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.
Subscribe to Journal
Get full journal access for 1 year
only $17.42 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
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).
Orgel, L.E. Prebiotic chemistry and the origin of the RNA world. Crit. Rev. Biochem. Mol. Biol. 39, 99–123 (2004).
White, H.B. III. Coenzymes as fossils of an earlier metabolic state. J. Mol. Evol. 7, 101–104 (1976).
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).
Fedor, M.J. & Williamson, J.R. The catalytic diversity of RNAs. Nat. Rev. Mol. Cell Biol. 6, 399–412 (2005).
Chen, X., Li, N. & Ellington, A.D. Ribozyme catalysis of metabolism in the RNA world. Chem. Biodivers. 4, 633–655 (2007).
Lincoln, T.A. & Joyce, G.F. Self-sustained replication of an RNA enzyme. Science 323, 1229–1232 (2009).
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).
McGinness, K.E. & Joyce, G.F. In search of an RNA replicase ribozyme. Chem. Biol. 10, 5–14 (2003).
Bartel, D.P. & Szostak, J.W. Isolation of new ribozymes from a large pool of random sequences. Science 261, 1411–1418 (1993).
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).
Ekland, E.H. & Bartel, D.P. RNA-catalysed RNA polymerization using nucleoside triphosphates. Nature 382, 373–376 (1996).
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).
Lawrence, M.S. & Bartel, D.P. New ligase-derived RNA polymerase ribozymes. RNA 11, 1173–1180 (2005).
Zaher, H.S. & Unrau, P.J. Selection of an improved RNA polymerase ribozyme with superior extension and fidelity. RNA 13, 1017–1026 (2007).
Wochner, A., Attwater, J., Coulson, A. & Holliger, P. Ribozyme-catalyzed transcription of an active ribozyme. Science 332, 209–212 (2011).
Shechner, D.M. et al. Crystal structure of the catalytic core of an RNA-polymerase ribozyme. Science 326, 1271–1275 (2009).
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).
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).
Serganov, A. et al. Structural basis for Diels-Alder ribozyme-catalyzed carbon-carbon bond formation. Nat. Struct. Mol. Biol. 12, 218–224 (2005).
Robertson, M.P. & Scott, W.G. The structural basis of ribozyme-catalyzed RNA assembly. Science 315, 1549–1553 (2007).
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).
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).
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).
Harding, M.M. Geometry of metal-ligand interactions in proteins. Acta Crystallogr. D Biol. Crystallogr. 57, 401–411 (2001).
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).
Steitz, T.A. A mechanism for all polymerases. Nature 391, 231–232 (1998).
Castro, C. et al. Nucleic acid polymerases use a general acid for nucleotidyl transfer. Nat. Struct. Mol. Biol. 16, 212–218 (2009).
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).
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).
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).
Basu, S. & Strobel, S.A. Thiophilic metal ion rescue of phosphorothioate interference within the Tetrahymena ribozyme P4–P6 domain. RNA 5, 1399–1407 (1999).
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).
Frederiksen, J.K. & Piccirilli, J.A. Identification of catalytic metal ion ligands in ribozymes. Methods 49, 148–166 (2009).
Saenger, W. Principles of Nucleic Acid Structure (Springer-Verlag, New York, 1984).
Jencks, W.P. Catalysis in Chemistry and Enzymology (McGraw-Hill, New York, 1969).
Venkatasubban, K.S. & Schowen, R.L. The proton inventory technique. CRC Crit. Rev. Biochem. 17, 1–44 (1984).
Schowen, K.B. & Schowen, R.L. Solvent isotope effects of enzyme systems. Methods Enzymol. 87, 551–606 (1982).
Das, S.R. & Piccirilli, J.A. General acid catalysis by the hepatitis delta virus ribozyme. Nat. Chem. Biol. 1, 45–52 (2005).
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).
Stahley, M.R. & Strobel, S.A. Structural evidence for a two-metal-ion mechanism of group I intron splicing. Science 309, 1587–1590 (2005).
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).
Yang, W. An equivalent metal ion in one- and two-metal-ion catalysis. Nat. Struct. Mol. Biol. 15, 1228–1231 (2008).
Fedor, M.J. Comparative enzymology and structural biology of RNA self-cleavage. Annu. Rev. Biophys. 38, 271–299 (2009).
Bevilacqua, P.C. & Yajima, R. Nucleobase catalysis in ribozyme mechanism. Curr. Opin. Chem. Biol. 10, 455–464 (2006).
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).
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).
Klein, D.J. & Ferre-D'Amare, A.R. Structural basis of glmS ribozyme activation by glucosamine-6-phosphate. Science 313, 1752–1756 (2006).
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).
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).
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).
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).
Emilsson, G.M., Nakamura, S., Roth, A. & Breaker, R.R. Ribozyme speed limits. RNA 9, 907–918 (2003).
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).
Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collection in oscillation mode. Methods Enzymol. 276, 307–326 (1997).
McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Adams, P.D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954 (2002).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).
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).
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.
The authors declare no competing financial interests.
Supplementary Figures 1–5, Supplementary Discussion and Supplementary Methods (PDF 2909 kb)
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)
About this article
Cite this article
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
Kinetic Effects of β,γ-Modified Deoxynucleoside 5′-Triphosphate Analogues on RNA-Catalyzed Polymerization of DNA
In vitro selection of ribozyme ligases that use prebiotically plausible 2-aminoimidazole–activated substrates
Proceedings of the National Academy of Sciences (2020)
Dynamics of the vesicles composed of fatty acids and other amphiphile mixtures: unveiling the role of fatty acids as a model protocell membrane
Biophysical Reviews (2020)
Chemical Society Reviews (2020)
Frontiers in Genetics (2019)