The factors that determine RNA structure formation, stability, and dynamics are inexorably linked to RNA function. The Hammerhead ribozyme (HHRz) has long served as a model for studying metal-dependent folding and catalysis in RNA. The HHRz consists of three helices meeting at a common junction of conserved nucleotides that form the active site of the ribozyme. Current models of metal-dependent HHRz function involve a requirement for divalent metals to globally fold the ribozyme at low metal concentrations, followed by a second metal-dependent process which activates the HHRz for catalysis. The exact role of metal ions in activating HHRz catalysis is still a subject of investigation. We used 2-aminopurine substitutions near the active site of the ribozyme to determine if this second metal-dependent process involves a conformational rearrangement in the core of the ribozyme. We find evidence for a conformational change beyond global folding in the core of the ribozyme that not only correlates with metal activated catalysis but is also sensitive to the identity of the metal ions used for folding. Though phosphorothioate substitutions indicate that a ground-state coordination of a catalytic metal to the scissile phosphate is required for efficient catalysis, our folding studies show that this coordination event is not absolutely required for folding of the HHRz core. To investigate possible roles for metal ions in general acid-base catalysis, we tested the pH dependence of the HHRz rate using a variety of metal ions. We find the pH dependent rate profile of the ribozyme is shifted by transition metal ions, whereas other group II metals show similar profiles to Mg
| Identifer | oai:union.ndltd.org:uoregon.edu/oai:scholarsbank.uoregon.edu:1794/12375 |
| Date | January 2012 |
| Creators | Ward, William, Ward, William |
| Contributors | DeRose, Victoria |
| Publisher | University of Oregon |
| Source Sets | University of Oregon |
| Language | en_US |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Rights | All Rights Reserved. |
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