Open Access Dissertation
Date of Award
Christine S. Chow
Nucleic acids undergo both global and local conformational changes that are important for their function. Structural studies have over the decades been invaluable in elucidation of various biomolecular mechanisms, hence contributing significantly to the understanding of biological events. However, a clear understanding of how molecules function in the cellular context requires investigation of their interconversion between multiple conformations, including mapping the folding landscape and any coupled changes in conformation. Work in this thesis focuses on fluorescence experiments, mainly at a single-molecule level to investigate such processes.
First, a novel single-molecule approach is described focusing on local dynamics within nucleic acids and taking advantage of the fluorescent properties of 2-aminopurine (2AP) and pyrrolo-cytosine (PC) to study local dynamics at single base resolution. A click chemistry-based, single-molecule immobilization methodology that enables sufficient minimization of background fluorescence to allow single-molecule detection was utilized. In the absence of stacking interactions, both PC and 2AP fluoresce steadily for several seconds hence demonstrating their sufficient photostability for single-molecule utilization. Local dynamics using 2AP across a DNA abasic-site mimic, SAM-1 riboswitch binding pocket reorganization, and tRNAPro 3' end are reported.
Additionally, RNA global motions in conditions with molecular crowding, which mimics the cellular environment, are reported using the cyclic-diguanylate monophosphate (c-di-GMP) riboswitch aptamer domain as a model. Riboswitches are examples of gene-regulating elements that normally act in cis to their host mRNAs and are hence located within their 5' untranslated regions. in vivo, the intracellular physiology, which is highly crowded, dictates the folding landscape of RNAs. Macromolecular crowding influences, and hence alters, the riboswitch folding pathway, and subsequently may change their mode of ligand/substrate recognition in comparison to experiments in a non-crowded medium. Single-molecule and steady-state FRET studies show that the presence of molecular crowding enhances docking of the ubiquitous bacterial c-di-GMP riboswitch aptamer region even in the absence of it's metabolite. The adoption of a structure reminiscent of the ligand-bound form implies that in vivo the riboswitch can fully fold, and hence, ligand binding may only require minimal rearrangements. This may further decrease the time between ligand-recognition, interaction, and transcription enhancement.
Patrick, Eric Muthuri, "Single-Molecule Studies Of Local And Global Nucleic-Acid Dynamics" (2013). Wayne State University Dissertations. 790.