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).

这个项目的长期目标是了解确保溶原性的机制 在温带噬菌体中维护，但确保必要时有效地切换到裂解。是这样的 了解这一点将有助于更好地控制噬菌体诱导的细菌。 发病机制。它也将是有价值的操纵诱导性设定点和使用噬菌体 在基因传递应用中。我们将使用噬菌体作为模型系统。最近的研究结果表明 稳定的溶原性和高效地转换为裂解都依赖于lambda的DNA环形成 抑制蛋白CI。CI介导的循环是一种最简单的转录调控机制 反馈机制，并决定噬菌体对发育生长的选择。然而，a 缺少CI介导的循环的特征。这项研究的结果也将是至关重要的 对于我们对转录调控和多蛋白介导的调控环的理解。 我们已经开始研究循环的分子机制，我们的结果表明：A 臭氧中心在环形成热力学中的关键作用，这对两个环都是一个复杂的动力学过程 形成和击穿，一个重要的，CI浓度依赖的臭氧位置在辅助作用中的作用 环形成高达20 nM的CI，这是一个重要的，与CI浓度无关的臭氧位置在 防止环路破裂，最后防止CI非特异性结合。总而言之，这些观察结果使得 形成和破裂的分子机制新假说的提出 监管环路。这一假说表明：(I)结合在臭氧上的CI二聚体起着“播种”作用 可能促进环路形成和干扰环路的更多二聚体的“招募”部位 (Ii)非特异性结合的CI二聚体的生理作用及其相互作用。 为了验证这一假说，我们提出：(1)理解CI介导的环路的机制 并确定与之相关的未成环物种。我们将通过以下方式完成此操作：(I)将 不同的、相关的未环物种及其对CI浓度的依赖(原子力 显微镜(AFM)和系留粒子显微镜(TPM))；(Ii)量化CI的非 特异性结合和探讨非特异性结合的CI二聚体之间协同作用的可能性 (磁性镊子的DNA拉取测量和理论建模)。(2)澄清 CI介导的环路崩溃的机制，以及与之相关的环路物种的特征。 我们将通过：(I)环状物种的可视化和它们的依赖关系的表征来实现这一点 时间化学计量学(AFM)；(2)对造成时间的机制进行表征 环路击穿动力学的依赖性(AFM和TPM)。(3)研究DNA的作用 在CI介导的环路(磁镊子)上的超线圈。