The lambda bacteriophage regulatory loop

lambda噬菌体调节环路

• 批准号: 8269952
• 负责人: Laura Finzi
• 金额: $ 25.83万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8269952
• 关键词: Acute; Address; Affinity; Atomic Force Microscopy; Bacteriophage lambda; Bacteriophages; Binding; Biological Models; Cell physiology; Chromatin Loop; Chromosomal translocation; Chromosomes, Human, Pair 3; Complex; Cytolysis; DNA; DNA-Directed RNA Polymerase; Dependence; Dependency; Development; Drug Formulations; Ensure; Epigenetic Process; Feedback; Gene Delivery; Genes; Genetic Recombination; Genetic Transcription; Goals; Growth; Homeostasis; Imagery; Kinetics; Lead; Left; Lysogeny; Lytic; Magnetism; Maintenance; Measurement; Mediating; Microscopy; Molecular; Operator Regions; Outcomes Research; Pathogenesis; Physiological; Proteins; Repression; Repressor Proteins; Research Design; Research Methodology; Role; Rupture; Site; Superhelical DNA; Telomere Maintenance; Testing; Theoretical model; Thermodynamics; Time; Transcriptional Regulation; Viral; Virus; Work; base; dimer; lambda repressor; novel; particle; prevent; promoter; research study; stoichiometry; tumor

The long-term goal of this project is to understand the mechanism that ensures lysogeny maintenance in temperate phages yet guaranteeing efficient switch to lysis when necessary. Such understanding will be useful in order to achieve a better control of phage-induced bacterial pathogenesis. It will also be valuable for manipulation of the inducibility set-point and use of phages in gene delivery applications. We will use ¿ bacteriophage as a model system. Recent findings showed that both stable lysogeny and efficient switch to lysis in ¿ rely on DNA loop formation by the lambda repressor protein CI. CI-mediated looping represents one of the simplest transcriptional regulatory feedback mechanisms and determines the choice of developmental growth by the phage. However, a characterization of CI-mediated looping is missing. The outcome of this research will also be pivotal for both our understanding of transcriptional regulation and multi-protein-mediated regulatoryloops. We have started investigating the molecular mechanism of ¿ looping and our results show: a pivotal role of the o3 sites for the thermodynamics of loop formation, a complex kinetics for both loop formation and breakdown, an important, CI concentration-dependent role of the o3 sites in aiding loop formation up to 20 nM CI, an important, CI concentration-independent role of the o3 sites in preventing loop rupture and, finally, CI non-specific binding. Together, these observations allow the formulation of a new hypothesis about the molecular mechanism for the formation and breakdown of the ¿ regulatory loop. This hypothesis suggests: (i) a "seeding" role for the CI dimers bound at the o3 sites in the "recruitment" of more dimers which may facilitate loop formation and interfere with loop breakdown; (ii) a physiological role for non-specifically bound CI dimers and their interaction. To test this hypothesis, we propose: (1) To understand the mechanism of CI-mediated loop formation and to identify the unlooped species relevant to it. We will do this by: (i) characterizing the different, relevant unlooped species and their dependence on CI concentration (Atomic Force Microscopy (AFM) and Tethered Particle Microscopy(TPM)); (ii) quantifying the extent of CI non- specific binding and probing the possibility of cooperativity between non-specifically bound CI dimers (DNA pulling measurements by magnetic tweezers and theoretical modeling). (2) To elucidate the mechanism of CI-mediated loop breakdown, and characterization of the looped species relevant to it. We will do this by: (i) visualization of looped species and characterization of the dependence of their stoichiometry on time (AFM); (ii) characterization of the mechanism responsible for the time dependency of the kinetics of loop breakdown (AFM and TPM). (3) To investigate the effect of DNA supercoiling on CI-mediated looping (magnetic tweezers).

该项目的长期目标是了解确保裂解性的机制 维持温度噬菌体，并确保必要时有效切换到裂解。这样的 理解对于更好地控制噬菌体诱导的细菌将是有用的 发病。这也将对于操纵诱导性设定点和噬菌体的使用有价值 在基因输送应用中。我们将使用„噬菌体作为模型系统。显示的最新发现 稳定的溶疗和有效切换到裂解»依赖于lambda的DNA环形成 抑制剂蛋白CI。 CI介导的循环代表最简单的转录调节之一 反馈机制并确定噬菌体的发展增长选择。但是， 缺少CI介导的循环的表征。这项研究的结果也将是关键 为了了解我们对转录调控和多蛋白介导的调节圈的理解。 我们已经开始研究循环的分子机制，我们的结果表明： 循环形成热力学的O3位点的关键作用，这是两种环的复杂动力学 形成和分解，这是O3位点在协助的重要的CI浓度依赖性作用 循环形成高达20 nm CI，这是O3位点的重要，CI浓度独立的作用 防止循环破裂，最后是CI非特异性结合。这些观察结果允许 形成了关于形成和崩溃的分子机制的新假设 调节循环。该假设表明：（i）CI二聚体结合在O3的“播种”作用 更多二聚体“招聘”中的地点，这可能有助于循环形成和干扰循环 分解; （ii）非特异性结合CI二聚体及其相互作用的物理作用。 为了检验该假设，我们提出：（1）了解CI介导的环的机制 形成并确定与之相关的无环物种。我们将通过：（i）表征 不同的，相关的无环物种及其对CI浓度的依赖（原子力 显微镜（AFM）和束缚粒子显微镜（TPM））; （ii）量化非CI非 - 特定的结合和探测非特异性结合CI二聚体之间协调的可能性 （通过磁性镊子和理论建模进行DNA拉测量）。 （2）阐明 CI介导的环分解的机理，以及与之相关的循环物种的表征。 我们将通过：（i）可视化循环物种的可视化和其依赖性的表征 时间计量计时（AFM）; （ii）特征的机制 循环分解动力学的依赖性（AFM和TPM）。 （3）研究DNA的效果 在CI介导的环（磁镊）上进行超螺旋。