Exploring the spatiotemporal functioning and regulation of RNA Polymerases in the live Escherichia coli bacterium

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Of the centrally dogmatic processes relating DNA to RNA to protein, transcription is the key step in which DNA is utilised to produce a complementary read in the form of RNA, enacted by RNA polymerases (RNAPs). Within the most prominent prokaryotic model, Escherichia coli, the spatiotemporal dynamics of transcription - i.e. the rate at which RNA is transcribed from DNA, exact localisations in the central mass of DNA (nucleoid) and in discrete DNA regions (plasmids) - have become increasingly studied with the advent of single-molecule, single-cell technologies such as superresolution microscopy. This project brings a two-pronged approach in developing and refining live cell tools utilising the specificity of DNA/RNA fluorescent probes and genetic engineering of DNA constructs to further refine existing techniques, and aims to apply these tools alongside superresolution microscopy to study the spatiotemporal regulation of RNAP in the contexts of RNAP subunit exchange in response to environmental stimuli and in the intrinsic supercoiling states of the nucleoid. Studying both the dynamics of the nucleoid via supercoiling-based regulation and RNAP enzymatic structural variations via sigma factor exchange in detail, in tandem with a broader observation of RNAP population distribution across the live cell at any one time via PALM superresolution microscopy will provide a more holistic appreciation for the multifactorial means of transcriptional regulation in the cell, underpinning the complexity, and indeed necessity of understanding, of basic science.