Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Physics and Astronomy." Includes bibliographical references (p. 100-108).

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (pages 49-54).

Thesis (Ph.D.)--University of Delaware, 2009. / Principal faculty advisors: Anne Skaja Robinson and Christopher J. Roberts, Dept. of Chemical Engineering. Includes bibliographical references.

Thesis (Ph. D.)--University of Chicago, Dept. of Pathology, Dec. 2003. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 112-126). Available also in a digital version from Dissertation Abstracts.

Structured RNAs and RNA-protein complexes (RNPs) are involved in many essential biological processes and the specific conformations of these RNAs are crucial to their various functions. However, in vitro studies have found that RNA has propensity for misfolding into inactive species that often consist of extensive secondary and tertiary interactions, which can be locally and globally stabilizing, resulting in long-lived non-native conformers. DEAD-box helicases are one class of proteins that have been found to accelerate folding and rearrangements of highly structured RNAs. While these proteins have been shown to use ATP to unwind short RNA helices, it is not known how they disrupt the tertiary interactions that often stabilize both native and misfolded RNA conformations. We used single molecule fluorescence to probe the mechanism by which DEAD-box proteins facilitate global unfolding of a structured RNA. DEAD-box protein CYT-19, a mitochondrial protein from Neurospora crassa, was found to destabilize a specific tertiary interaction with the Tetrahymena group I intron ribozyme using a helix capture mechanism. The protein molecule binds to a helix within the structured RNA only after the helix spontaneously loses its tertiary contacts, and then uses ATP to unwind the helix, liberating the product strands. Ded1, a multi-functional DEAD-box protein found in Saccharomyces cerevisiae, gives analogous results with small but reproducible differences that may reflect its in vivo roles. The requirement for spontaneous dynamics likely targets DEAD-box proteins toward less stable RNA structures, which are likely to experience greater dynamic fluctuations, and provides a satisfying explanation for previous correlations between RNA stability and CYT-19 unfolding efficiency. Biologically, the ability to sense RNA stability probably biases DEAD-box proteins to act preferentially on misfolded structures and thereby to promote native folding while minimizing spurious interactions with stable, natively-folded RNAs. In addition, this straightforward mechanism for RNA remodeling does not require any specific structural environment of the helicase core and is likely to be relevant for DEAD-box proteins that promote RNA rearrangements of RNP complexes including the spliceosome and ribosome. / text

DExD/H-box proteins are a diverse class of proteins that are implicated in RNA and RNP remodeling. They have sequence homology to DNA helicases and share conserved ATPase domains, suggesting that they use the energy of ATP binding and hydrolysis to mediate conformational rearrangements in RNAs. In the past, the action of DExD/H-box proteins has been characterized primarily on simple model substrates such as small RNA duplexes. It is not known how DExD/H-box proteins manipulate structured RNA, what determines target specificity and what molecular events follow their action. Here, using the well-characterized Tetrahymena group I intron ribozyme, I performed kinetic and thermodynamic studies to understand the mechanism of CYT-19 and related DExD/Hbox proteins. CYT-19 has been shown previously to facilitate the folding of several group I and group II introns. I demonstrated that CYT-19 acts as a chaperone, accelerating the re-folding of a long-lived misfolded species of the Tetrahymena group I ribozyme to its native state. Further characterization of this reaction gave insights into how CYT-19 achieves this action; CYT-19 partially unfolds the misfolded ribozyme and allows it to fold again along the same pathway that exists in the absence of CYT-19. In addition to acting on the misfolded state, CYT-19 also acts on the native state, but this action is largely obscured under stabilizing conditions for the native state because the action is inefficient under such conditions. However, under conditions where the native state is destabilized, the native ribozyme was indeed shown to be partially unfolded by CYT-19. By acting on either species, CYT-19 sets up a steady state of unfolding, and the distribution is shifted from equilibrium to kinetic control, increasing the relative populations of conformations that are kinetically preferred during folding. The efficiency of action seems to correlate with the stability of the ribozyme. These activities are not restricted to CYT-19; the DExD/H-box proteins Mss116p and Ded1 were demonstrated to possess similar activities. Together, these studies give important insights into the mechanisms of action for this ubiquitous class of proteins and have implications for all structured RNAs in cells. / text

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