The role of transcription-coupled DNA supercoiling in the coordination of temporal geneexpression.

理解基因调控和基因组结构的概念是为工业目的设计人工染色体的核心。我们正面临着从遗传学基础研究向工程研究过渡的新时代的到来。序列组装技术是可用的，但是基因的相互作用是进化优化仍然远未被理解。最近的研究表明，基因插入原生染色体的位置对插入基因的表达以及周围基因的表达都有重大影响。因此，对于染色体的从头设计，了解这种邻域依赖性的来源是很重要的。在这一建议中，我们提出了惊人的初步数据，揭示转录偶联DNA超卷曲（TCDS），由转录rna聚合酶引入的扭转张力，是位置效应的主要来源。对于单个基因，已经证明TCDS影响邻近基因的基因表达。我们展示的证据表明，基因在整个大肠杆菌染色体上的排列是由TCDS和基因对TCDS的敏感性决定的。TCDS的调控概念为理解细菌和病毒致病性或自发耐药的遗传灵活性开辟了新的视角。此外，它将允许使用特定基因安排构建人工调节回路。因此，我们建议对模式生物大肠杆菌中TCDS介导的调控进行全面研究。我们计划在不同的组织水平上解决这个问题。在全球范围内，我们将使用最先进的时间分辨基因表达数据来研究邻近基因转录活性的相互依赖性。利用这些数据，我们将构建一个局部基因表达相互依赖的TCDS网络并分析其结构。此外，我们计划整合TCDS网络和由监管机构与其目标之间的联系组成的经典监管网络。我们的初步数据表明，通过调节因子和它们的靶标对DNA超卷曲变化的敏感性，这些网络之间存在联系。为了更深入地了解单个基因的局部相互作用，我们将研究大肠杆菌染色体上基因排列的表达，改变其基因排列，并与我们的模型进行比较。通过本地和全球的方法，我们确信能够阐明TCDS的复杂性。由于TCDS是由任何生物体中发现的基本过程引起的，我们对揭示远远超出大肠杆菌模式生物和细菌范围的重要遗传见解持高度乐观态度。

Understanding the concepts of genetic regulation and genome architecture is central for the design of artificial chromosomes for industrial purposes.We are facing the advent of a new era of transition from basic research in genetics to engineering.The technologies for sequence assembly are available, however the interplay of genes that was evolutionary optimized is still far from understood.Recent studies have shown that the position of a gene insertion into a native chromosome has a major impact on expression of the inserted gene as well as on the expression of the surrounding genes.Hence, for a de novo design of a chromosome it is important to understand the sources of such neighborhood dependencies.In this proposal we present striking preliminary data that reveals transcription-coupled DNA supercoiling (TCDS), a torsional tension introduced by transcribing RNA-polymerase, as a major source of positional effects.For individual genes it has been shown that TCDS affects gene expression of neighboring genes.We show evidence that the arrangement of genes on the whole E.coli chromosome is dictated by TCDS and the sensitivity of genes to TCDS.The regulatory concept of TCDS opens a new perspective to understand genetic flexibility in bacterial and viral pathogenicity or spontaneous drug resistance.Furthermore, it will allow for the construction of artificial regulatory circuits using particular gene arrangements.We therefore propose a comprehensive study of TCDS mediated regulation in the model organism E.coli.We plan to address the problem at different levels of organization.At a global level we will investigate the interdependencies of transcriptional activity of neighboring genes using state-of-the-art time-resolved gene expression data.With this data we will construct a TCDS network of local gene expression interdependencies and analyse its structure.Furthermore, we plan to integrate the TCDS network and the classical regulatory network consisting of links between regulators and their targets. Our preliminary data suggests a connection of the networks via the sensitivity of regulators and their targets to alterations in DNA supercoiling.For a deeper understanding of the local interactions of individual genes, we will study the expression of gene arrangements taken from the E.coli chromosome, alter its gene arrangement and compare the effects with our models.With the local and global approach we are convinced to elucidate the complexity of TCDS. As TCDS is caused by fundamental processes found in any organism we are highly optimistic to reveal important genetic insights far beyond the scope of the E.coli model organism and bacteria.