Long Range Interactions in Mu and Bacterial DNA

Mu 和细菌 DNA 的长程相互作用

• 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
• 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

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）：染色体动力学和体内引导 DNA 运动的蛋白质是细胞复制、基因表达、遗传重组和达尔文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 个独立的结构域，限制了超螺旋扩散。未来四年我们研究的主要重点将是识别关键蛋白质并剖析控制细菌染色体结构和活细胞内超螺旋运动的机械机制。我们有具体的目标。 1) 将旋转酶和细菌压缩蛋白 MukB 的活性与类核压缩联系起来。这一目标包括一个新的组成部分，涉及 DNA 旋转酶生物化学和遗传方法，这些方法是从大肠杆菌/沙门氏菌物种比较中演变而来的。遗传选择将用于鉴定在 400 结构域染色体中形成随机和序列特异性结构域边界的蛋白质。候选“域蛋白”或控制染色体结构片段的蛋白质将通过 4 项测试来测量域行为。这些测试包括分析特定基因在八个不同位置改变超螺旋密度和位点特异性解析效率的能力，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结构域大小。将结构域和 DNA 运动连接起来。我们将测试域结构的环模型并定义高度转录的核糖体 RNA 操纵子的动态特征。随着细胞在丰富的培养基中快速生长，所有 RNA 合成的 70% 都致力于稳定的 RNA（核糖体和 tRNA 基因）。新的实验将测试这些区域是否形成特定的转录环，并确定高度转录的基因在折叠基因组中的位置。我们还将确定野生型细菌中的转录是否可以产生“正超螺旋波”。 2) 将领域和运动连接到结构。我们将测试一个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 操纵子的循环模型。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与单细胞行为联系起来。将分析作为染色体行为的细胞生物标记的 Lac 和 Tet 操纵子模块的结构域。研究将确定模块在未占据时、用不同水平的荧光结合蛋白修饰时如何作为体内染色体动力学元件表现，以及诱导物如何改变具有结合阻遏物的位点的超螺旋动力学。在这些实验过程中，我们将使用上次资助期间开发的高效重组方法将荧光蛋白结合模块放置到大肠杆菌和沙门氏菌染色体的20个不同位置，并确定当一段染色体通过位点特异性重组与主体分离时会发生什么。公共健康相关性：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 动态。许多参与细菌细胞分裂的必需蛋白质也存在于真核生物乃至人类中。这项工作为开发新的抗生素来对抗病原微生物和解决细胞生长过程中染色体如何解开的老问题提供了理论基础。