Structural Dynamics of the Hepatitis Delta Virus and Hairpin Ribozymes: Implications for Function.

Small catalytic RNAs (ribozymes) are a class of RNAs less than 200 nucleotides in length, which are able to catalyze both cleavage and subsequent re-ligation of their own phosphodiester backbones. Cleavage results in formation of 2’,3’ cyclic phosphate and 5’ hydroxyl termini. There are five naturally occurring members of this group: the hepatitis delta virus (HDV) ribozyme, the hairpin ribozyme, the hammerhead ribozyme, the Varkud Satellite (VS) ribozyme, and the glmS ribozyme. All of these RNAs employ a distinct active site fold and combination of mechanisms to perform their chemical function. Despite extensive biochemical and structural characterization, the structural requirements of catalysis for the HDV and hairpin ribozymes have not been ascertained. The work in this thesis interrogates the conformational dynamics of these ribozymes to provide deeper insight into their structure-function relationships. In Chapter 2, we utilize a combination of ensemble fluorescence resonance energy transfer (FRET) experiments and molecular dynamics (MD) simulations to outline a portion of the free energy folding pathway of the HDV ribozyme, rationalizing all extant crystal structures. Our MD simulations in Chapter 3 define specific structural roles for the two GU wobble pairs in the HDV ribozyme active site through mutational analysis. We show in Chapter 4, with the aid of newly developed constant pH MD simulations for nucleic acids, that experimental pKa measurements of an active site residue in the hairpin ribozyme reflect a minor conformational population. Taken together, the results in this thesis reveal the complex network of noncovalent interactions within the HDV and hairpin ribozymes that can have either favor or disfavor catalysis. Our results thus provide insights into the function of these two catalytic RNAs through interrogations of their structural dynamics. Such knowledge consequently provides a deeper understanding of catalytic RNAs, and all non-coding RNAs in general. A thorough knowledge of these complex biomolecules is of growing importance given the increasing recognition of the crucial roles played by RNA in all three domains of life.PhDMedicinal ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/107339/1/ksripath_1.pd