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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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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 Text and Figures

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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