Investigating the catalytic mechanism of the hepatitis delta virus ribozyme

The hepatitis delta virus (HDV) ribozyme is a small self-processing RNA motif involved in HDV replication. In this work, we used functional assays to identify the roles that components of the ribozyme play to enhance the rate of the phosphodiester bond cleavage reaction. We developed a ligation-based method to incorporate atomic modifications site-specifically into a well-characterized experimental system to study catalysis by the HDV ribozyme. The key role of 5' leaving group activation by a specific atom in the ribozyme, the N3 imino nitrogen of C76, had previously been established in the laboratory; this work expands outward from that point to probe the functional role of other groups in or near the active site of the ribozyme.;We exploited the fact that ribozyme-catalyzed cleavage of a substrate containing a 5'-sulfur leaving group can be used to distinguish between the effects of a modification on the ability of C76 to act as a general acid, and structural perturbations of the modification. We incorporated a variety of cytosine analogs into position 76 of the ribozyme and found, using Bronsted analysis, that proton transfer to the 5' leaving group in the transition state is nearly complete, consistent with a late transition state. We also found that, under the reaction conditions tested, removal of the O2 keto group of C76 appears to have no effect on catalysis. This functional group has been posited to serve as an outer-sphere ligand for a catalytic divalent metal ion.;A variety of the functional evidence we obtained suggests that the transition state of the HDV ribozyme-catalyzed cleavage reaction resembles the self-cleaved product state. We also tested the functional relevance of two different aspects in which the two crystal structures of the HDV ribozyme, self-cleaved product and C76U inactive precursor, vary. In both cases, the conformation of the G28 nucleobase and the proximity of the 2' hydroxyl group of C76 and the N4 exocyclic amino group of C25, our kinetic data support a transition state model that more closely resembles the self-cleaved product crystal structure than the precursor crystal structure. This work suggests that further structure-based functional studies of the HDV ribozyme-catalyzed reaction would be better served by using the crystal structure of the self-cleaved product as a starting point rather than the C76U inactive precursor form.;The work performed here on analyzing catalysis by the HDV ribozyme could also be extended to studying the mechanism of the other small nucleolytic ribozymes. These ribozymes also appear to utilize one or both of nucleobases or divalent metal ions in the active site to effect general acid-base catalysis. Although none of the other small ribozymes utilize a cytosine nucleobase in their active sites, the systematic efforts to probe the functions of the C76 nucleobase and other active site functional groups done here can also be applied with judicious modifications to study the catalytic mechanism of these other endonucleolytic ribozymes.