Date of Award

Spring 1-1-2011

Document Type


Degree Name

Doctor of Philosophy (PhD)



First Advisor

David J. Nesbitt

Second Advisor

Arthur Pardi

Third Advisor

W. Carl Lineberger

Fourth Advisor

Thomas Perkins

Fifth Advisor

Margaret Murnane


RNA functional diversity is coupled with its ability to fold into unique structures, a process that is generally hierarchical--tertiary interactions occur between preformed secondary structural elements, e.g., loops and helices. For RNA to fold into compact, biochemically competent shapes, counterion neutralization of the negatively charged-phosphate backbone is required. The objective of this thesis is to investigate the physical principles that dictate how an RNA molecule achieves and maintains its tertiary structure. Toward this end, single-molecule fluorescence resonance energy transfer methods are combined with temperature control to probe the mediation of RNA folding landscapes by cation-facilitated tertiary interactions.

The primary focus of this thesis is kinetic and thermodynamic characterization of the ubiquitous GAAA tetraloop-receptor tertiary interaction using freely diffusing and immobilized single-molecule assays. The apparent first-order rate constants (kdock and kundock) for the intramolecular docking/undocking of the tetraloop and receptor are measured as function of monovalent, divalent, and trivalent cation concentration. We observe that the [cation] needed to promote folding is correlated with charge density of the ion, which we interpret in terms of counterion condensation on the RNA. The temperature dependence of kdock and kundock are also determined, which yield the standard state and transition state free energies, enthalpies, and entropies for docking and undocking. At physiological conditions, the transition state for tetraloop-receptor docking is early, with its formation rate-limited by an entropic barrier. The overall docking reaction is exothermic and entropically costly, consistent with the large number of hydrogen bonding and base-stacking interactions formed by the tertiary contact. Surprisingly, we reveal an entropic origin of Mg2+-facilitated RNA folding, which conflicts with the common expectation that increasing [Mg2+] aids folding by reducing electrostatic repulsions of the RNA backbone. We propose instead that higher [Mg2+] promotes folding by decreasing the entropic penalty of counterion uptake in the folding transition state and by reducing disorder in the unfolded conformational ensemble. This work elucidates potential RNA folding paradigms, such as early transitions states and an entropic origin of tertiary cooperativity and cation-facilitated folding.