Dissertation/Thesis Abstract

tRNA Processing and Quality Control in Bacteria
by Alluri, Ravi Kumar, Ph.D., Florida Atlantic University, 2013, 159; 3571440
Abstract (Summary)

In this work, we report that the only exoribonuclease in M. genitalium , RNase R, is able to generate mature 3'-ends. The aminoacyl-acceptor stem, CCA terminus and discriminator residue plays an important role in stopping RNase R digestion at the mature 3'-end. Disruption of the stem causes partial or complete degradation of the pre-tRNA, whereas extension of the stem results in the formation of a mature 3’-end. CC residues in CCA terminus and A or G residues at discriminator position are the most preferred residues for precise stopping of RNase R at mature 3’ end. The significance of this works shows that M. genitalium RNase R generates mature tRNA in a single step by recognizing features in the terminal domains of tRNA, a process requiring multiple RNases in most bacteria.

Our studies with in vivo and in vitro oxidation of Escherichia coli tRNA revealed that 8-hydroxyguanosine (8-oxo-G) levels of tRNA increased upon treatment with hydrogen peroxide (H2O2) in a dose dependent manner. Interestingly, native tRNA was oxidized to higher level than its corresponding heat-denatured tRNA. This demonstrates that higher order structures in tRNA promote tRNA oxidative damage.

E. coli tRNA pseudouridine synthase TruD catalyzes pseudouridylation of U13 of tRNAGlu. TruD protein binds to oxidized RNA with high affinity and protects cells under oxidative stress. Extensive RNA binding experiments revealed that only wild type TruD can differentiate normal and oxidized RNA. Surprisingly active site mutant proteins D80N and D80T did not show such differential specificity demonstrating the importance of charged aspartic acid in the active site. Studies to show the biochemical evidence behind protective role of TruD under oxidative stress revealed no concrete evidence. Our attempts have shown that under oxidative stress conditions, in cells lacking TruD aminoacylation of tRNAGlu was unaffected and tRNAGlu was stable. TruD was also not involved in the specific removal of 8-oxo-G containing RNA and oxidized base repair. The significance of this work shows the ability of TruD binding to oxidized RNA with high affinity and protects cells under oxidative stress. This demonstrates the existence of unknown biochemical mechanism behind the protective role of TruD which is yet to be elucidated.

Indexing (document details)
Advisor: Li, Zhongwei
Commitee: Ayyamithan, Kasirajan, Caputi, Massimo, Prentice, Howard M.
School: Florida Atlantic University
Department: Integrative Biology
School Location: United States -- Florida
Source: DAI-B 74/11(E), Dissertation Abstracts International
Subjects: Molecular biology, Microbiology, Biochemistry
Keywords: Bacteria, Oxidative stress, Quality control, RNA oxidation, RNase R, tRNA processing
Publication Number: 3571440
ISBN: 978-1-303-22817-9
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