| The role of cation size in the RNA folding pathway is not well understood, and very little is known about the structure of the RNA rate limiting transition state. This study investigates the role of cation charge density in RNA folding by means of native gel electrophoresis, using the Tetrahymena ribozyme as a model system. The structure of the transition state is investigated by fluorescence spectroscopy using the P5abc domain of Tetrahymena ribozyme.;The data presented in this thesis shows that small polyamines and small group IIA metal ions have an entropic advantage over large polyamines and large group II A metal ions in folding the Tetrahymena ribozyme. In addition, small cations create highly stable native structures. The investigation of the role of polyamine size in the folding pathway of the Tetrahymena ribozyme demonstrates that small polyamines increase the energy difference between folded and unfolded structure, and they move the transition state for U to I conversion closer to that of unfolded RNA. Conversely, small polyamines are able to better stabilize the kinetic intermediate and as a consequence the activation energy for the conversion of I to N increases and the observed rate of folding decreases in the presence of small polyamines.;Comparisons of equilibrium and kinetics data show that the folding of P5abc in Mg2+ and the unfolding of P5abc in urea is not a simple two state folding process. The transition from the intermediate to the native RNA is not sensitive to Mg2+ or to the monovalent ions in the solution, but it is sensitive to urea. Moreover, measurements of the temperature dependence of the observed rate of folding for I to N conversion shows that this transition state has a high activation energy. Clearly, the rate limiting transition state for the I to N transition must involve the rearrangement of P5abc secondary structure.;One consequence of this work is the improved understanding of the RNA rate limiting transition state structure. This study also demonstrates that both polyamine concentration and polyamine type change the folding landscape of RNA. Finally, the work systematically and experimentally demonstrates for the first time that the size of cations is a key factor in RNA folding. |
