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

在DNA、RNA和蛋白质相关的中心教条过程中，转录是关键步骤，其中DNA被用来产生RNA形式的互补读本，由RNA聚合酶（rnap）发挥作用。在最著名的原核生物模型大肠杆菌中，随着超分辨率显微镜等单分子、单细胞技术的出现，转录的时空动力学——即RNA从DNA转录的速率、DNA中心质量（类核）和离散DNA区域（质粒）的精确定位——已经得到越来越多的研究。本项目采用双管齐下的方法开发和改进活细胞工具，利用DNA/RNA荧光探针的特异性和DNA构建的基因工程来进一步改进现有技术，并旨在将这些工具与超分辨率显微镜一起应用于RNAP亚基交换的背景下研究RNAP的时空调节，以响应环境刺激和在类核的固有超卷曲状态下。通过基于超卷曲的调控研究类核动力学和通过sigma因子交换详细研究RNAP酶结构变化，同时通过PALM超分辨率显微镜对活细胞中RNAP种群分布进行更广泛的观察，将为细胞中转录调控的多因子手段提供更全面的认识，支持基础科学的复杂性和理解的必要性。

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.