Intracellular studies of ribozymes and riboswitches

• Main Author: Watson, Peter Y
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2012
• Subjects: Biochemistry; Biophysics
• ISBN: 9781267909206; 126790920X

This work explores the biological functions of structured RNAs in the living cell. RNA folding studies of the hairpin ribozyme, functional exploration of the glmS ribozyme, and discovery of the ydaO ATP riboswitch are described herein. We expressed the hairpin ribozyme in a yeast model system to report intracellular folding outcomes. We determined that a threshold of structural stability exists; structures with stabilities weaker than -17 kcal/mol undergo exchange, while more stable structures resist equilibration. Using the same system, we investigated the folding of a regulatory RNA, or riboswitch, that controls expression of glucosamine-6-phosphate synthase. Expanding upon the traditional view that riboswitches respond to a specific metabolite ligand, we determined that the glmS riboswitch is also subject to inhibition by endogenous metabolites. We further confirmed this finding using reporter experiments in Bacillus subtilis , the natural context of the glmS riboswitch. These insights increased our understanding of RNAs’ regulatory capacity by demonstrating that they are capable of integrating information from multiple chemical signals. These results prompted an increased interest in the role of riboswitches in sensing central components of energy metabolism. We therefore performed a novel biochemical screen to determine whether “orphan” riboswitches of unknown function might bind ATP. We discovered that the ydaO riboswitch, a representative of a widespread class of riboswitches, binds ATP in vitro and responds to ATP levels in vivo. This motif controls diverse genes across a variety of Gram-positive bacteria, demonstrating that a single riboswitch can regulate disparate pathways in response to energy availability. These studies lay the framework for understanding the complex and diverse regulatory functions of RNAs.