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General acid catalysis by the hepatitis delta virus ribozyme

Nature Chemical Biology volume 1, pages 45–52 (2005)Cite this article

Abstract

Recent crystallographic and functional analyses of RNA enzymes have raised the possibility that the purine and pyrimidine nucleobases may function as general acid-base catalysts. However, this mode of nucleobase-mediated catalysis has been difficult to establish unambiguously. Here, we used a hyperactivated RNA substrate bearing a 5′-phosphorothiolate to investigate the role of a critical cytosine residue in the hepatitis delta virus ribozyme. The hyperactivated substrate specifically suppressed the deleterious effects of cytosine mutations and pH changes, thereby linking the protonation of the nucleobase to leaving-group stabilization. We conclude that the active-site cytosine provides general acid catalysis, mediating proton transfer to the leaving group through a protonated N3-imino nitrogen. These results establish a specific role for a nucleobase in a ribozyme reaction and support the proposal that RNA nucleobases may function in a manner analogous to that of catalytic histidine residues in protein enzymes.

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Figure 1: Kinetically equivalent roles proposed for the active-site cytosine in the HDV ribozyme reaction.
Figure 2: The wild-type HDV ribozyme catalyzes cleavage of the hyperactivated substrate S5′-S.
Figure 3: S5′-S suppresses the C76U mutant defect.
Figure 4: The single-atom-mutant ribozyme C76c3C shows nearly wild-type activity against S5′-S.
Figure 5: Kinetically equivalent models from two independent, offsetting titrations simulate the pH dependence of the HDV ribozyme.
Figure 6: The pH dependence of the C76z6C mutant ribozyme implicates C76 in general acid catalysis.

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Acknowledgements

We thank A. Ke and J.A. Doudna for helpful discussions and J. Staley, M.D. Been, P.C. Bevilacqua, D. Herschlag and members of the Piccirilli laboratory for critical comments and suggestions on the manuscript. We also thank J. Olvera for T4 DNA ligase and E. Duguid for assistance with MALDI mass spectroscopy. This work was supported by the Howard Hughes Medical Institute.

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  1. Departments of Biochemistry & Molecular Biology and Chemistry, Howard Hughes Medical Institute, University of Chicago, 5841 S. Maryland Avenue, MC1028, Chicago, 60637, Illinois, USA

    Subha R Das & Joseph A Piccirilli

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Correspondence to Joseph A Piccirilli.

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Supplementary information

Supplementary Fig. 1

1H-NMR spectrum of 2′-O-TBDMS-5′-O-DMTr-N4-phenoxyacetyl-3-deazacytidine which was phosphitylated to obtain the phosphoramidite analogue (31P-NMR peaks at δ 150.4 ppm and 149.8 ppm; H3PO4 external standard) for solid phase synthesis. (PDF 5340 kb)

Supplementary Fig. 2

1H-NMR spectrum of 2′-OTBDMS-5′-O-DMTr-N4-acetyl-6-azacytidine which was phosphitylated to obtain the phosphoramidite analogue (31P-NMR peaks at δ 150.6 ppm and 149.6 ppm; H3PO4 external standard) for solid phase synthesis. (PDF 5527 kb)

Supplementary Note (PDF 245 kb)

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Das, S., Piccirilli, J. General acid catalysis by the hepatitis delta virus ribozyme. Nat Chem Biol 1, 45–52 (2005). https://doi.org/10.1038/nchembio703

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