Date of Award

12-1-2010

Degree Name

Master of Science

Department

Molecular Biology Microbiology and Biochemistry

First Advisor

Gupta, Ramesh

Abstract

Pseudouridine synthases are enzymes that catalyze the isomerization of uridine to pseudouridine (Ψ) in RNA. The other common modification of RNA is the 2'-O-ribose methylation. The pseudouridine synthases from the three domains of life namely, bacterial TruB, archaeal Pus10 and eukaryal Pus4, are responsible for the formation of the universally conserved Ψ55 in the T-arm of tRNAs. We have carried out a comparative analysis of their activities on two different tRNA substrates namely tRNATrp and tRNAMet. Our findings support the observation that TruB absolutely requires the U54:A58 reverse Hoogsteen base pairing when it encounters the minimal T-arm-substrate. However, this requirement is relaxed when the length of the tRNA substrate is increased. Pus10 does not require this reverse Hoogsteen pair for its activity when tested in lower salt. However, as the salt concentration is increased, the Ψ formation is lost in the minimal substrate lacking this base pairing. Pus4, on the other hand, can modify the T-arm substrate and does not have an absolute requirement for the U54:A58 pairing. The Ψ54 and Ψ55 activity of Pus10 and TruB was also compared and it was seen that as the length of the substrate increased, the Pus10 activity became more specific for Ψ54 formation while TruB did not show any Ψ54 formation as reported earlier. Additionally, the effect of Cm56 methylation on the activities of TruB, Pus10 and Pus4 was determined in vitro. TruB shows a decrease in Ψ55 formation in the C56 methylated tRNAMet transcript but does not seem to affect the C56 methylated tRNATrp transcript. Pus10 and Pus4, on the other hand show a lower Ψ55 formation in both the C56 methylated transcripts. In vivo analysis of the effect of Cm56 methylation on the activity of TruB in E. coli was not conclusive and requires further study.

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