RNA catalysis is of fundamental importance in many biological functions, such as the peptidyl transferase activity of the ribosome and genetic control by riboswitches, among others. Small ribozymes are a convenient system to increase our understanding about the structure, folding and catalytic mechanism of ribozymes. This dissertation includes analysis of certain aspects of the catalytic mechanism in the hairpin and hammerhead ribozyme. In the hairpin ribozyme, we studied the functional consequences of molecular substitutions at two conserved positions, A9 and A10. These nucleotides are located close to the scissile phosphate but their exact function is unclear since they do not appear to be making any essential interactions with other nucleotides in the catalytic core. G, C, U, 2-aminopurine, 2, 6-diaminopurine, purine, and inosine were substituted at A9 and A10 and their effects on cleavage and ligation rates were analyzed. The effect of the variations on tertiary structure and docking was monitored by hydroxyl radical footprinting and native gel electrophoresis. It was observed that all the variants that exhibited poor docking and/or tertiary structure formation were also ligation challenged whereas they performed normally in the cleavage reaction. We found a unique variant, A10G that cleaved five times faster than A10 but did not exhibit any ligation. Results suggested that ligation required a more kinetically stable core than that needed to carry out cleavage. The hammerhead ribozyme field featured extensive disagreements between the crystal structure of the minimal hammerhead released in the mid 90s and the accumulating biochemical data. Much of the conflict was resolved with the new crystal structure of the extended hammerhead ribozyme. This structure confirmed many of the biochemical findings and brought out some new interactions, notably the G8·C3 base pair. We studied numerous base substitutions to establish the importance of the base pair for cleavage and ligation. Catalysis requires the formation of the base pair but even the fastest base paired variant was 10-fold slower than G8·C3 base pair. Docking and tertiary structure analysis by hydroxyl radical footprinting and native gel electrophoresis emphasized the importance of having a purine at position 8 and a pyrimidine at 3. Catalysis in the unmodified ribozyme was uniquely accompanied by hydrolysis of the 2′, 3′- cyclic phosphate ring present on one of the cleavage products, leading to the generation of non-ligatable products during a ligation assay. We determined the ligation rate-pH profile for unmodified ribozyme that differed from the cleavage rate-pH profile only at high pH.

The hairpin ribozyme is a small catalytic RNA that cleaves a phosphodiester bond. In order for cleavage to occur, the hairpin ribozyme must properly fold into its docked conformation, in which the two loops interact to form the active site. Metal ions and the four-way junction play critical roles in the stabilization of the docked conformation. The work presented in this thesis attempts to investigate the metal-ion dependence of the docking of the four-way junction construct of the hairpin ribozyme. In addition, the activity of the hairpin ribozyme in the presence of Mn2+ was observed. Initially, a four-stranded four-way junction construct of the hairpin ribozyme and a loopless mutant were characterized by native gel electrophoresis and thermal denaturation to verify ribozyme formation. A novel interaction between the sulfur of a phosphorothioate-substituted mononucleotide, such as adenosine thiomonophosphate (AMPS) or adenosine thiotrisphoshate (ATPgS), and Cd2+ has been characterized by UV-vis spectroscopy. A feature at 208 nm was identified to be a result of sulfur-to-Cd2+ transfer. The apparent binding affinities, the apparent extinction coefficients, and the binding ratios were determined for each complex.