| The discovery of RNA catalysis in the early 1980's marked a shift in a long standing paradigm in enzymology; no longer were proteins considered the sole catalytic cellular agents, but RNA was now included in that category. Following this initial discovery, numerous other catalytic RNAs, ribozymes, were soon identified. Even twenty years later new ribozymes continue to be identified or generated. Clearly, the field of catalytic RNA study continues to grow and broaden in scope and focus. In order to obtain a better overall understanding of RNA catalytic function it is necessary to examine specific ribozymes.;The hammerhead is a well studied ribozyme that causes a site specific transesterification in the RNA backbone leading to a 2', 3'-cyclic phosphate and 5'-hydroxyl termini. Until four years ago minimal hammerheads were primarily used to investigate the requirements of the reaction. However, in 2003 the importance of added sequence elements, present in the natural sequences where the hammerhead motif was originally identified, as able to enhance catalysis was appreciated. This discovery again caused another shift in focus of study, this time from "minimal" hammerheads towards naturally derived "extended" hammerheads.;The work included in this thesis focuses on understanding the enhancement of the extended hammerhead on catalysis and comparing its enhancement to that which is observed for the minimal hammerhead. For this purpose it is necessary to establish the experimental validity of using hybrid hammerheads, hammerheads containing both minimal and extended sequences.;This system, once validated, is then used to test the metal ion binding properties of an extended hammerhead with phosphorothioate substitutions. Kapp [Cd2+] values are obtained for extended hammerheads with a phosphorothioate substitution at either P1.1 or P.9. The same experiments are performed on a mutant version of the extended hammerhead as well as on a minimal hammerhead. The results show that while the extended hammerhead binds cadmium ions tighter than the minimal hammerhead, the trends in K app [Cd2+] values for the different phosphorothioate substitutions compared to the all oxygen control in both backgrounds are very similar.;Furthermore, a recent crystal structure of an extended hammerhead showed a novel core interaction between residues G8 and C3. This interaction is explored in both the extended and minimal background by introducing single point mutations followed by the compensatory double mutation. Additionally, the possible synergistic relationship between the identity of the cleavage site nucleotide and the different N8-N3 nucleotides is explored. The similar behavior in response to this set of mutations for the minimal and extended hammerhead suggests that the two share a dynamic pathway. Therefore, in the final chapter, the large existing body of biochemical data collected in the minimal hammerhead is evaluated in the background of the extended hammerhead structure where it maybe better understood. |
