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

在DNA、RNA和蛋白质之间的中心教条过程中，转录是关键的一步，在这一过程中，DNA被用来产生由RNA聚合酶(RNAP)调控的RNA形式的互补读数。在最著名的原核生物模型--大肠杆菌中，随着超分辨显微镜等单分子、单细胞技术的出现，转录的时空动力学--即从DNA转录RNA的速度，DNA中心质量(类核)和离散DNA区域(质粒)的准确定位--已经得到越来越多的研究。该项目采用了一种双管齐下的方法来开发和提炼活细胞工具，利用DNA/RNA荧光探针的特异性和DNA结构的基因工程来进一步改进现有的技术，并旨在将这些工具与超分辨率显微镜一起应用于研究RNAP亚单位交换对环境刺激的响应和在类核的内在超螺旋状态下RNAP的时空调节。通过超螺旋调控的类核动态和通过Sigma因子交换的RNAP酶结构变化的详细研究，结合通过Palm超分辨率显微镜对RNAP群体在任何时候在活细胞中的分布的更广泛的观察，将提供对细胞中转录调控的多因素手段的更全面的认识，从而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.