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An in vitro evolved glmS ribozyme has the wild-type fold but loses coenzyme dependence

Nature Chemical Biology volume 9, pages 805–810 (2013)Cite this article

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Abstract

Uniquely among known ribozymes, the glmS ribozyme-riboswitch requires a small-molecule coenzyme, glucosamine-6-phosphate (GlcN6P). Although consistent with its gene-regulatory function, the use of GlcN6P is unexpected because all of the other characterized self-cleaving ribozymes use RNA functional groups or divalent cations for catalysis. To determine what active site features make this ribozyme reliant on GlcN6P and to evaluate whether it might have evolved from a coenzyme-independent ancestor, we isolated a GlcN6P-independent variant through in vitro selection. Three active site mutations suffice to generate a highly reactive RNA that adopts the wild-type fold but uses divalent cations for catalysis and is insensitive to GlcN6P. Biochemical and crystallographic comparisons of wild-type and mutant ribozymes show that a handful of functional groups fine-tune the RNA to be either coenzyme or cation dependent. These results indicate that a few mutations can confer new biochemical activities on structured RNAs. Thus, families of structurally related ribozymes with divergent function may exist.

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Figure 1: Comparison of the pre-cleavage active site structures and catalytic strategies of three natural nucleolytic ribozymes.
Figure 2: glmSAAA is a stably folded metal ion–dependent ribozyme.
Figure 3: A cation-binding site in glmSAAA important for catalysis.
Figure 4: Effect of active site residues in GlcN6P-dependent and metal ion-dependent catalysis.
Figure 5: Chemical and structural analyses correlate metal ion utilization by glmSAAA and glmSWT.

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Acknowledgements

We thank the staff at beamlines 5.0.1 and 5.0.2 of the Advanced Light Source and of 12-ID (BESSRC CAT) of the Advanced Photon Source for crystallographic and SAXS data collection support, respectively; X. Fang and Y.-X. Wang for access to SAXS beamtime; D.-Y. Lee and R. Levine for access to MS; J. Sellers for access to a rapid quench apparatus; N. Baird for performing analysis of SAXS data; K. Deigan, J. Posakony and J. Zhang for discussions; and an anonymous referee for motivating the phosphorothioate interference analysis of glmSAAG and glmSUAG. M.W.L.L. was a recipient of the Croucher Foundation Fellowship. This work was supported in part by the intramural program of the US National Heart, Lung and Blood Institute–National Institutes of Health.

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  1. National Heart, Lung and Blood Institute, Bethesda, Maryland, USA

    Matthew W L Lau & Adrian R Ferré-D'Amaré

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  1. Matthew W L Lau
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M.W.L.L. and A.R.F.-D. designed experiments, M.W.L.L. performed all of the biochemistry, M.W.L.L. collected SAXS data and grew crystals and M.W.L.L. and A.R.F.-D. collected diffraction data, solved and refined the crystal structures and wrote the manuscript.

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Correspondence to Adrian R Ferré-D'Amaré.

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Lau, M., Ferré-D'Amaré, A. An in vitro evolved glmS ribozyme has the wild-type fold but loses coenzyme dependence. Nat Chem Biol 9, 805–810 (2013). https://doi.org/10.1038/nchembio.1360

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