Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Dr. David Haniford

Abstract

Transposons play an integral role in bacterial adaptation, therefore it is important to understand the chemical steps and regulatory factors that govern their mobility. The properties that drive insertion of transposons into subsequent DNA sites have been studied for few elements. The transposon Tn5 in particular has few defined insertion sites and little is known about how its targets are selected. An objective of this thesis was to determine the molecular details of target insertion in Tn5 transposition in vitro. To this end, I assessed whether Tn5 could preferentially insert into short oligonucleotide substrates. I detected a Tn5 transpososome complex bound to a short target molecule in vitro and observed specific insertion into target sites. I also determined that distortion of the target backbone at precise locations is a determinant for insertion efficiency. This flexibility may facilitate a unique mechanism for specific target selection in Tn5. In addition, the regulation of transposons by bacterial proteins remains elusive for many transposons. A secondary objective of this thesis was to investigate the impact of the host proteins H-NS and Hfq on Tn5 transposition in Escherichia coli. H-NS and Hfq were determined to regulate Tn5 transposition in vivo in opposing directions. An Δhns strain displayed lower transposition than an hns+ strain and an Δhfq strain supported higher levels than an hfq+ strain. H-NS was found to specifically bind the transpososome in vitro and stimulate transpososome assembly under conditions that would normally disfavour assembly. I determined potential H-NS-DNA binding sites in the transpososome, and found that these sequences were critical for the interaction, and for H-NS up-regulation of Tn5 in vivo. Modeling of the H-NS protein onto the structure of the Tn5 transpososome revealed potential interactions. Hfq was found to strongly inhibit transposase expression under certain growth conditions, specifically from a transposase-lacZ translational fusion. Hfq also bound to the 5’ region of transposase RNA in vitro, providing evidence that Hfq acts directly to repress transposase expression and down-regulate Tn5 transposition. As these host proteins are key mediators of stress responses and virulence, they may act to link cellular physiology to transposition rates.

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