An integrated picture of HDV ribozyme catalysis

Barbara L. Golden, Sharon Hammes-Schiffer, Paul R. Carey, Philip C. Bevilacqua

Research output: Chapter in Book/Report/Conference proceedingChapter

12 Scopus citations

Abstract

The hepatitis delta virus (HDV) ribozyme, a small self-cleaving RNA originally identified in the human pathogen HDV, has been found to be broadly dispersed throughout life. In this article, we describe an integrated approach to understand the catalytic mechanism of this ribozyme that combines kinetics, crystallography, Raman spectroscopy, and calculations. Kinetics studies provide rate and binding parameters for protons and metal ions, and allow for design of properly folded and catalytically relevant RNAs for crystallography. Raman studies on these crystals provide direct evidence that the nucleobase of C75 has a shifted pK a. Moreover, Raman crystallography and solution kinetics demonstrate that proton binding to the N3 of C75 couples anticooperatively with binding of a Mg2+ ion, suggesting that the two species are close in space. Extensive structural studies on this ribozyme suggest that the cleavage reaction proceeds through a combination of Lewis acid catalysis by a catalytic Mg2+ ion and general acid catalysis by the nucleobase of C75. Molecular dynamics and electrostatics calculations support the above mechanism and reveal an intensely electronegative pocket that plays key roles in positioning the catalytic metal ion and C75 for catalysis. Integrating the results of kinetics, X-ray crystallography, Raman crystallography, and molecular dynamics suggests that there is a second Mg2+ ion in the active site that is bound diffusely and may play a structural role. In sum, these four disparate approaches provide for a robust kinetic mechanism for the HDV ribozyme that lays groundwork for future studies into its detailed mechanism of dynamics and cleavage.

Original languageEnglish (US)
Title of host publicationBiophysics of RNA Folding
PublisherSpringer New York
Pages135-167
Number of pages33
ISBN (Electronic)9781461449546
ISBN (Print)9781461449539
DOIs
StatePublished - Jan 1 2013

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Fingerprint Dive into the research topics of 'An integrated picture of HDV ribozyme catalysis'. Together they form a unique fingerprint.

  • Cite this

    Golden, B. L., Hammes-Schiffer, S., Carey, P. R., & Bevilacqua, P. C. (2013). An integrated picture of HDV ribozyme catalysis. In Biophysics of RNA Folding (pp. 135-167). Springer New York. https://doi.org/10.1007/978-1-4614-4954-6_8