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

探索活大肠杆菌中 RNA 聚合酶的时空功能和调节

基本信息

批准号：
2446099
负责人：
金额：
--
依托单位：
University of Oxford
依托单位国家：
英国
项目类别：
Studentship
财政年份：
2020
资助国家：
英国
起止时间：
2020 至无数据
项目状态：
未结题

来源：
https://gtr.ukri.org/projects?ref=studentship-2446099
关键词：
Exploring spatiotemporal functioning regulation RNA

项目摘要

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.

在将DNA与RNA与蛋白质相关联的中心教条式过程中，转录是关键步骤，其中DNA用于产生由RNA聚合酶（RNAP）产生的RNA形式的互补读段。在最著名的原核模型大肠杆菌中，转录的时空动力学-即RNA从DNA转录的速率，在DNA中心质量（类核）和离散DNA区域（质粒）中的精确定位-随着单分子，单细胞技术（如超分辨率显微镜）的出现而越来越多地被研究。该项目带来了一种双管齐下的方法，利用DNA/RNA荧光探针的特异性和DNA构建体的基因工程来开发和完善活细胞工具，以进一步完善现有技术，旨在将这些工具与超分辨率显微镜一起应用于研究RNAP亚基交换对环境刺激的响应以及类核内在超螺旋状态下RNAP的时空调节。详细研究通过基于超卷曲的调控的类核动力学和通过sigma因子交换的RNAP酶结构变异，同时通过PALM超分辨率显微镜在任何时候对活细胞中RNAP群体分布进行更广泛的观察，将为细胞中转录调控的多因素手段提供更全面的认识，支持复杂性，以及理解基础科学的必要性。