A catalytic metal ion interacts with the cleavage site G·U Wobble in the HDV ribozyme

Jui Hui Chen, Bo Gong, Philip C. Bevilacqua, Paul R. Carey, Barbara L. Golden

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

The HDV ribozyme self-cleaves by a chemical mechanism involving general acid-base catalysis to generate 2′,3′-cyclic phosphate and 5′-hydroxyl termini. Biochemical studies from several laboratories have implicated C75 as the general acid and hydrated magnesium as the general base. We have previously shown that C75 has a pKa shifted >2 pH units toward neutrality [Gong, B., Chen, J. H., Chase, E., Chadalavada, D. M., Yajima, R., Golden, B. L., Bevilacqua, P. C., and Carey, P. R. (2007) J. Am. Chem. Soc. 129, 13335-13342], while in crystal structures, it is well-positioned for proton transfer. However, no evidence for a hydrated magnesium poised to serve as a general base in the reaction has been observed in high-resolution crystal structures of various reaction states and mutants. Herein, we use solution kinetic experiments and parallel Raman crystallographic studies to examine the effects of pH on the rate and Mg 2+ binding properties of wild-type and 7-deazaguanosine mutants of the HDV ribozyme. These data suggest that a previously unobserved hydrated magnesium ion interacts with N7 of the cleavage site G·U wobble base pair. Integrating this metal ion binding site with the available crystal structures provides a new three-dimensional model for the active site of the ribozyme that accommodates all available biochemical data and appears competent for catalysis. The position of this metal is consistent with a role of a magnesium-bound hydroxide as a general base as dictated by biochemical data.

Original languageEnglish (US)
Pages (from-to)1498-1507
Number of pages10
JournalBiochemistry
Volume48
Issue number7
DOIs
StatePublished - Feb 24 2009

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Catalytic RNA
Magnesium
Metal ions
Metals
Ions
Catalysis
Crystal structure
Magnesium Hydroxide
Acids
Base Pairing
Hydroxyl Radical
Proton transfer
Protons
Catalytic Domain
Phosphates
Binding Sites
Kinetics
Experiments

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Chen, Jui Hui ; Gong, Bo ; Bevilacqua, Philip C. ; Carey, Paul R. ; Golden, Barbara L. / A catalytic metal ion interacts with the cleavage site G·U Wobble in the HDV ribozyme. In: Biochemistry. 2009 ; Vol. 48, No. 7. pp. 1498-1507.
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A catalytic metal ion interacts with the cleavage site G·U Wobble in the HDV ribozyme. / Chen, Jui Hui; Gong, Bo; Bevilacqua, Philip C.; Carey, Paul R.; Golden, Barbara L.

In: Biochemistry, Vol. 48, No. 7, 24.02.2009, p. 1498-1507.

Research output: Contribution to journalArticle

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T1 - A catalytic metal ion interacts with the cleavage site G·U Wobble in the HDV ribozyme

AU - Chen, Jui Hui

AU - Gong, Bo

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