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Pistol ribozyme adopts a pseudoknot fold facilitating site-specific in-line cleavage

Abstract

The field of small self-cleaving nucleolytic ribozymes has been invigorated by the recent discovery of the twister, twister-sister, pistol and hatchet ribozymes. We report the crystal structure of a pistol ribozyme termed env25, which adopts a compact tertiary architecture stabilized by an embedded pseudoknot fold. The G-U cleavage site adopts a splayed-apart conformation with in-line alignment of the modeled 2′-O of G for attack on the adjacent to-be-cleaved P-O5′ bond. Highly conserved residues G40 (N1 position) and A32 (N3 and 2′-OH positions) are aligned to act as a general base and a general acid, respectively, to accelerate cleavage chemistry, with their roles confirmed by cleavage assays on variants, and an increased pKa of 4.7 for A32. Our structure of the pistol ribozyme defined how the overall and local topologies dictate the in-line alignment at the G-U cleavage site, with cleavage assays on variants revealing key residues that participate in acid-base-catalyzed cleavage chemistry.

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Figure 1: Secondary and tertiary structure of the env25 pistol ribozyme.
Figure 2: Structural details of the env25 pistol ribozyme in the vicinity of the G53-U54 cleavage site.
Figure 3: In-line alignment at the G53-U54 cleavage site in the env25 pistol ribozyme and comparison with the structure of the RzB hammerhead ribozyme.
Figure 4: Self-cleavage of the env25 pistol ribozyme.
Figure 5: NMR spectroscopic analysis of the env25 pistol ribozyme in solution.
Figure 6: Cleavage kinetics of the env25 pistol ribozyme analyzed by Ap fluorescence.

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Acknowledgements

We acknowledge assistance by staff at Northeastern Collaborative Access Team (NE-CAT) beamlines at the Advanced Photon Source and the experimental station 14-1 at the Stanford Synchrotron Radiation Light Source. X-ray diffraction studies were conducted at the Advanced Photon Source on the Northeastern Collaborative Access Team beamlines, which are supported by a grant from the National Institute of General Medical Sciences (P41 GM103403) from the US National Institutes of Health (NIH). Use of the Advanced Photon Source, an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the US DOE under contract DE-AC02-06CH11357. A.R. was supported in part by new faculty start-up funds from Zhejiang University and the Thousand Young Talents Plan of China. The research was supported by US NIH grant 1 U19 CA179564 to D.J.P., by the Memorial Sloan-Kettering Cancer Center Core Grant (P30 CA008748), and the Austrian Science Fund FWF (P27947, I1040 to R.M., P26550, P28725 to C.K.), and the Swiss National Foundation SNF (Early Postdoc.Mobility to J.G.).

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A.R. undertook all of the crystallographic experiments and the structure analysis with the assistance of P.G. under the supervision of D.J.P. while N.V., J.G. and M.J. were involved in nucleoside phosphoramidite synthesis, RNA preparation and cleavage assays under the supervision of R.M. C.K. designed and performed NMR spectroscopy measurements. The paper was written jointly by A.R., N.V., C.K., R.M. and D.J.P. with input from the remaining authors.

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Correspondence to Ronald Micura or Dinshaw J Patel.

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Supplementary Results, Supplementary Table 1 and Supplementary Figures 1–5. (PDF 2825 kb)

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Ren, A., Vušurović, N., Gebetsberger, J. et al. Pistol ribozyme adopts a pseudoknot fold facilitating site-specific in-line cleavage. Nat Chem Biol 12, 702–708 (2016). https://doi.org/10.1038/nchembio.2125

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