Control of Sigma B Activity in B subtilis

描述：（由申请人提供）：为了应对环境压力， sigma B，B的转录调节因子。枯草芽孢杆菌，是从一个 与抗σ B蛋白（RsbW）抑制性结合以激活 细菌的一般应激调节子的表达。压力产生的 激活sigma B的信号是未知的;然而，最近的证据表明， 细菌的核糖体和小GTP结合蛋白（Obg）在这个过程中。 该提案旨在确定核糖体和Obg在应激中的作用 诱导激活σ B。Obg和非必需核糖体蛋白 L11是环境胁迫激活sigma B所必需的。我们将执行 这些编码序列中的每一个的定向和随机改变 蛋白质，以鉴定修饰的诱导的区域或活性， 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。分析应确定Obg和L11的区域， 重要的生物化学活动，并建议这些 特性是σ B诱导所必需的。应力的几个分量 已观察到激活级联反应（RsbR、S和T）与 核糖体我们将用速度来检验这种假定的联系 离心和凝胶过滤分析的条件下，可能会 导致部分或全部的分离。特定的核糖体组分 并且将鉴定参与所述关联的Rsb蛋白。的 发现的复合物类型和直接参与的蛋白质的身份可以 提供线索的作用，该协会的压力信号和西格玛B 诱导最后，将对rsbT进行详细的突变分析， σ B胁迫诱导途径中最上游的正调节因子 而其产物最有可能直接受 压力信号预计rsbT及其相关基因的变化 由此产生的表型也将为RsbT的拟议活性提供测试 作为鉴定应激定向信号改变RsbT活性的位点。这项工作 将不仅探索细胞如何 识别并对恶劣环境做出反应，但是，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剂， 单核细胞增生李斯特菌和结核分枝杆菌，它可能具有实用性 应用，确定这些病原体的压力适应弱点 对宿主防御的反应。

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.