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Direct measurement of the ionization state of an essential guanine in the hairpin ribozyme

Nature Chemical Biology volume 5, pages 351–357 (2009)Cite this article

Abstract

Active site guanines are critical for self-cleavage reactions of several ribozymes, but their precise functions in catalysis are unclear. To learn whether protonated or deprotonated forms of guanine predominate in the active site, microscopic pKa values were determined for ionization of 8-azaguanosine substituted for G8 in the active site of a fully functional hairpin ribozyme in order to determine microscopic pKa values for 8-azaguanine deprotonation from the pH dependence of fluorescence. Microscopic pKa values above 9 for deprotonation of 8-azaguanine in the active site were about 3 units higher than apparent pKa values determined from the pH dependence of self-cleavage kinetics. Thus, the increase in activity with increasing pH does not correlate with deprotonation of G8, and most of G8 is protonated at neutral pH. These results do not exclude a role in proton transfer, but a simple interpretation is that G8 functions in the protonated form, perhaps by donating hydrogen bonds.

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Figure 1: Structure and function of G8 in the hairpin ribozyme active site.
Figure 2: RNAs and complexes used for fluorescence experiments.
Figure 3: pH dependence of ribozyme cleavage kinetics and 8-azaguanine fluorescence emission intensity under various salt conditions.
Figure 4: pH dependence of 8azaG fluorescence emission intensity under various salt conditions.

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Acknowledgements

We thank the Fedor lab for help with manuscript preparation. This work was supported by US National Institutes of Health grant GM046422 and by a postdoctoral fellowship provided by the Skaggs Institute for Chemical Biology to L.L.

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Authors and Affiliations

  1. Department of Chemical Physiology, Department of Molecular Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA

    Lu Liu, Joseph W Cottrell & Martha J Fedor

  2. Cassia LLC, San Diego, California, USA

    Lincoln G Scott

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  1. Lu Liu
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  2. Joseph W Cottrell
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  3. Lincoln G Scott
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  4. Martha J Fedor
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Contributions

L.L., J.W.C. and M.J.F. designed the experiments, interpreted the results and prepared the manuscript. L.G.S. synthesized 8azaGTP and developed methods for using 8azaGTP in transcription reactions. L.L. and J.W.C. prepared the RNAs and carried out self-cleavage and fluorescence assays.

Corresponding author

Correspondence to Martha J Fedor.

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Liu, L., Cottrell, J., Scott, L. et al. Direct measurement of the ionization state of an essential guanine in the hairpin ribozyme. Nat Chem Biol 5, 351–357 (2009). https://doi.org/10.1038/nchembio.156

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