The structural basis of RNA-catalyzed RNA polymerization

Abstract

Early life presumably required polymerase ribozymes capable of replicating RNA. Known polymerase ribozymes best approximating such replicases use as their catalytic engine an RNA-ligase ribozyme originally selected from random RNA sequences. Here we report 3.15-Å crystal structures of this ligase trapped in catalytically viable preligation states, with the 3′-hydroxyl nucleophile positioned for in-line attack on the 5′-triphosphate. Guided by metal- and solvent-mediated interactions, the 5′-triphosphate hooks into the major groove of the adjoining RNA duplex in an unanticipated conformation. Two phosphates and the nucleophile jointly coordinate an active-site metal ion. Atomic mutagenesis experiments demonstrate that active-site nucleobase and hydroxyl groups also participate directly in catalysis, collectively playing a role that in proteinaceous polymerases is performed by a second metal ion. Thus artificial ribozymes can use complex catalytic strategies that differ markedly from those of analogous biological enzymes.

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Figure 1: The class I ligase ribozyme preligation complex.
Figure 2: The class I ligase active site.
Figure 3: Biochemical interrogation of C47.
Figure 4: Catalytic roles of active-site functional groups.
Figure 5: Transition-state stabilization by protein and RNA active sites.

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References

  1. 1

    Joyce, G.F. & Orgel, L.E. Prospects for understanding the origin of the RNA world. in The RNA World 2nd edn. (eds. Gesteland, R.F., Cech, T.R. & Atkins, J.F.) 49–77 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 1999).

  2. 2

    Orgel, L.E. Prebiotic chemistry and the origin of the RNA world. Crit. Rev. Biochem. Mol. Biol. 39, 99–123 (2004).

    CAS  Article  PubMed  Google Scholar 

  3. 3

    White, H.B. III. Coenzymes as fossils of an earlier metabolic state. J. Mol. Evol. 7, 101–104 (1976).

    CAS  Article  PubMed  Google Scholar 

  4. 4

    Nissen, P., Hansen, J., Ban, N., Moore, P.B. & Steitz, T.A. The structural basis of ribosome activity in peptide bond synthesis. Science 289, 920–930 (2000).

    CAS  Article  PubMed  Google Scholar 

  5. 5

    Fedor, M.J. & Williamson, J.R. The catalytic diversity of RNAs. Nat. Rev. Mol. Cell Biol. 6, 399–412 (2005).

    CAS  Article  PubMed  Google Scholar 

  6. 6

    Chen, X., Li, N. & Ellington, A.D. Ribozyme catalysis of metabolism in the RNA world. Chem. Biodivers. 4, 633–655 (2007).

    CAS  Article  PubMed  Google Scholar 

  7. 7

    Lincoln, T.A. & Joyce, G.F. Self-sustained replication of an RNA enzyme. Science 323, 1229–1232 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8

    Bartel, D.P. Re-creating an RNA replicase. in The RNA World 2nd edn. (eds. Gesteland, R.F., Cech, T.R. & Atkins, J.F.) 143–162 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 1999).

  9. 9

    McGinness, K.E. & Joyce, G.F. In search of an RNA replicase ribozyme. Chem. Biol. 10, 5–14 (2003).

    CAS  Article  PubMed  Google Scholar 

  10. 10

    Bartel, D.P. & Szostak, J.W. Isolation of new ribozymes from a large pool of random sequences. Science 261, 1411–1418 (1993).

    CAS  Article  Google Scholar 

  11. 11

    Ekland, E.H., Szostak, J.W. & Bartel, D.P. Structurally complex and highly active RNA ligases derived from random RNA sequences. Science 269, 364–370 (1995).

    CAS  Article  Google Scholar 

  12. 12

    Ekland, E.H. & Bartel, D.P. RNA-catalysed RNA polymerization using nucleoside triphosphates. Nature 382, 373–376 (1996).

    CAS  Article  PubMed  Google Scholar 

  13. 13

    Johnston, W.K., Unrau, P.J., Lawrence, M.S., Glasner, M.E. & Bartel, D.P. RNA-catalyzed RNA polymerization: accurate and general RNA-templated primer extension. Science 292, 1319–1325 (2001).

    CAS  Article  Google Scholar 

  14. 14

    Lawrence, M.S. & Bartel, D.P. New ligase-derived RNA polymerase ribozymes. RNA 11, 1173–1180 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15

    Zaher, H.S. & Unrau, P.J. Selection of an improved RNA polymerase ribozyme with superior extension and fidelity. RNA 13, 1017–1026 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16

    Wochner, A., Attwater, J., Coulson, A. & Holliger, P. Ribozyme-catalyzed transcription of an active ribozyme. Science 332, 209–212 (2011).

    CAS  Article  PubMed  Google Scholar 

  17. 17

    Shechner, D.M. et al. Crystal structure of the catalytic core of an RNA-polymerase ribozyme. Science 326, 1271–1275 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18

    Bagby, S.C., Bergman, N.H., Shechner, D.M., Yen, C. & Bartel, D.P. A class I ligase ribozyme with reduced Mg2+ dependence: selection, sequence analysis, and identification of functional tertiary interactions. RNA 15, 2129–2146 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19

    Stuhlmann, F. & Jaschke, A. Characterization of an RNA active site: interactions between a Diels-Alderase ribozyme and its substrates and products. J. Am. Chem. Soc. 124, 3238–3244 (2002).

    CAS  Article  PubMed  Google Scholar 

  20. 20

    Serganov, A. et al. Structural basis for Diels-Alder ribozyme-catalyzed carbon-carbon bond formation. Nat. Struct. Mol. Biol. 12, 218–224 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21

    Robertson, M.P. & Scott, W.G. The structural basis of ribozyme-catalyzed RNA assembly. Science 315, 1549–1553 (2007).

    CAS  Article  Google Scholar 

  22. 22

    Xiao, H., Murakami, H., Suga, H. & Ferre-D'Amare, A.R. Structural basis of specific tRNA aminoacylation by a small in vitro selected ribozyme. Nature 454, 358–361 (2008).

    CAS  Article  Google Scholar 

  23. 23

    Pitt, J.N. & Ferre-D'Amare, A.R. Structure-guided engineering of the regioselectivity of RNA ligase ribozymes. J. Am. Chem. Soc. 131, 3532–3540 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24

    Glasner, M.E., Bergman, N.H. & Bartel, D.P. Metal ion requirements for structure and catalysis of an RNA ligase ribozyme. Biochemistry 41, 8103–8112 (2002).

    CAS  Article  PubMed  Google Scholar 

  25. 25

    Harding, M.M. Geometry of metal-ligand interactions in proteins. Acta Crystallogr. D Biol. Crystallogr. 57, 401–411 (2001).

    CAS  Article  PubMed  Google Scholar 

  26. 26

    Chen, J.H. et al. A 1.9 A crystal structure of the HDV ribozyme precleavage suggests both Lewis acid and general acid mechanisms contribute to phosphodiester cleavage. Biochemistry 49, 6508–6518 (2010).

    CAS  Article  PubMed  Google Scholar 

  27. 27

    Steitz, T.A. A mechanism for all polymerases. Nature 391, 231–232 (1998).

    CAS  Article  Google Scholar 

  28. 28

    Castro, C. et al. Nucleic acid polymerases use a general acid for nucleotidyl transfer. Nat. Struct. Mol. Biol. 16, 212–218 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29

    Hsiao, C. et al. Complexes of nucleic acids with group I and II cations. in Nucleic Acid-Metal Ion Interactions Vol. 1 (ed. Hud, N.V.) 1–38 (RSC Publishing, Cambridge, UK, 2009).

  30. 30

    Harding, M.M. The geometry of metal-ligand interactions relevant to proteins. II. Angles at the metal atom, additional weak metal-donor interactions. Acta Crystallogr. D Biol. Crystallogr. 56, 857–867 (2000).

    CAS  Article  PubMed  Google Scholar 

  31. 31

    Glasner, M.E., Yen, C.C., Ekland, E.H. & Bartel, D.P. Recognition of nucleoside triphosphates during RNA-catalyzed primer extension. Biochemistry 39, 15556–15562 (2000).

    CAS  Article  PubMed  Google Scholar 

  32. 32

    Basu, S. & Strobel, S.A. Thiophilic metal ion rescue of phosphorothioate interference within the Tetrahymena ribozyme P4–P6 domain. RNA 5, 1399–1407 (1999).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33

    Sträter, N., Lipscomb, W.N., Klabunde, T. & Krebs, B. Two-metal ion catalysis in enzymatic acyl- and phosphoryl-transfer reactions. Angew. Chem. Int. Edn Engl. 35, 2024–2055 (1996).

    Article  Google Scholar 

  34. 34

    Frederiksen, J.K. & Piccirilli, J.A. Identification of catalytic metal ion ligands in ribozymes. Methods 49, 148–166 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35

    Saenger, W. Principles of Nucleic Acid Structure (Springer-Verlag, New York, 1984).

  36. 36

    Jencks, W.P. Catalysis in Chemistry and Enzymology (McGraw-Hill, New York, 1969).

  37. 37

    Venkatasubban, K.S. & Schowen, R.L. The proton inventory technique. CRC Crit. Rev. Biochem. 17, 1–44 (1984).

    CAS  Article  Google Scholar 

  38. 38

    Schowen, K.B. & Schowen, R.L. Solvent isotope effects of enzyme systems. Methods Enzymol. 87, 551–606 (1982).

    CAS  Article  PubMed  Google Scholar 

  39. 39

    Das, S.R. & Piccirilli, J.A. General acid catalysis by the hepatitis delta virus ribozyme. Nat. Chem. Biol. 1, 45–52 (2005).

    CAS  Article  PubMed  Google Scholar 

  40. 40

    Gong, B. et al. Direct measurement of a pKa near neutrality for the catalytic cytosine in the genomic HDV ribozyme using Raman crystallography. J. Am. Chem. Soc. 129, 13335–13342 (2007).

    CAS  Article  PubMed  Google Scholar 

  41. 41

    Stahley, M.R. & Strobel, S.A. Structural evidence for a two-metal-ion mechanism of group I intron splicing. Science 309, 1587–1590 (2005).

    CAS  Article  PubMed  Google Scholar 

  42. 42

    Toor, N., Keating, K.S., Taylor, S.D. & Pyle, A.M. Crystal structure of a self-spliced group II intron. Science 320, 77–82 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. 43

    Yang, W. An equivalent metal ion in one- and two-metal-ion catalysis. Nat. Struct. Mol. Biol. 15, 1228–1231 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  44. 44

    Fedor, M.J. Comparative enzymology and structural biology of RNA self-cleavage. Annu. Rev. Biophys. 38, 271–299 (2009).

    CAS  Article  PubMed  Google Scholar 

  45. 45

    Bevilacqua, P.C. & Yajima, R. Nucleobase catalysis in ribozyme mechanism. Curr. Opin. Chem. Biol. 10, 455–464 (2006).

    CAS  Article  PubMed  Google Scholar 

  46. 46

    Ke, A., Zhou, K., Ding, F., Cate, J.H. & Doudna, J.A. A conformational switch controls hepatitis delta virus ribozyme catalysis. Nature 429, 201–205 (2004).

    CAS  Article  PubMed  Google Scholar 

  47. 47

    Lupták, A., Ferre-D'Amare, A.R., Zhou, K., Zilm, K.W. & Doudna, J.A. Direct pKa measurement of the active-site cytosine in a genomic hepatitis delta virus ribozyme. J. Am. Chem. Soc. 123, 8447–8452 (2001).

    Article  PubMed  Google Scholar 

  48. 48

    Klein, D.J. & Ferre-D'Amare, A.R. Structural basis of glmS ribozyme activation by glucosamine-6-phosphate. Science 313, 1752–1756 (2006).

    CAS  Article  Google Scholar 

  49. 49

    Cochrane, J.C., Lipchock, S.V. & Strobel, S.A. Structural investigation of the GlmS ribozyme bound to Its catalytic cofactor. Chem. Biol. 14, 97–105 (2007).

    CAS  Article  Google Scholar 

  50. 50

    Nesbitt, S., Hegg, L.A. & Fedor, M.J. An unusual pH-independent and metal-ion-independent mechanism for hairpin ribozyme catalysis. Chem. Biol. 4, 619–630 (1997).

    CAS  Article  PubMed  Google Scholar 

  51. 51

    Rupert, P.B., Massey, A.P., Sigurdsson, S.T. & Ferre-D'Amare, A.R. Transition state stabilization by a catalytic RNA. Science 298, 1421–1424 (2002).

    CAS  Article  PubMed  Google Scholar 

  52. 52

    Salter, J., Krucinska, J., Alam, S., Grum-Tokars, V. & Wedekind, J.E. Water in the active site of an all-RNA hairpin ribozyme and effects of Gua8 base variants on the geometry of phosphoryl transfer. Biochemistry 45, 686–700 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  53. 53

    Emilsson, G.M., Nakamura, S., Roth, A. & Breaker, R.R. Ribozyme speed limits. RNA 9, 907–918 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. 54

    Ferré-D'Amaré, A.R. & Doudna, J.A. Crystallization and structure determination of a hepatitis delta virus ribozyme: use of the RNA-binding protein U1A as a crystallization module. J. Mol. Biol. 295, 541–556 (2000).

    Article  Google Scholar 

  55. 55

    Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collection in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  56. 56

    McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  57. 57

    Adams, P.D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954 (2002).

    Article  PubMed  Google Scholar 

  58. 58

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  59. 59

    Bullard, D.R. & Bowater, R.P. Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4. Biochem. J. 398, 135–144 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank F. Eckstein for the gift of GTP analogs, R.A. Grant, D. Lim, T. Schwartz and K.R. Rajashankar for assistance with data collection and processing, H. Mackie, E. Roesch and J. De Luca for advice on oligonucleotide synthesis, J. Chen, A. Ricardo, K. Frederick, N. Yoder and W. Johnston for help with HPLC purification, E. Spooner for mass spectrometry, and U. RajBhandary, C. Drennan, J. Piccirilli, J. Szostak and members of the Bartel laboratory for helpful discussions. Supported by US National Institutes of Health (NIH) grant GM061835 to D.B. This work is also based upon research conducted at the Northeastern Collaborative Access Team (NE-CAT) beamlines of the Advanced Photon Source (APS), supported by award RR-15301 from the National Center for Research Resources at the NIH. Use of the APS is supported by the US Department of Energy, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357.

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D.M.S. and D.P.B. designed the experiments and wrote the manuscript. D.M.S. carried out the experiments.

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Correspondence to David P Bartel.

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Supplementary Figures 1–5, Supplementary Discussion and Supplementary Methods (PDF 2909 kb)

Supplementary Movie 1

Simulated conformational changes and catalysis by the Class I ligase. A series of linear morphs between the Ca2+–Sr2+ preligation, Mg2+–Sr2+ preligation and product structures. Atoms are colored as in Figure 2a,b; the red dotted line indicates the proposed line of nucleophilic attack. (MP4 914 kb)

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Shechner, D., Bartel, D. The structural basis of RNA-catalyzed RNA polymerization. Nat Struct Mol Biol 18, 1036–1042 (2011). https://doi.org/10.1038/nsmb.2107

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