Riboswitches as Versatile Scaffolds for Ligand Recognition and as Tools for Drug Discovery
Date of Award
Doctor of Philosophy (PhD)
Molecular, Cellular, and Developmental Biology
Riboswitches are highly structured, noncoding RNAs that bind specific ligands to regulate gene expression. To date, over fifty riboswitch classes have been validated to control fundamental biological processes and recognize various coenzymes, nucleotides, signaling molecules, ions, and other important ligands. As described in Chapter 1, careful study of these various riboswitches reveals an incredible diversity in their structural and biochemical characteristics as well as their function. Herein is detailed the efforts to validate long-standing orphan riboswitch classes involved in essential cellular functions such as maintaining nucleotide pools, managing ion toxicity, osmoregulation, and ATP production.The ykkC family of riboswitches are a set of at least five variant classes that share a conserved sequence and structure, but have accrued specific mutations in their ligand binding pockets to diverge and sense distinct ligands. Previous efforts to ascertain the ligand identities of these classes has resulted in the validation of the guanidine-I, ppGpp and PRPP riboswitches. These studies have revealed previously unknown biological activities, signaling networks, and complex gene regulatory systems. Chapter 2 describes the validation of the fourth variant class of the ykkC riboswitches, which promiscuously binds (d)ADP and (d)CDP to regulate hydrolase enzymes which presumably function to balance cellular nucleotide pools. The complexity of the molecular recognition of these molecules by the RNA serves to demonstrate the incredible adaptability of riboswitch sensors. Also reported in this chapter, are the efforts towards validating the last known ykkC riboswitch class, and the challenges faced in this endeavor. In Chapters 3 and 4, the first four riboswitch classes that specifically recognize the monovalent cations Na+ or Li+ are described. Many of the genes regulated by these riboswitches encode metal ion transporters that could help bacteria address ion toxicity. Other proteins whose expression are regulated by Na+-sensing riboswitches mitigate osmotic stress or harness Na+ gradients for ATP production. Some bacteria use a Na+ riboswitch in tandem with a riboswitch that recognizes the bacterial second messenger c-di-AMP to form a Boolean logic gate. This sophisticated device integrates information regarding Na+ concentration with c-di-AMP signaling to adapt to changing osmotic conditions. To date, only one protein factor has been experimentally validated to sense Na+ and regulate gene expression. Furthermore, the mechanisms of Li+ toxicity are poorly understood in general. Thus, the study of these riboswitches substantially advances our understanding of how cells sense and respond to these ions. Riboswitches are widely utilized by cells to perform sensory and regulatory tasks, but they also have great utility for various other applications. Chapter 5 describes the use of the S-adenosylhomocysteine (SAH) riboswitch as a biosensor for cellular levels of SAH in a high-throughput screen for small molecules that inhibit SAH nucleosidase, an enzyme responsible for recycling this toxic molecule. This example serves as another reminder of the amazing versatility of riboswitches as biological sensors.
Sadeeshkumar, Harini, "Riboswitches as Versatile Scaffolds for Ligand Recognition and as Tools for Drug Discovery" (2022). Yale Graduate School of Arts and Sciences Dissertations. 518.