The lambda bacteriophage regulatory loop

lambda噬菌体调节环路

• 批准号: 8072532
• 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/8072532
• 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; Health; 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

DESCRIPTION (provided by applicant): 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 regulatory loops. 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 nonspecific 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). PUBLIC HEALTH RELEVANCE The proposal aims to characterize the molecular bases of the epigenetic switch in lambda bacteriophage; an acute understanding of this mechanism will provide a better control of phage-induced bacterial pathogenesis and allow the use of inducible viruses for gene delivery and/or therapy. The lambda switch is also a paradigm of long-range interactions and multi-protein assemblages which if altered can lead to anomalies and tumors.

描述(由申请人提供)：这个项目的长期目标是了解确保温带噬菌体保持溶源的机制，同时确保必要时有效地切换到裂解。这样的了解将有助于更好地控制噬菌体诱导的细菌致病。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。我们会用什么？以噬菌体为模型系统。最近的研究结果表明，稳定的溶原性和有效的转换为裂解在？依赖于Lambda阻遏蛋白CI形成的DNA环。CI介导的环路是最简单的转录调控反馈机制之一，它决定了噬菌体对发育生长的选择。然而，缺少CI介导的循环的特征。这项研究的结果也将是我们理解转录调控和多蛋白介导的调控环的关键。我们已经开始研究？的分子机制。环形成和我们的结果表明：臭氧中心对于环形成的热力学起着关键作用，环形成和破裂的复杂动力学，臭氧中心在高达20 nM CI的帮助环形成中的重要的CI浓度依赖的作用，03中心在防止环破裂以及最后的CI非特异性结合方面的重要的CI浓度无关的作用。综上所述，这些观察结果允许提出一种新的假说，即？监管环路。这一假说表明：(I)结合在臭氧位点上的CI二聚体在更多二聚体的“招募”中起“播种”作用，这可能促进环的形成并干扰环的破裂；(Ii)非特异性结合的CI二聚体及其相互作用的生理作用。为了验证这一假说，我们提出：(1)了解CI介导的环形成机制，并识别与其相关的未环物种。我们将通过以下方式完成这项工作：(I)表征不同、相关的未环物种及其对CI浓度的依赖(原子力显微镜(AFM)和拴系粒子显微镜(TPM))；(Ii)量化CI非特异性结合的程度，并探索非特异性结合CI二聚体之间协同作用的可能性(利用磁钳测量DNA拉动和理论建模)。(2)阐明CI介导的环路破裂机制，以及与之相关的环路物种的特征。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化学计量比对时间的依赖性(AFM)；(Ii)环状击穿动力学(AFM和TPM)对时间依赖性的机理的表征。(3)研究DNA超螺旋对CI介导的环化(磁钳)的影响。与公共卫生的相关性该提案旨在表征Lambda噬菌体表观遗传开关的分子基础；对这一机制的敏锐理解将有助于更好地控制噬菌体诱导的细菌发病机制，并允许使用可诱导病毒进行基因传递和/或治疗。波长开关也是一种长程相互作用和多蛋白质组合的范例，如果改变，可能会导致异常和肿瘤。