| Summary: | <p>Protein-DNA interactions are critical to many important biological functions, from transcription to DNA replication. To better understand these processes we need to look at molecular details, such as the stoichiometries and binding kinetics of these proteins. However, focusing on the molecular level can miss the bigger picture; we also need to understand how protein-DNA interactions shape the organisation of chromosomes and cause phenotypical changes over the whole cell.</p> <p>In this thesis I describe the construction of a super-resolution fluorescence microscope to image single molecules in live bacteria, and show how analysis with tools like single-particle tracking allow individual proteins specifically bound to DNA to be distinguished from mobile molecules, offering a new perspective on protein-DNA interactions, from the molecular level to the length scale of whole bacterial cells. I detail how I have applied these techniques to answer key questions in transcription, chromosome organisation, and DNA segregation in <em>Escherichia coli.</em></p> <p>Firstly, I looked at RNA polymerase (RNAP) to study how transcription affects the organisation of the nucleoid. Discriminating specifically bound RNAPs showed that low levels of transcription can occur throughout the nucleoid, but clustering analysis and 3D Structured Illumination Microscopy (SIM) showed that dense clusters of transcribing RNAPs format the nucleoid periphery, indicating a movement of gene loci out of the bulk of DNA as levels of transcription increase. Furthermore, I developed an assay to characterise the search process and non-specific DNA interactions of RNAP, which I also apply to a diverse selection of other DNA-binding proteins.</p> <p>I also characterized the <em>in vivo</em> behaviour of the type II topoisomerase, TopoIV. Imaging both subunits of TopoIV, combined with over-expression of unlabelled subunits, allowed the fraction of functional enzymes to be determined. Measuring the duration of catalytic events indicated that the majority of active TopoIV molecules catalyse decatenation. Finally, I studied MukBEF, an SMC (Structural Maintenance of Chromosomes) complex that acts in chromosome segregation, to show that TopoIV and MukB interact directly <em>in vivo</em> and determine the dissociation constant and turnover of this TopoIV-MukB complex.</p>
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