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Observation of internal cleavage and ligation reactions of a ribozyme

Nature Structural & Molecular Biology volume 11, pages 1107–1113 (2004)Cite this article

An Erratum to this article was published on 01 December 2004

Abstract

We have used single-molecule spectroscopy to untangle conformational dynamics and internal chemistry in the hairpin ribozyme. The active site of the ribozyme is stably formed by docking two internal loops, but upon cleavage undocking is accelerated by two orders of magnitude. The markedly different kinetic properties allow us to differentiate cleaved and ligated forms, and thereby observe multiple cycles of internal cleavage and ligation of a ribozyme in a uniquely direct way. The position of the internal equilibrium is biased toward ligation, but the cleaved ribozyme undergoes several undocking events before ligation, during which products may dissociate. Formation of the stably docked active site, rapid undocking after cleavage, and a strong bias toward ligation should combine to generate a stable circular template for the synthesis of the viral (+) strand and thus ensure a productive replication cycle.

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Figure 1: Analysis of cleavage and ligation in the hairpin ribozyme.
Figure 2: Simple cleavage experiments.
Figure 3: Multiple cleavage and ligation reactions.
Figure 4: Assigning the cleaved and ligated states.
Figure 5: pH-dependence of ligation and cleavage rates.
Figure 6: Structural dynamics of cleaved, nonligatable ribozymes.
Figure 7: Ribozyme dynamics and the replication cycle of viral satellite RNA.

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Acknowledgements

Funding was provided by the US National Science Foundation (NIH) (PHY-0134916, DBI-0215869, T.H.), by the US National Institutes of Health (GM065367, T.H.), and by Cancer Research-UK (D.M.J.L.). M.K.N. was partially supported by the NIH molecular biophysics training grant (T32GM008276).

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Authors and Affiliations

  1. Physics Department, University of Illinois, Urbana-Champaign, Urbana, 61801, Illinois, USA

    Michelle K Nahas, Sungchul Hohng & Taekjip Ha

  2. Cancer Research-UK Nucleic Acid Structure Research Group, MSI/WTB Complex, The University of Dundee, Dundee, DD1 5EH, UK

    Timothy J Wilson, Kaera Jarvie & David M J Lilley

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  1. Michelle K Nahas
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  2. Timothy J Wilson
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Correspondence to David M J Lilley or Taekjip Ha.

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Supplementary information

Supplementary Fig. 1

Histograms of the structural dynamics of the cleaved ribozyme obtained from the simple ligation experiment. (PDF 16 kb)

Supplementary Fig. 2

Duration of the cleaved state in the multiple cycles experiment. (PDF 11 kb)

Supplementary Fig. 3

pH-dependence of docking and undocking rates of the cleaved ribozyme. (PDF 22 kb)

Supplementary Fig. 4

pH-dependence of the apparent cleavage rate. (PDF 11 kb)

Supplementary Note (PDF 14 kb)

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Nahas, M., Wilson, T., Hohng, S. et al. Observation of internal cleavage and ligation reactions of a ribozyme. Nat Struct Mol Biol 11, 1107–1113 (2004). https://doi.org/10.1038/nsmb842

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