Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor(s)

David Haniford

Abstract

Bacterial transposons typically exist in a mutually beneficial relationship with the host cell. Limited transposition can benefit the host while also ensuring the survival of the element. An important component of this relationship is that transposition must be tightly regulated. In this thesis I explore ways that the host and transposon each control transposition in E. coli and provide evidence that a transposon can also control host gene expression in S. enterica Typhimurium. Post-transcriptional regulation with small non-coding RNAs (sRNA) has emerged as a key way that bacteria respond to stress and regulate many cellular processes. The RNA-binding protein Hfq is the nexus of sRNA regulatory networks and acts by promoting base-pairing interactions between sRNAs and their target mRNAs. Previous work found that Hfq is a potent negative regulator of IS10 transposition in E. coli and suggested that Hfq inhibited transposase translation using an IS10-encoded sRNA (RNA-OUT) as well as an undefined mechanism that was independent of RNA-OUT. I show that Hfq promotes base-pairing between RNA-OUT and IS10 transposase mRNA (RNA-IN) by melting the secondary structure of both RNAs to expose residues involved in intermolecular base-pairing. I also investigated how Hfq can repress translation of RNA-IN in the absence of RNA-OUT and demonstrate that Hfq-binding to an mRNA can directly repress translation in the absence of any sRNA. The data suggested Hfq may regulate other transposons and I show that the unrelated IS200 element is also subject to Hfq regulation. In contrast to the IS10 system, Hfq represses IS200 transposase (tnpA) translation completely independent of the IS200-encoded sRNA (art200). Translation initiation on tnpA is inhibited >350-fold by the cooperation of Hfq, art200, and an RNA structural element in the tnpA 5’UTR illustrating how host- and transposon-encoded factors can coordinate to repress transposition. Lastly, I demonstrate that tnpA is processed to produce an sRNA that alters transcript abundance >2-fold for 73 S. enterica Typhimurium genes, which provides a new twist on our understanding of host-transposon interactions. Taken together, this work suggests that RNA transactions play an important role in governing host-transposon relationships in bacteria.

Additional Table 4.1 - Mapped sequencing reads.xls (960 kB)
Excel spreadsheet with mapped reads for RNA-Seq experiment described in Chapter 5

Additional Table 4.2 - ALDEx Output.xlsx (2677 kB)
Excel spreadsheet with results of differential expression analysis (ALDEx2) of RNA-Seq data for Chapter 5

Additional Table 4.3 - Differentially Expressed Genes.xlsx (119 kB)
Excel spreadsheet with list of genes identified as differentially expressed (Effect>2) in RNA-Seq experiment described in Chapter 5


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