Identification and Biophysical Characterization of Factors Influencing the Thermostability of Biological RNAs

Open Access
- Author:
- Whitman, Elisabeth E
- Graduate Program:
- Chemistry
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- June 24, 2011
- Committee Members:
- Philip C. Bevilacqua, Thesis Advisor/Co-Advisor
Philip C. Bevilacqua, Thesis Advisor/Co-Advisor - Keywords:
- RNA folding
macromolecular crowding - Abstract:
- The popular model of RNA folding is that its highly stable and organized secondary structure allows for pre-organization of the tertiary structure, which drives RNA to fold on a biological timescale. The crowded conditions of the cell may also play a role in facilitating RNA folding, reducing the stability of any misfolded intermediates. In addition, metal ions (and in particular divalent metal ions) stabilize RNA folding by neutralizing the inherent clash of negatively charged phosphate backbones. Studies in this thesis focus on the effects of RNA folding under macromolecular crowding conditions, as well as the role of divalent metals on RNA secondary structural elements. Although the folding of functional RNAs under idealized conditions of dilute solution and high ionic strength has been well studied, much less is known about RNA folding under the more in vivo conditions of low Mg2+ concentrations and high macromolecular crowding. It is believed that while RNA may fold hierarchically, a certain cooperativity of folding transitions might be necessary to fold the RNA on a biological timescale. We propose that a functional RNA (transfer RNA) can fold cooperatively in the presence of a molecular crowder and physiological Mg2+ concentrations (Chapter 2). In addition, through a study of secondary structure strengthening mutants, we established that under cooperative (two-state-like) conditions, thermostability of a functional RNA can be increased through base pairing. In Chapter 3, the divalent metal ion interactions with several GAAA tetraloops were studied using thermal denaturation by UV absorbance (referred to as “melting” throughout the thesis) to compare thermodynamic parameters, in particular, Gibbs free energy values (∆G37). GAAA tetraloops with both cg and gc closing base pairs were studied in the presence of divalent alkaline earth metals to determine if there is preference for a particular divalent metal ion to stabilize the GAAA tetraloop with a gc closing base pair. Preliminary results suggest that, surprisingly, both closing base pairs prefer larger ions for stabilization. An inner-sphere coordination site for the metal ion in the tetraloops may explain these results. Further studies for both Chapters 2 and 3 are discussed in Chapter 4. After studying the thermodynamics of RNA folding under crowding conditions, folding kinetics of a functional RNA can be studied using stopped-flow spectroscopy as well as fluorescence resonance energy transfer (FRET) single molecule studies. We hypothesize that macromolecular crowding conditions should cause slower unfolding of tertiary structure under cooperative conditions. In addition, Raman spectroscopy and phosphorothioate substitutions can be used to further probe GAAA tetraloops to determine the nature of a metal-coordination site.