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Pseudouridine synthases (Ψ synthases) are the enzymes that catalyze the isomerization of uridine (U) to pseudouridine (Ψ), which is the most prevalent post-transcriptional modification of RNA. The Ψ synthases fall into six different families that share no significant global sequence similarity; however, they all involve a conserved aspartic acid residue which is absolutely essential for activity. Tyrosine is a conserved residue in the active site in five of the six families of Ψ synthases (phenylalanine in the TruD family) and was hypothesized as the general base for the isomerization reaction. To confirm the function of Tyr-96, Y96F RluA was assayed with both ASL and [F5U]ASL. U is converted to Ψ and F5U to F5U products. These results argue against the role of Tyr serving as general base. However, the slow rates of reactions and higher concentration of Y96F RluA needed for any reaction indicates that Tyr-96 does facilitates at least one step of the reaction. The major product of F5U from the action of Ψ synthases is a ribo isomer whereas the minor product is arabino, and its generation requires epimerization at C2′. The deprotonation at C2′ can be achieved by the conserved Asp or O2. To test if O2 is the general base, the isomerized U was replaced by 4-thiouridine (s4U). As an essential first step, RNA containing s4U needs to be verified as a good substrate for Ψ synthases, so RluA and TruB were incubated with [s4U]RNA. Intact [s4U]RNA shifted to later and shorter retention times after incubation with RluA and TruB, respectively. Traces of the digestion products of [s4U]RNA after incubation with the two enzymes also showed the new peaks that absorbed more strongly at 330 nm than 260 nm. These results indicate that [s4U]RNA can be handled as a substrate.
Duong, Uyen T., "Mechanism investigation of pseudouridine synthases TruB and RluA with RNA containing 5-fluorouridine and 4-thiouridine." (2017). Electronic Theses and Dissertations. Paper 2781.