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Supercoiling in genome topology and transcription

基因组拓扑和转录中的超螺旋

基本信息

批准号：
10159293
负责人：
Laura Finzi
金额：
$ 35.37万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-01 至 2022-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10159293
关键词：
Address Affect Affinity Architecture Area Bacterial Chromosomes Binding Binding Proteins Biological Assay Cell physiology Characteristics Chromatin Loop Chromosomes Collaborations Complex DNA DNA-Binding Proteins DNA-Directed RNA Polymerase Data Dependence Diffuse Dissociation Drug Design Elements Enzymes Escherichia coli Eukaryota Genetic Recombination Genetic Transcription Genome Genomic DNA Genomics Handedness In Vitro Individual Knowledge Lac Repressors LacZ Genes Length Leucine Location Magnetism Maps Measurement Mediating Methods Modeling Nucleosome Core Particle Outcome Phase Transition Plasmids Play Polymerase Probability Prokaryotic Cells Proteins Repression Repressor Proteins Role Structure Superhelical DNA System Testing Topoisomerase Transact Twin Multiple Birth Variant base experimental study genetic regulatory protein improved in vivo promoter single molecule wound

项目摘要

PROJECT SUMMARY DNA supercoiling is a ubiquitous feature of genomes, generated by the activity of translocating enzymes and by the binding of many proteins that wrap or change the twist of sequences to which they bind. Thus, genomic supercoiling is dynamic and is a sensitive regulator of genome-based activities, such as transcription and recombination. Most recent efforts have focused on transcription-generated supercoiling, the twin domain model, and the activity of topoisomerases. Largely unexplored is instead the impact of changes in DNA supercoiling on major aspects of cell physiology such as (i) long distance genomic interactions and (ii) the binding of architectural proteins to DNA. A satisfactory strategy to map genomic supercoiling (iii) is also lacking, since current approaches utilize probes that alter the structure of the double helix. Therefore, leveraging our expertise with magnetic tweezers, protein-mediated DNA looping, nucleoid associated proteins, and well established collaborations for in vitro and in vivo transcription assays, we have developed aims that will advance significantly our knowledge in these three fundamental areas: (1) Test the hypothesis that DNA supercoiling significantly facilitates the formation of topological structures, such as protein-mediated loops. Using the lac repressor protein (LacI) as a DNA looping protein, a range of loop lengths, and complementary in vitro and in vivo assays, we will establish the levels of supercoiling, tension and nucleoid associated proteins which most likely catalyze in vivo looping. (2) Test the hypothesis that DNA supercoiling affects the binding of proteins that define the architecture of the genome. We will establish the dependence on DNA topology of the (i) dissociation constant, and (ii) DNA compaction by representative, abundant nucleoid associated proteins (NAPs) that bind DNA non-specifically. (3) Test the hypothesis that promoter activity is affected, in a distance- and supercoiling-dependent manner, by an upstream protein-mediated loop.

项目概要 DNA 超螺旋是基因组普遍存在的特征，由易位酶和通过许多蛋白质的结合来包裹或改变它们所结合的序列的扭曲。因此，基因组超螺旋是动态的，是基于基因组的活动的敏感调节器，例如转录和重组。最近的努力集中在转录生成的超螺旋，即孪生结构域模型和拓扑异构酶的活性。相反，DNA 变化的影响很大程度上未被探索超螺旋在细胞生理学的主要方面，例如（i）长距离基因组相互作用和（ii）结构蛋白与 DNA 的结合。绘制基因组超螺旋图谱 (iii) 的令人满意的策略也是缺乏，因为目前的方法利用改变双螺旋结构的探针。所以，利用我们在磁力镊子、蛋白质介导的 DNA 环、类核相关蛋白等方面的专业知识，以及在体外和体内转录测定方面建立的良好合作，我们制定的目标是将显着提高我们在这三个基本领域的知识： (1) 检验 DNA 超螺旋显着促进拓扑结构形成的假设，例如蛋白质介导的环。使用 lac 阻遏蛋白 (LacI) 作为 DNA 环蛋白，一系列环长度以及互补的体外和体内测定，我们将确定超螺旋的水平，最有可能催化体内循环的张力和类核相关蛋白。 (2) 检验 DNA 超螺旋影响定义 DNA 结构的蛋白质结合的假设基因组。我们将建立 (i) 解离常数和 (ii) DNA 对 DNA 拓扑的依赖性通过非特异性结合 DNA 的代表性丰富的核相关蛋白 (NAP) 进行压缩。 (3) 检验启动子活性受到距离和超螺旋依赖性影响的假设方式，通过上游蛋白质介导的环。