RNA plays important roles in diverse cellular processes ranging from transcription and translation to protein translocation across membranes. In addition, catalytic RNAs or ribozymes are known to be required for the viability of certain plant pathogens and the human hepatitis delta virus (HDV). Interestingly, ribozymes found in the genomic and antigenomic strands of the HDV have similar but not identical sequences and secondary structures. Knowledge of the detailed three-dimensional architecture of these RNAs is indispensable for understanding their mechanism of action, evolution and relation to other classes of biological catalysts. Progress in RNA structure determination has been hampered by the difficulty in obtaining well-ordered crystals suitable for structural analysis. In recent years, the Doudna group developed powerful techniques to facilitate crystallization of RNA. These techniques led to the structure determination of the HDV genomic ribozyme. Although the structure revealed a novel fold and offered hints about the catalytic mode of action of the ribozyme, the reaction mechanism is still unresolved due to the lack of information on the orientation of the substrate and the position of active site residues prior to reaction. My proposed research will be aimed at determining the structure and function of the antigenomic HDV ribozyme. The first step of the research involves solving the structure of the antigenomic ribozyme and comparing it to that of the genomic ribozyme. As a second step the ribozyme will be inhibited with non-cleavable analogues. Structures of the cleaved and precursor forms will reveal the position of active site residues prior to reaction, providing important clues to the structure of the transition state.
