REGULATION OF GENE EXPRESSION BY OXYGEN

氧气对基因表达的调节

• 批准号: 2183452
• 负责人: PATRICIA J KILEY
• 金额: $ 14.23万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2183452
• 关键词: DNA binding protein; DNA footprinting; Escherichia coli; biological signal transduction; cell growth regulation; cellular respiration; conformation; gene expression; genetic mapping; hypoxia; microorganism metabolism; mutant; oxygen tension; transcription factor

For many organisms, the ability to sense and adapt to changes in oxygen tension in the environment is crucial to their survival. For example, excess oxygen creates reactive oxygen species that can damage most macromolecules, whereas oxygen deprivation can result in energy starvation and consequently inhibit cell growth. Although oxygen plays a pivotal role in many biological systems, the molecular mechanism by which oxygen is sensed by cells is poorly understood. Our long range goal is to identify how cells sense and respond to changes in environmental oxygen. The Escherichia coli transcription factor Fnr provides an ideal system in which to investigate this question since it globally regulates gene expression in response to oxygen deprivation. Under anaerobic conditions, Fnr stimulates transcription of anaerobic respiratory enzymes, and it also represses synthesis of at least two aerobic respiratory complexes. Fnr levels are not oxygen regulated, but Fnr activity is regulated by oxygen deprivation. The central questions in this field are the identities of the physiological signal which results from oxygen deprivation and the effector molecule which converts Fnr from its inactive form in aerobic cells to an active transcription under anaerobic conditions. Therefore, the ultimate goal of our experiments is to biochemically define how Fnr activity is regulated by oxygen deprivation. The eventual realization of this goal will require identifying the effector for Fnr, developing an in vitro system to monitor Fnr-specific DNA interactions, and determining if the conformation of Fnr is altered by effector binding. I have selected Fnr* mutants that activate transcription of an Fnr target operon in the presence of oxygen. These Fnr* mutants provide a unique opportunity to develop an in vitro system to study Fnr-DNA interactions because they bypass the need for the unknown effector which has made previous analysis of the wild type protein difficult. I will confirm that the phenotype of these Fnr* mutants is correlated with their unique ability to bind to target sequences in the presence of oxygen in vivo. I will purify Fnr* proteins to determine if they contain any bound prosthetic groups and identify the effector. In addition, I will develop an in vitro system to determine if effector binding alters wild type Fnr conformation. A genetic analysis will begin tracing steps in the proposed signal transduction pathway from the physiological signal induced by oxygen deprivation to effector binding and Fnr activation. Finally, analysis of Fnr mutants will allow us to determine regions of this protein that allow it to respond to oxygen deprivation. This interdisciplinary approach will allow me to demonstrate how oxygen deprivation regulates Fnr activity.

对于许多生物来说，感知和适应氧气变化的能力 环境中的紧张局势对它们的生存至关重要。 比如说， 过量的氧气会产生活性氧，这会损害大多数人的健康 大分子，而缺氧可导致能量饥饿 从而抑制细胞生长。 尽管氧气在 在许多生物系统中，氧被吸收的分子机制 对细胞的感知知之甚少。 我们的长期目标是 细胞是如何感知和响应环境氧气的变化的 的 大肠杆菌转录因子Fnr提供了一个理想的系统， 来研究这个问题，因为它在全球范围内调节基因表达， 对缺氧的反应。 在厌氧条件下，Fnr 刺激厌氧呼吸酶的转录， 抑制至少两种需氧呼吸复合物的合成。 FNR 水平不受氧调节，但Fnr活性受氧调节 剥夺 这一领域的中心问题是 生理信号是由缺氧和效应器引起的 一种将Fnr从有氧细胞中的非活性形式转化为 在厌氧条件下的活性转录。 因此，最终 我们实验的目的是从生物化学上确定Fnr活性是如何影响 由缺氧调节。 这一目标的最终实现 将需要确定Fnr的效应子，开发体外 系统来监测Fnr特异性DNA相互作用，并确定 Fnr的构象被效应物结合改变。 我选择了FNR* 突变体，其在存在下激活Fnr靶操纵子的转录， 氧气。 这些Fnr* 突变体提供了一个独特的机会， 研究Fnr-DNA相互作用的体外系统，因为它们绕过了 对于未知效应子，其先前已对野生型进行了分析， 蛋白质难。 我将证实这些Fnr* 突变体的表型 与它们独特的结合靶序列的能力相关， 体内存在氧气。 我将纯化Fnr* 蛋白，以确定 它们含有任何结合的辅基并识别效应物。 在 此外，我将开发一个体外系统，以确定是否效应 结合改变野生型Fnr构象。 基因分析将开始 跟踪步骤，在拟议的信号转导途径，从 由氧剥夺诱导的生理信号与效应物结合， FNR激活。 最后，对Fnr突变体的分析将使我们能够 确定这种蛋白质的区域，使其能够对氧气做出反应， 剥夺 这种跨学科的方法将使我能够证明 缺氧如何调节Fnr活性