGGUp, and ACCGGUp. Biochemistry. 1983 Jan 18;22(2):256–263. doi: 10.1021/bi00271a004. [DOI] [PubMed] [Google Scholar]
  34. Pyle A. M., Cech T. R. Ribozyme recognition of RNA by tertiary interactions with specific ribose 2'-OH groups. Nature. 1991 Apr 18;350(6319):628–631. doi: 10.1038/350628a0. [DOI] [PubMed] [Google Scholar]
  35. Semrad K., Schroeder R. A ribosomal function is necessary for efficient splicing of the T4 phage thymidylate synthase intron in vivo. Genes Dev. 1998 May 1;12(9):1327–1337. doi: 10.1101/gad.12.9.1327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Spedding G., Draper D. E. Allosteric mechanism for translational repression in the Escherichia coli alpha operon. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4399–4403. doi: 10.1073/pnas.90.10.4399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Staley J. P., Guthrie C. Mechanical devices of the spliceosome: motors, clocks, springs, and things. Cell. 1998 Feb 6;92(3):315–326. doi: 10.1016/s0092-8674(00)80925-3. [DOI] [PubMed] [Google Scholar]
  38. Séraphin B., Simon M., Faye G. MSS18, a yeast nuclear gene involved in the splicing of intron aI5 beta of the mitochondrial cox1 transcript. EMBO J. 1988 May;7(5):1455–1464. doi: 10.1002/j.1460-2075.1988.tb02963.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Walstrum S. A., Uhlenbeck O. C. The self-splicing RNA of Tetrahymena is trapped in a less active conformation by gel purification. Biochemistry. 1990 Nov 20;29(46):10573–10576. doi: 10.1021/bi00498a022. [DOI] [PubMed] [Google Scholar]
  40. Wilson K. S., Noller H. F. Molecular movement inside the translational engine. Cell. 1998 Feb 6;92(3):337–349. doi: 10.1016/s0092-8674(00)80927-7. [DOI] [PubMed] [Google Scholar]
  41. Woodson S. A., Cech T. R. Alternative secondary structures in the 5' exon affect both forward and reverse self-splicing of the Tetrahymena intervening sequence RNA. Biochemistry. 1991 Feb 26;30(8):2042–2050. doi: 10.1021/bi00222a006. [DOI] [PubMed] [Google Scholar]
  42. Woodson S. A., Emerick V. L. An alternative helix in the 26S rRNA promotes excision and integration of the Tetrahymena intervening sequence. Mol Cell Biol. 1993 Feb;13(2):1137–1145. doi: 10.1128/mcb.13.2.1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Woodson S. A. Exon sequences distant from the splice junction are required for efficient self-splicing of the Tetrahymena IVS. Nucleic Acids Res. 1992 Aug 11;20(15):4027–4032. doi: 10.1093/nar/20.15.4027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yanofsky C. Attenuation in the control of expression of bacterial operons. Nature. 1981 Feb 26;289(5800):751–758. doi: 10.1038/289751a0. [DOI] [PubMed] [Google Scholar]
  45. Zaug A. J., Grosshans C. A., Cech T. R. Sequence-specific endoribonuclease activity of the Tetrahymena ribozyme: enhanced cleavage of certain oligonucleotide substrates that form mismatched ribozyme-substrate complexes. Biochemistry. 1988 Dec 13;27(25):8924–8931. doi: 10.1021/bi00425a008. [DOI] [PubMed] [Google Scholar]
  46. Zaug A. J., McEvoy M. M., Cech T. R. Self-splicing of the group I intron from Anabaena pre-tRNA: requirement for base-pairing of the exons in the anticodon stem. Biochemistry. 1993 Aug 10;32(31):7946–7953. doi: 10.1021/bi00082a016. [DOI] [PubMed] [Google Scholar]
  47. Zavanelli M. I., Britton J. S., Igel A. H., Ares M., Jr Mutations in an essential U2 small nuclear RNA structure cause cold-sensitive U2 small nuclear ribonucleoprotein function by favoring competing alternative U2 RNA structures. Mol Cell Biol. 1994 Mar;14(3):1689–1697. doi: 10.1128/mcb.14.3.1689. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES