Long Range Interactions in Mu and Bacterial DNA

Mu 和细菌 DNA 的长程相互作用

基本信息

批准号：
7923644
负责人：
NORMAN P. HIGGINS
金额：
$ 21.97万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
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：
2009-09-30 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7923644
关键词：
Accounting Antibiotics Bacteria Bacterial Chromosomes Bacterial DNA Bacteriophages Behavior Binding Binding Proteins Biochemical Biochemistry Biological Assay Biological Markers C-terminal Cell division Cell model Cells Characteristics Chromosome Structures Chromosomes Chromosomes, Human, Pair 10 Chromosomes, Human, Pair 15 Complement DNA DNA Gyrase DNA Sequence DNA biosynthesis Data Diffusion Elements Escherichia coli Escherichia coli K12 Evolution Funding Gene Expression Genes Genetic Genetic Recombination Genetic Transcription Genome Goals Grant Human Life Ligation Location Measures Mechanics Mediating Methods Modeling Molecular Conformation Molecular Models Movement Mutagenesis Operon Organism Pattern Personal Communication Positioning Attribute Protein Binding Protein Dynamics Proteins Publishing Reporting Research Resolution Resolvase Running Salmonella Site Structure Superhelical DNA Surveys System Techniques Technology Testing Tetanus Helper Peptide Time Transfer RNA United States National Institutes of Health Work base cell behavior cell growth condensin daughter cell density design fighting gene synthesis genetic selection genome-wide in vivo microorganism molecular modeling new technology promoter public health relevance research study single molecule

项目摘要

DESCRIPTION (provided by applicant): Chromosome dynamics and the proteins that channel DNA movement in vivo are critical determinants of cell replication, gene expression, genetic recombination, and Darwinian evolution. Recent studies have demonstrated that bacterial chromosomes are organized into about 400 independent domains that limit supercoiling diffusion. The primary focus of our research over the next four years will be to identify the critical proteins and dissect the mechanical mechanisms that control bacterial chromosome structure and supercoil movement inside living cells. We have specific aims. 1) Connect the activities of gyrase and the bacterial condensin, MukB, to nucleoid compaction. This aim includes a new component involving DNA gyrase biochemistry and genetic methods that evolved from the E. coli/Salmonella species comparison. Genetic selections will be used to identify the proteins that form both the stochastic and sequence-specific domain boundaries in the 400 domain chromosome. Candidate "Domainins" or proteins that control a segment of chromosome structure will be run through a gauntlet of 4 tests to measure domain behavior. These tests include analyzing specific genes for their ability to change supercoil density and site-specific resolution efficiency at eight different locations, testing their effect on ribosomal RNA operons, and measuring the average domain size for each gene. Connect domains and DNA movement to structure. We will test a loop model for domain structure and define the dynamic characteristics of highly transcribed ribosomal RNA operons. As cells grow rapidly in rich media, 70% of all RNA synthesis is devoted to stable RNAs (ribosomal and tRNA genes). New experiments will test whether these regions form specific transcription loops and determine where the highly transcribed genes are in the folded genome. We will also establish whether or not transcription in WT bacteria can generate "waves of positive supercoils." 2) Connect domains and movement to structure. We will test a loop model for organizing highly transcribed protein-encoding genes, ribosomal RNA operons, and tRNA operons. We will also exploit the chromosome conformation capture technology to prove our hypothesis on looping. 3) Connect the average chromosome structure to single cell behavior using fluorescent cell technology. The domain structure of Lac- and Tet-operator modules that serve as cell biological markers of chromosome behavior will be analyzed. Studies will determine how modules behave as chromosome dynamics elements in vivo when unoccupied, when decorated with different levels of fluorescent binding protein, and how inducer changes supercoil dynamics for sites with bound repressors. In the course of these experiments, we will place fluorescent protein binding modules into the E. coli and Salmonella chromosome at 20 different positions using efficient recombination methods developed in the last grant period, and determine what happens when a segment of chromosome is separated from the main body by site specific recombination. PUBLIC HEALTH RELEVANCE: This project aims to develop a molecular model of chromosome organization and measure DNA dynamics inside living cells. Many essential proteins that participate in bacterial cell division are also found in eukaryotic organisms up to humans. This work provides a rationale for developing new antibiotics to fight pathogenic microorganisms and to solve old problems about how chromosomes become disentangled during cell growth.

描述（由申请人提供）：体内通道DNA运动的染色体动力学和蛋白质是细胞复制，基因表达，遗传重组和达尔文式进化的关键决定因素。最近的研究表明，细菌染色体被组织成大约400个限制超螺旋扩散的独立域。在接下来的四年中，我们的研究的主要重点是鉴定关键蛋白质，并剖析控制细菌染色体结构和活细胞内超盘运动的机械机制。我们有具体的目标。 1）将回旋酶的活性和细菌冷凝蛋白MUKB连接到核苷压实。该目的包括一个新成分，涉及从大肠杆菌/沙门氏菌物种比较进化的DNA旋酶生物化学和遗传方法。遗传选择将用于识别在400个结构域染色体中同时形成随机和序列特异性结构域边界的蛋白质。候选“域”或控制染色体结构段的蛋白质将通过4个测试的手套进行测量域行为。这些测试包括分析特定基因在八个不同位置改变超副盘密度和特定地点的分辨率效率的能力，测试它们对核糖体RNA操纵子的影响，并测量每个基因的平均域大小。将域和DNA运动连接到结构。我们将测试域结构的循环模型，并定义高度转录的核糖体RNA操纵子的动态特性。随着细胞在富培养基中的迅速生长，所有RNA合成中有70％用于稳定的RNA（核糖体和TRNA基因）。新实验将测试这些区域是否形成特定的转录环，并确定高度转录基因在折叠基因组中的位置。我们还将确定WT细菌中的转录是否可以产生“阳性超级锅波”。 2）将域和运动连接到结构。我们将测试一个循环模型，用于组织高度转录的蛋白质编码基因，核糖体RNA操纵子和tRNA操纵子。我们还将利用染色体构象捕获技术来证明我们关于循环的假设。 3）使用荧光细胞技术将平均染色体结构连接到单细胞行为。将分析用作染色体行为的细胞生物学标志物的LAC和TET-操作器模块的结构结构。研究将确定模块在未居住时在体内的染色体动力学元素的表现，当用不同水平的荧光结合蛋白装饰时，以及诱导剂如何改变具有绑定抑制剂的位点的超级旋转动力学。在这些实验的过程中，我们将使用在上一个赠款期间开发的有效重组方法将荧光蛋白结合模块放入20个不同位置的大肠杆菌和沙门氏菌染色体中，并确定当染色体部分通过现场重组与主体与主体分离时会发生什么。公共卫生相关性：该项目旨在开发染色体组织的分子模型并测量活细胞内的DNA动力学。在人类的真核生物中也发现了许多参与细菌细胞分裂的必需蛋白质。这项工作为开发新的抗生素来抗击致病性微生物，并解决有关在细胞生长过程中如何散布染色体的旧问题的基本原理。