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.

项目摘要 DNA超螺旋是基因组的普遍特征，是由易位酶和活性产生的通过包裹或改变其结合序列的扭曲的许多蛋白质的结合。因此，基因组超螺旋是动态的，是基于基因组活动的敏感调节剂，例如转录和重组。最近的最新努力集中在转录生成的超螺旋，双域模型和拓扑异构酶的活性。相反，在很大程度上没有探索的是DNA变化的影响在细胞生理学的主要方面进行超密集，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（III）的令人满意的策略也是缺少，因为当前的方法利用了改变双螺旋结构的探针。所以，利用磁镊子，蛋白质介导的DNA环，核苷相关蛋白的专业知识，以及为体外和体内转录分析的合作良好的合作，我们开发了目标将在这三个基本领域中显着提高我们的知识：（1）检验以下假设：DNA超串联显着促进拓扑结构的形成，例如蛋白质介导的环。使用LAC阻遏蛋白（LACI）作为DNA环蛋白，一系列循环长度以及体外和体内分析的互补，我们将建立超串联的水平，张力和核苷相关蛋白，最有可能在体内循环中催化。（2）检验以下假设：DNA超螺旋影响蛋白质的结合，以定义的结构基因组。我们将建立对（i）解离常数的DNA拓扑的依赖性，并且（ii）DNA 通过代表性的，丰富的核苷相关蛋白（NAP）的压实，该蛋白质（NAP）与DNA非特异性结合。（3）检验启动子活性在距离和超螺旋依赖性的假设通过上游蛋白质介导的环的方式。