Control of Sigma B Activity in B subtilis

描述：(申请人提供)：为应对环境压力， 枯草杆菌的转录调节子Sigma B从一个 与抗Sigma B蛋白(RsbW)联合抑制激活 细菌一般应激调节基因的表达。由压力产生的 激活Sigma B的信号尚不清楚；然而，最近的证据表明 细菌的核糖体和一个小的GTP结合蛋白(Obg)在这个过程中。 该提案试图确定核糖体和obg在应激中的作用。 诱导Sigma B激活OBG和非必需核糖体蛋白 L11是环境压力激活sigma B所必需的。我们将执行 其中每一个的编码序列的定向和随机改变 蛋白质识别改变诱导性的区域或活动 Sigma B，并确定这些变化如何影响这些蛋白质中的每一个 已知的功能。分析应该识别Obg和L11的区域 对它们的生化活动很重要，并建议这些 对于西格玛B入职，属性是必需的。压力的几个组成部分 活化级联(RsbR、S和T)被观察到与 核糖体。我们将使用速度来检验这种假定的关联。 离心和凝胶过滤分析在可能的条件下 导致部分或全部分离。特定的核糖体组分组成 并将确定参与这些关联的RSB蛋白。这个 发现的复合体类型和直接涉及的蛋白质的身份可能 提供关于该关联在压力信号和Sigma B中的作用的线索 归纳法。最后，将对RSBT进行详细的突变分析， Sigma B应激诱导途径中最上游的正调控因子 其产物最有可能直接受到 压力信号。预计RSBT的变化及其 由此产生的表型也将为RSBT拟议的活动提供测试 识别应激信号改变RSBT活性的部位。这部作品 不仅探索基本的生物学问题，即细胞如何 识别并对恶劣环境做出反应，但考虑到西格玛B的存在 及其在人类病原体金黄色葡萄球菌中的主要调节因子， 单核细胞增多性李斯特菌和结核分枝杆菌，它可能有实用价值 应用，识别这些病原体在应激适应中的弱点 对主机防御的反应。

DESCRIPTION: (provided by applicant): In response to environmental stress, sigma B, a transcriptional regulator of B. subtilis, is released from an inhibitory association with an anti-sigma B protein (RsbW) to activate expression of the bacterium's general stress regulon. The stress-generated signal that activates sigma B is unknown; however, recent evidence implicates the bacterium's ribosome and a small GTP binding protein (Obg) in this process. The proposal seeks to determine the roles of the ribosome and Obg in the stress induced activation of sigma B. Both Obg and the non-essential ribosome protein L11 are needed for environmental stress to activate sigma B. We will perform directed and random alterations of the coding sequences for each of these proteins to identify regions or activities that modify the inducibility of sigma B and determine how these changes influence each of these proteins' other known functions. The analyses should identify regions of Obg and L11 that are important for their biochemical activities and suggest which of these properties are required for sigma B induction. Several components of the stress activation cascade (RsbR, S and T) have been observed to cofractionate with ribosomes. This putative association will be examined, using velocity centrifugation and gel filtration analyses under conditions that are likely to cause partial or total dissociations. The specific ribosome fraction components and Rsb proteins that are involved in the associations will be identified. The types of complexes found and the identities of proteins directly involved could give clues as to the role of the association in stress signaling and sigma B induction. Finally, a detailed mutational analysis will be undertaken of rsbT, the most upstream positive regulator in the sigma B stress induction pathway and the gene whose product is the most likely to be directly influenced by stress signaling. It is anticipated that the changes in rsbT and their resulting phenotypes will provide a test of RsbT's proposed activities as well as identify sites where stress directed signals alter RsbT activity. This work will not only explore the fundamental biological question of how cells recognize and react to hostile environments, but, given the presence of sigma B and its principal regulators in the human pathogens Staphylococcus aureus, Listeria monocytogenes and Mycobacterium tuberculosis, it may have practical applications, identifying weaknesses in these pathogens' stress adaptation responses to host defenses.