2008 Rustbelt RNA Meeting
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Talk on Saturday 09:00-09:20am submitted by Jui-Hui Chen

A catalytic metal ion interacts with the cleavage site GU wobble in the HDV ribozyme

Jui-Hui Chen (Department of Biochemistry, Purdue University, 175 South University Street, West Lafayette, Indiana 47907), Bo Gong (Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106), Philip C. Bevilacqua (Department of Chemistry, The Pennsylvania State University, 104 Chemistry Building, University Park, Pennsylvania 16802), Paul R. Carey (Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106), Barbara L. Golden (Department of Biochemistry, Purdue University, 175 South University Street, West Lafayette, Indiana 47907)

Abstract:
The hepatitis delta virus (HDV) ribozyme is a self-cleaving RNA motif found on the genomic and antigenomic strands of the HDV RNA and within the mammalian transcriptosome. The cleavage reaction is believed to proceed through a general acid/general base mechanism in which C75 serves as a general acid and a magnesium-bound water serves as the general base. Naturally occurring variants of the HDV ribozyme contain a semi-conserved GU wobble base pair at the cleavage site. We propose that the N7 of cleavage site guanosine (G1) provides a ligand to the catalytic metal ion. The evidence for this includes: 1) the deleterious effect of a 7-deaza modification at G1 on catalysis, 2) apparent reduction in magnesium affinity resulting from a 7-deaza modification at G1, 3) observation of magnesium binding and guanine N7 coordination upon C75 deprotonation in Raman spectra, and 4) failure to observe the Raman features corresponding to magnesium binding and guanine N7 coordination when a 7-deaza modification is introduced at G1. Together with previous biochemical studies, a model of the catalytic metal ion bound within the HDV active site is proposed. This model explains the solution kinetic and Raman spectroscopy results presented here and is in agreement with published biochemical data on the wild-type HDV ribozyme.

Keywords: Hepatitis delta virus (HDV) ribozyme, Raman spectroscopy, 7-deazaguanosine