The goal of the proposed research is to define and model features of the tertiary structure of the hepatitis delta virus (HDV) ribozyme. HDV is a human pathogen and an understanding of the structure and function of the ribozyme could aid in the design of small molecules that would block wiral replication by inhibiting ribozyme activity. Data from several different experiemtns will provide constraints for tertiary structure analysis. 2'-hydroxyl groups and pro-Rp phosphate oxygens essentials for cleavage activity will be identified by random substittution and mapping those positions that interfere with cleavage. Photactivatable cross- linking agents will be used to determine the relative positions and orientations of helical regions. Cross-linking agenst will be introduced into a substrate oligonucleotide capable of binding the trans-cleaving ribozyme to provide information on the orientation of the substrate to the binding domain of the ribozyme. To explore alternative helical stacking possibilities, psoralen will be intercalated at the base of helices. Subsequent to UV irridation, sites of cross-links will be identified by altered mobility on polyacrylamide gels. Finally, in vitro selection experiments will be used to identify non-canonical tertiary contacts: Specific mutations which reduce cleavage activity will be introduced along with randomized positions, and compensatory mutations which restore activity will be identified by enriching for those sequences. Both a low resolution tertiary structure model of the entire HDV ribozyme and a high resolution all-atom model of the catalytic core of the HDV ribozyme wil be generated with computer modeling programs using the data obtained from these experiments as constraints and checks on the accuracy of a model.
