Date of Award
Fall 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Molecular, Cellular, and Developmental Biology
First Advisor
Breaker, Ronald
Abstract
Riboswitches are regulatory RNA elements commonly present in the 5′ untranslated regions (UTRs) of certain mRNA molecules. They carry a conserved aptamer region that binds to metabolites or elemental ions. The binding of ligand promotes distinct folding pathway in the RNA structure which is translated into a regulatory response that either activates or inhibits the expression of downstream genes. Over 55 different classes of riboswitches have been experimentally validated so far that govern various aspects of biology in bacteria, archaea, and select eukaryotes. These RNA elements are thought have played crucial roles in gene regulation during the RNA World era, an evolutionary period before the emergence of proteins. Herein, I present the discovery and characterization of five riboswitch variant classes, including an engineered variant, based on the predominant guanine-I riboswitch class. All these classes share a common three-way junction architecture but comprise nucleotide variations in the core regions of the aptamer. The variants selectively recognize purine-derived metabolites and some have implications in the purine degradation pathways in Firmicutes. These findings signify how RNAs can evolve to diversify their ligand recognition characteristics without having to depend on entirely new structures. Additionally, I also pursue the exploration of a novel riboswitch class represented by terC motif RNAs that are potentially involved in metal ion-sensing. Preliminary genetics data suggest that these riboswitch candidates may respond to sodium or potassium ions, however, additional investigations are needed to validate this hypothesis. Furthermore, I also present the optimized process of generating a ssDNA ladder utilizing a self-cleaving DNA enzyme, I-R3. An improved version of the in-vitro selection method for generating self-hydrolyzing DNA enzymes is also reported. Lastly, the search for natural examples of I-R3-like deoxyribozymes is presented.
Recommended Citation
Hamal Dhakal, Siddhartha, "Investigation of Variant Guanine Riboswitches and Other Nucleic Acid Motifs that Sense Metal Ions" (2023). Yale Graduate School of Arts and Sciences Dissertations. 1177.
https://elischolar.library.yale.edu/gsas_dissertations/1177
