Title
Biochemical Analysis of Dual T-Loop RNA Motifs that Perform Shape Recognition of the tRNA Elbow
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.