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Translocation of an RNA duplex on a ribozyme

Nature Structural Biology volume 1, pages 13–17 (1994)Cite this article

Abstract

RNA cleavage by the Tetrahymena ribozyme requires recognition of the reaction–site helix by the catalytic apparatus. This binding can occur in several registers, each of which results in reaction at a different nucleotide in the helix. We now identify commensurate sets of 2′–hydroxyl interactions on both strands of the reaction–site helix that account for its translocation into alternative binding registers. These results indicate that the ribozyme has a relatively rigid substrate–binding pocket into which the helix can bind in different alignments. A similar mechanism of reaction site recognition is proposed to occur during intron circularization and ribozyme polymerase activity. Translocation of the reaction site duplex provides an example of structural heterogeneity in packing of helices during the teritary folding of RNA.

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References

  1. 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. 267, 17479–17482 (1992).

    CAS  PubMed  Google Scholar 

  2. 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 27, 8924–8931 (1988).

    Article  CAS  Google Scholar 

  3. Herschlag, D. & Cech, T.R. Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site. Biochemistry 29, 10159–10171 (1990).

    Article  CAS  Google Scholar 

  4. Herschlag, D. Evidence for processivity and two-step binding of the RNA substrate from studies of J1/2 mutants of the Tetrahymena ribozyme. Biochemistry 31, 1386–1399 (1992).

    Article  CAS  Google Scholar 

  5. Bevilacqua, P.C., Kierzek, R., Johnson, K.A. & Turner, D.H. Dynamics of ribozyme binding of substrate revealed by fluorescence-detected stopped-flow methods. Science 258, 1355–1358 (1992).

    Article  CAS  Google Scholar 

  6. 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 47, 207–216 (1986).

    Article  CAS  Google Scholar 

  7. Waring, R.B., Towner, P., Minter, S.J. & Davies, R.W. Splice-site selection by a self-splicing RNA of Tetrahymena. Nature 321, 133–139 (1986).

    Article  CAS  Google Scholar 

  8. Pyle, A.M. & Cech, T.R. Ribozyme recognition of RNA by tertiary interactions with specific ribose 2′-OH groups. Nature 350, 628–631 (1991).

    Article  CAS  Google Scholar 

  9. Bevilacqua, P.C. & Turner, D.H. Comparison of binding of mixed ribose-deoxyribose analogues of CUCU to a ribozyme and to GGAGAA by equilibrium dialysis: evidence for ribozyme specific interaction with 2′-OH groups. Biochemistry 30, 10632–10640 (1991).

    Article  CAS  Google Scholar 

  10. 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 32, 8299–8311 (1993).

    Article  CAS  Google Scholar 

  11. Strobel, S.A. & Cech, T.R. Tertiary interactions with the internal guide sequence mediate docking of the P1 helix into the catalytic core of the Tetrahymena ribozyme. Biochemistry (in the press).

  12. Been, M.D. & Cech, T.R. Selection of circularization sites in a group I IVS RNA requires multiple alignments of an internal template-like sequence. Cell 50, 951–961 (1987).

    Article  CAS  Google Scholar 

  13. Doudna, J.A., Cormack, B.P. & Szostak, J.W. RNA structure, not sequence, determines the 5′ splice-site specificity of a group I intron. Proc. natn. Acad. Sci. U.S.A. 86, 7402–7406 (1989).

    Article  CAS  Google Scholar 

  14. Moore, M.J. & Sharp, P.A. Site-specific modification of pre-mRNA: the 2′-hydroxyl groups at the splice sites. Science 256, 992–997 (1992).

    Article  CAS  Google Scholar 

  15. Herschlag, D., Piccirilli, J.A. & Cech, T.R. Ribozyme-catalyzed and non-enzymatic reactions of phosphate diesters: rate effects upon substitution of sulfur for a nonbridging phosphoryl oxygen atom. Biochemistry 30, 4844–4854 (1991).

    Article  CAS  Google Scholar 

  16. Barfod, E.T. & Cech, T.R. The conserved U·G pair in the 5′ splice site duplex of a group I intron is required in the first but not the second step of self-splicing. Molec. cell. Biol. 9, 3657–3666 (1989).

    Article  CAS  Google Scholar 

  17. Young, B., Herschlag, D. & Cech, T.R. Mutations in a nonconserved sequence of the Tetrahymena ribozyme increase activity and specificity. Cell 67, 1007–1019 (1991).

    Article  CAS  Google Scholar 

  18. Downs, W.D. & Cech, T.R. An ultraviolet-inducible adenosine-adenosine crosslink reflects the catalytic structure of the Tetrahymena ribozyme. Biochemistry 29, 5605–5613 (1990).

    Article  CAS  Google Scholar 

  19. Doudna, J.A. & Szostak, J.W. RNA-catalysed synthesis of complementary-strand RNA. Nature 339, 519–522 (1989).

    Article  CAS  Google Scholar 

  20. Been, M.D. & Cech, T.R. RNA as an RNA polymerase: net elongation of an RNA primer catalyzed by the Tetrahymena ribozyme. Science 239, 1412–1416 (1988).

    Article  CAS  Google Scholar 

  21. Doudna, J.A., Couture, S. & Szostak, J.W. A multisubunit ribozyme that is a catalyst of and template for complementary strand RNA synthesis. Science 251, 1605–1608 (1991).

    Article  CAS  Google Scholar 

  22. Holbrook, S.R., Cheong, C., Tinoco, I. & Kim, S.H. Crystal structure of an RNA double helix incorporating a track of non-Watson-Crick base pairs. Nature 353, 579–581 (1992).

    Article  Google Scholar 

  23. Michel, F., Hanna, M., Green, R., Bartel, D.P. & Szostak, J.W. The guanosine binding site of the Tetrahymena ribozyme. Nature 342, 391–395 (1989).

    Article  CAS  Google Scholar 

  24. Yarus, M., Illangesekare, M. & Christian, E. An axial binding site in the Tetrahymena precursor RNA. J. molec. Biol. 222, 995–1012 (1991).

    Article  CAS  Google Scholar 

  25. Pyle, A.M., Murphy, F.L. & Cech, T.R. RNA substrate binding site in the catalytic core of the Tetrahymena ribozyme. Nature 358, 123–128 (1992).

    Article  CAS  Google Scholar 

  26. Michel, F. & Westhof, E. Modelling of the three-dimensional architecture of Group I catalytic introns based on comparative sequence analysis. J. molec. Biol. 216, 585–610 (1990).

    Article  CAS  Google Scholar 

  27. Wang, J.F. & Cech, T.R. Tertiary structure around the guanosine-binding site of the Tetrahymena ribozyme. Science 256, 526–529 (1992).

    Article  CAS  Google Scholar 

  28. Wang, J.F., Downs, W.D. & Cech, T.R. Movement of the guide sequence during RNA catalysis by a group I ribozyme. Science 260, 504–508 (1993).

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Howard Hughes Medical Institute Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO, 80309-0215, USA

    Scott A. Strobel & Thomas R. Cech

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  1. Scott A. Strobel
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  2. Thomas R. Cech
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Strobel, S., Cech, T. Translocation of an RNA duplex on a ribozyme. Nat Struct Mol Biol 1, 13–17 (1994). https://doi.org/10.1038/nsb0194-13

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  • DOI: https://doi.org/10.1038/nsb0194-13

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