Structures, Dynamics, and Ribozymes: An Investigation of RNA Structural Dynamics with the Hepatitis Delta Virus and Hairpin Ribozymes.

Abstract

The function of catalytic ribonucleic acid (RNA), or ribozymes, depends on the structural dynamics of the molecule. Although the main function of RNA was once thought to be mainly in the production of proteins, it is now known to be involved in many crucial processes in biology. Small ribozymes, less than 200 nucleotides in length, are useful model systems for studying RNA due to their easily detectable functional activity. Knowledge gained from these small ribozymes is applicable to the wider range of non-protein coding RNAs and in the design of therapeutics.

In this thesis, the impact of structural dynamics on RNA function was investigated by studying two small ribozymes. The first aim analyzed the global and local structural dynamics of the hepatitis delta virus (HDV) ribozyme. From fluorescence resonance energy transfer (FRET) experiments, it is seen that at physiological pH, conversion of the precursor HDV ribozyme to the product form is accompanied by an approximately 8 angstrom global end-to-end lengthening and that the precursor population displays heterogeneity. Local, active site dynamics were probed using molecular dynamics (MD) simulations, which can be linked to observations from solution experiments by the in-line fitness parameter. Together, experimental and MD data suggest that the HDV ribozyme has a rugged folding landscape along its catalytic pathway. The second aim involved MD simulations to determine hairpin ribozyme variants with a disrupted active site water chain. The robustness of the water chain is such that it displays disruption only for variants with two single-atom modified nucleobases. For the third aim, the impact on the hairpin ribozyme be- havior by an intracellular-mimic buffer, macromolecular crowding and interactions with cellular components was explored. While the intracellular-mimic buffer favors the undocked state, both crowding by PEG-8000 and the presence of yeast or HeLa cell extract favors the docked state. The interactions with cellular components are also more potent in promoting the docked state compared to crowding by PEG. This thesis exemplifies two trends in RNA research: the integration of computational and experimental work, and an interest in understanding the effects on RNA by various features of the intracellular environment.