Structural and functional studies of the hepatitis delta virus ribozyme
Abstract
The HDV ribozyme self-cleaves by a mechanism involving general acid-base catalysis. Although high-resolution crystal structures of this ribozyme have been determined, the active sites of these structures do not agree fully with key findings from biochemical studies implicating C75 as the general acid and hydrated magnesium as the general base. In order to clarify the role of the hydrated metal ion in the catalytic reaction, we re-examine the possible binding site for the catalytic metal ion. The first G˙U wobble pair in the active site tends to be conserved and prefers to be a purine-pyrimidine combination. Since the GU pair is usually involved in coordinating metal ions, we use 7-deaza G1 HDV ribozyme mutants to probe the function of this GU pair. Our results from kinetic and Raman spectroscopic studies all supported that the N7 of G1 has direct interaction with hydrated magnesium. Combining our biochemical result with published structural studies, we propose a model for the active site that accommodates all available biochemical data and appears competent for catalysis. The second attempt was to solve the crystal structure by using a weaker nucleophile to achieve the pre-cleavage state. The original ribozyme construct, which was used to carry out the kinetic studies, was thought to be twinned and could not provide useful information to solve the structure. A new construct with one base-pair extension at P2 region was able to provide a molecular replacement result and to allow a solving of the structure. The new structure reveals similar overall conformation to the published results. However, the electron density of the critical U-1 residue cannot be observed in the map. The cleavage reaction may still occur when using the weaker nucleophile. The future work will focus on using 2'-deoxy modification on three continuous residues, U-1, G1, and G2, to inhibit the cleavage reaction. Hopefully the new modified oligo can prevent the cleavage and retain the real pre-cleavage state.
Degree
Ph.D.
Advisors
Golden, Purdue University.
Subject Area
Molecular biology|Virology|Biophysics
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