The lambda bacteriophage regulatory loop

这个项目的长期目标是了解确保溶原性的机制 维持在温和的温度下，同时保证在必要时有效地切换到裂解。等 理解将是有用的，以实现更好的控制噬菌体诱导的细菌 发病机制这对电感调定点的控制和电感的使用也是有价值的 基因传递的应用。我们将使用噬菌体作为模型系统。最近的调查结果显示， 稳定的溶原性和有效的裂解转换都依赖于λ-DNA环的形成， 阻遏蛋白C1。CI介导的成环是最简单的转录调控机制之一。 反馈机制，决定了噬菌体发育生长的选择。但 缺少CI介导的成环的表征。这项研究的结果也将是至关重要的 对于我们理解转录调控和多蛋白质介导的调节环。 我们已经开始研究环的分子机制，我们的结果表明： 关键作用的o3网站的热力学环路的形成，一个复杂的动力学为两个环 形成和分解，一个重要的，CI浓度依赖的作用，O3网站，帮助 环形成高达20 nM CI，这是o3位点在 防止环断裂，最后，防止Cl非特异性结合。总之，这些观察结果允许 形成和分解的分子机制的新假说 调节循环。这一假设表明：（i）在α 3位结合的Cl二聚体的“播种”作用， 更多二聚体的“募集”中的位点，其可促进环形成并干扰环形成。 分解;（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，并确定与之相关的非循环物种。我们将通过以下方式做到这一点：（i）表征 不同的、相关的未成环物质及其对CI浓度的依赖性（原子力 （ii）定量C1非-C1结构的程度; 特异性结合和探测非特异性结合的Cl二聚体之间的协同性的可能性 (DNA通过磁镊和理论建模的牵引测量）。(2)阐明本 CI介导的环断裂的机制，以及与之相关的环状物质的表征。 我们将通过以下方式做到这一点：（i）循环物种的可视化和其依赖性的表征 化学计量时间（AFM）;（ii）表征的机制负责的时间 环破裂动力学的依赖性（AFM和TPM）。(3)为了研究DNA的影响， CI介导的成环（磁镊）上的超螺旋。

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).