Effects of Chromosomal Topology and Organization on E. coli Gene Expression

项目摘要/摘要 细菌染色体在细胞内以空间和拓扑的方式组织，呈现出构象。 这种变化是细胞过程、生长速度和细胞外条件的函数。类核- 相关蛋白质通过弯曲、环化和包裹DNA形成拓扑结构来促进这种组织 不同长度级别的域。在最低水平，大肠杆菌染色体形成小的环状结构域，或 染色体拓扑隔离结构域(TID)，大小约为10kbp。这个染色体组织 在拓扑隔离的结构域中，超螺旋定位于染色体的不同部分。这个 这与转录之间是一种耦合关系，其中超线圈密度决定了 转录活性，但转录活性也改变超螺旋密度。为了探索这些影响并 为了测试我们的假设，即TID的形成显著调节基因表达谱，我们将使用 单分子成像和计算建模相结合的方法。 为了更多地了解DNA拓扑对转录的影响，我们必须能够分离 一系列合成系统中的各种组件。在目标1中，我将构建一个体内合成环路 结构域的形成对两个基因转录和表达动力学的影响 不同条件下结构域内的基因。表达将通过单分子进行监测 合成环结构域的荧光原位杂交和活蛋白表达。在目标2中， 定量了解TID的拓扑状态如何影响RNAP的转录动力学， 我将监测单个RNAP分子在环状模板DNA上的起始和延伸速率 用单分子蛋白诱导的荧光增强(SmPIFE)在体外模拟TID。在《目标3》中， 用反应扩散模型模拟DNA-蛋白质，合成这两种尺度的模型 相互作用和超级盘绕。用这种方法研究细菌的染色体组织可能会揭示 细菌在存在扰动时维持转录的机制以及它们可能 利用DNA拓扑效应进行基因调控。

Project Summary/Abstract Bacterial chromosomes are organized spatially and topologically within cells, taking on conformations that change as a function of cellular processes, growth rates and conditions external to the cell. Nucleoid- associated proteins facilitate this organization by bending, looping, and coating DNA to form topological domains at different length levels. At the lowest level, the E. coli chromosome forms small looping domains, or chromosomal topologically isolated domains (TIDs), on the order of 10 kbp. This chromosomal organization into topologically isolated domains localizes supercoiling to different portions of the chromosome. The relationship between this and transcription is one of coupling, where supercoiling density determines transcriptional activity, but transcriptional activity also changes supercoiling density. To probe these effects and to test our hypothesis that the formation of TIDs significantly modulates gene expression profiles we will use a combined single-molecule imaging and computational modeling approach. To learn the most about the DNA topological effects on transcription as we must be able to isolate the various components in a series of synthetic systems. In Aim 1, I will construct an in vivo synthetic looping domain to investigate the effect of domain formation on the transcription and expression dynamics of two genes inside the domain under different conditions. Expression will be monitored via single molecule fluorescence in situ hybridization and live protein expression from synthetic looping domains. In Aim 2, to obtain a quantitative understanding of how the topological state of a TID impacts RNAP’s transcription kinetics, I will monitor the initiation and elongation rates of single RNAP molecules on a circular template DNA mimicking a TID using single molecule protein induced fluorescent enhancement (smPIFE) in vitro. In Aim 3, to synthesize the models at these two size scales by using a reaction-diffusion model to simulate DNA-protein interactions and supercoiling. Studying bacterial chromosomal organization in this way may reveal mechanisms that bacteria use to maintain transcription in the presence of perturbations and ways they may exploit DNA topology effects for gene regulation.