Combining in silico and in vitro studies on tRNA dynamics and pseudouridine formation by TruB

Thumbnail Image
Friedt, Jenna
Journal Title
Journal ISSN
Volume Title
Lethbridge, Alta. : University of Lethbridge, Dept. of Chemistry and Biochemistry
RNA is an important molecule in the cell capable of carrying sequence information for protein production and acting as a functional component in essential cellular processes. In order to perform its many roles, RNA must often adopt specific three-dimensional structures that can be stabilized by the introduction of chemical modifications. Pseudouridine (Ψ) is the most abundant post-transcriptional RNA modification and is formed by enzymes known as pseudouridine synthases. These enzymes share a common core fold and active site structure, but the exact mechanism of pseudouridine formation is still undetermined. Here, a combination of in silico and in vitro studies were used to identify residues involved in catalysis to better understand the mechanism of pseudouridine formation. Molecular dynamics (MD) simulations of the Escherichia coli pseudouridine synthase TruB, responsible for the formation of the conserved Ψ55 in tRNA, were performed that revealed an interaction network of three active site residues. In vitro mutational studies showed that these residues are not required for substrate binding, but are highly important for catalysis. Additionally, MD simulations of unmodified tRNA were performed to establish a foundation for nucleic acid simulation by monitoring the effect of Mg2+ ions on the stability of tRNA in silico. These simulations will allow for a complex of tRNA and TruB to be simulated in order to complement the experimental studies and better understand the role of protein-RNA interactions. Together, computational and experimental work will assist in determining the mechanism of pseudouridine formation that may be common to all pseudouridine synthases.
ix, 134 leaves : ill. (some col.) ; 29 cm
Transfer RNA -- Research , Pseudouridine -- Research , Post-translational modification -- Research , Dissertations, Academic