The lambda bacteriophage regulatory loop

lambda噬菌体调节环路

• 批准号: 8463214
• 负责人: Laura Finzi
• 金额: $ 24.92万
• 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 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8463214
• 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溶源的机制 在温带噬菌体中维持，但保证在必要时有效切换到裂解。 了解将有助于更好地控制噬菌体诱导的细菌 它对于操纵诱导设定点和噬菌体的使用也很有价值。 在基因传递应用中，我们将使用 ¿最近的研究结果显示噬菌体作为模型系统。 稳定的溶源性和高效的裂解能力 ¿依赖于 lambda 形成 DNA 环 阻遏蛋白 CI 介导的环化是最简单的转录调控之一。 然而，反馈机制并决定噬菌体发育生长的选择。 CI 介导的循环的特征缺失，这项研究的结果也将是关键。 为了我们对转录调控和多蛋白介导的调控环路的理解。 我们已经开始研究 ¿循环，我们的结果显示： o3 位点对于环形成热力学的关键作用，这是两个环的复杂动力学 形成和分解，o3 位点在协助中发挥重要的 CI 浓度依赖性作用 环形成高达 20 nM CI，o3 位点的一个重要的、与 CI 浓度无关的作用 防止环破裂，最后，CI 非特异性结合，这些观察结果使得 提出关于形成和分解的分子机制的新假设 这 该假设表明：(i) CI 二聚体在 o3 上的“播种”作用。 更多二聚体“招募”中的位点可能促进环形成并干扰环 分解；(ii) 非特异性结合的 CI 二聚体的生理作用及其相互作用。 为了检验这一假设，我们建议：（1）了解 CI 介导的循环机制 形成并识别与之相关的未环物种，我们将通过以下方式做到这一点：（i）表征 不同的、相关的未环物质及其对 CI 浓度的依赖性（原子力 (ii) 量化 CI 非的程度 特异性结合并探讨非特异性结合 CI 二聚体之间协同的可能性 （通过磁力镊子进行 DNA 牵引测量和理论建模） (2) 阐明。 CI 介导的环破坏机制，以及与其相关的环物种的表征。 我们将通过以下方式做到这一点：（i）环状物种的可视化及其依赖性的表征 (ii) 负责时间的机制的特征 (3) 研究DNA的作用 CI 介导的循环（磁镊子）上的超螺旋。