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

染色体拓扑和组织对大肠杆菌基因表达的影响

• 批准号: 10744700
• 负责人: Nicolas Naguib Yehya
• 金额: $ 4.77万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-30 至 2024-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10744700
• 关键词: Affect; Bacteria; Bacterial Chromosomes; Behavior; Binding; Cell physiology; Cells; Chromatin Loop; Chromosome Structures; Chromosomes; Computer Models; Coupled; Coupling; DNA; DNA-Directed RNA Polymerase; DNA-Protein Interaction; Data; Diffusion; Escherichia coli; Eukaryotic Cell; Feedback; Fluorescent Dyes; Fluorescent in Situ Hybridization; Gene Expression; Gene Expression Regulation; Gene Order; Generations; Genes; Genetic Transcription; Growth; Image; In Situ Hybridization; In Vitro; Individual; Kinetics; Knowledge; Lead; Learning; Length; Light; Maps; Measurement; Mechanics; Messenger RNA; Modeling; Molecular; Molecular Conformation; Monitor; Movement; Phase; Phenotype; Polymers; Positioning Attribute; Process; Production; Property; Proteins; Reaction; Relaxation; Series; Signal Transduction; Superhelical DNA; System; Testing; Time; Topoisomerase; Training; Work; antagonist; cell fixing; density; design; experimental study; in vivo; information processing; insight; mechanical properties; molecular imaging; premature; protein expression; single molecule; transcription factor

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.

项目总结/摘要 细菌染色体在细胞内按空间和拓扑结构进行组织， 这些变化是细胞过程、生长速率和细胞外部条件的函数。类核- 相关蛋白质通过弯曲、成环和包覆DNA以形成拓扑结构， 不同长度的域。在最低层，E.大肠杆菌染色体形成小环域，或 染色体拓扑分离结构域（TID），大约10 kbp。这种染色体组织 将超螺旋定位于染色体的不同部分。的 这和转录之间的关系是一个耦合，其中超螺旋密度决定 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， 为了验证我们的假设，即TID的形成显著调节基因表达谱，我们将使用 结合单分子成像和计算建模方法。 为了了解DNA拓扑学对转录的影响，我们必须能够分离出 一系列合成系统中的各种成分。在目标1中，我将构建一个体内合成环， 结构域，以研究结构域形成对两个转录和表达动力学的影响。 基因在不同条件下的变化。将通过单分子监测表达 荧光原位杂交和来自合成环结构域的活蛋白表达。目标2： 获得TID的拓扑状态如何影响RNAP的转录动力学的定量理解， 我将监测单个RNAP分子在环状模板DNA上的起始和延伸速率 在体外使用单分子蛋白诱导的荧光增强（smPIFE）模拟TID。在目标3中， 用反应扩散模型模拟DNA-蛋白质， 相互作用和超螺旋。以这种方式研究细菌染色体组织可能会揭示 细菌用来在干扰存在下维持转录的机制以及它们可能 利用DNA拓扑学效应进行基因调控。