Date of Award
Spring 2021
Document Type
Thesis
Degree Name
Master of Science (MS)
Department
Chemistry
First Advisor
Reiter, Nicholas
Second Advisor
Clark, Joseph R.
Third Advisor
Yi, Chae
Abstract
T-loops are highly abundant, structurally conserved five nucleotide motifs that facilitate long range intra- and intermolecular interactions and function to stabilize the tertiary fold of RNA. Interlocked or dual T-loop motifs provide additional thermodynamic stability and perform long-range docking of other RNA molecules. Dual T-loop RNA structures are present within the multi-turnover ribonuclease P-based ribozyme, the ribosome, and the bacterial tRNA-sensing riboswitch genetic element. In all three systems, a dual T-loop structure recognizes TYC/D-loop region of tRNA. Interestingly, sequence conservation at specific positions in the T-loops suggest a conserved interlocked RNA architecture. While X-ray crystallography provides insight into the putative hydrophobic stacking interactions, little or no information exists regarding hydrogen bonding or the conformational flexibility of the dual T-loop motifs. Through extensive NMR experiments, we examine the pre-assembly of the dual T-loop motif and define the hydrophobic stacking and hydrogen bonding environment of the secondary structural elements of a minimal construct containing the dual T-loop motif in a bacterial Bacillus subtilis glyQS T-box riboswitch. To better understand the structural diversity and functional roles of interlocked T-loop RNA motifs, the thermodynamic parameters and recognition properties of the dual-T loop RNA – tRNA interaction will be determined using a combined UV- melt analysis and isothermal titration calorimetry (ITC) approach. Results from these experiments suggest weak binding of the truncated constructs with a dissociation constant of 2.00±0.47 µM. In addition, preliminary data on the thermodynamic stability of con- structs has been obtained. RNA structural motifs, such as the dual T-loop motif, play an important role in the gene regulation and understanding the recognition patterns could lead to potential application in pharmaceuticals.
