Chemical and structural analysis of RNA-based gene regulation

Thesis (Ph. D.)--University of Rochester. Dept. of Chemistry, 2009.Non-protein coding RNA (ncRNA), once thought to be a functional spectator in biological systems have recently been recharacterized as a critical component of many essential life processes. Due to the emergent pervasiveness of these vital biomolecules, structure/function studies aimed at elucidating their molecular basis of action are becoming increasingly important to the scientific community. To that end, this thesis is aimed at understanding the chemical and structural basis of function in ncRNA macromolecules, and to compare their molecular basis of action with proteins. In order to accomplish this, the thesis focuses on two ncRNA macromolecules: the hairpin ribozyme and the PreQ1 riboswitch. The hairpin ribozyme is an autocatalytic RNA motif found in tobacco ringspot virus satellite RNA. It serves to process concatentated RNA transcripts during rolling-circle RNA. Unlike other catalytic RNA motifs, the hairpin ribozyme relies solely on the localized nucleobases of the catalytic core. This all-RNA based mode of catalysis makes it an ideal system to elucidate the chemical principles employed by RNA for rate acceleration. To shed light on the mode of activity of this RNA construct, a synergistic approach using organic synthesis, enzymology and X-ray crystallography has been undertaken. From these studies, we have begun to understand how this RNA uses substrate orientation and functional group complimentarity to initiate activity. Additionally, parallels between RNA and protein enzymes have been derived, highlighting similar mechanisms of RNA processing. The second goal of the thesis is aimed at understanding the mode of metabolite recognition in the PreQ1 riboswitch aptamer domain. The PreQ1 riboswitch aptamer domain functions to regulate bacterial genes encoding proteins that are critical for exogenous preQ1 uptake or de novo biosynthesis. Notably, the essential aptamer domain was characterized to be ~3-times smaller than other analogous purine-sensing riboswitches. To understand how PreQ1 is recognized by the aptamer, an examination was performed employing X-Ray crystallographic techniques; this study provided a molecular glimpse into the mode of metabolite recognition. From these studies, we have expanded the knowledge of structure-based functional RNA, and have been able to compare modes of metabolite recognition between RNA and protein macromolecules