Date of Award

January 2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Ann M. Flower

Abstract

During the process of ribosome assembly and maturation, the rRNA is heavily modified. The process of modification is ubiquitous throughout all kingdoms of life and includes cleavage by RNases, methylation and pseudouridylation of RNA species. Interestingly, while rRNA modification is ubiquitous and modification locations are frequently conserved, in many species the modification enzymes are dispensable for normal growth. This non-essentiality has made understanding the functions of RNA modification difficult.

Pseudouridines are one of the most common rRNA modifications and, in Escherichia coli, comprise nearly one third of the modifications found within the ribosome. Pseudouridylation is the post-transcriptional base isomerization of uridine to pseudouridine and is completed by a group of enzymes called pseudouridine synthases. In E. coli, the 6 synthases responsible for modifying the 23S rRNA are RluA, RluB, RluC, RluD, RluE and RluF, and the single synthase responsible for modifying the 16S rRNA is RsuA. Currently the few hypotheses as to the function of these modifications involve the effects of the unique chemical properties of a pseudouridine base on the RNA structure, but few studies have shown any effect on ribosome profiles in vivo.

Through construction of single pseudouridine synthase deletion strains we were able to identify several new phenotypes associated with deletion of either rluC or rluE. All the identified phenotypes were associated with the ability of the bacteria to tolerate low-oxygen conditions including altered glucose fermentation products, increased ability to utilize nitrate

in oxidative metabolism, and increased growth in low oxygen environments. Additionally, a series of multiple deletion mutants were constructed in order to investigate cumulative effects that might occur from loss of multiple pseudouridines from a single ribosomal RNA. Interestingly, no new growth alterations were noted, even in strains lacking all 7 of the rRNA pseudouridine synthase genes, indicating that under the tested conditions, there are not likely to be ribosomal assembly or function defects. While we were not able to determine a specific function for pseudouridines from this work, the additional phenotypes and the construction of a strain lacking all known synthase genes will provide new tools for furthering our understanding.

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