REGULATION OF GENE EXPRESSION BY OXYGEN

对于许多生物体来说，感知和适应氧气变化的能力 环境的紧张对它们的生存至关重要。例如, 过量的氧气会产生活性氧物种，对人体造成最大的伤害 大分子，而缺氧会导致能量匮乏 从而抑制细胞生长。尽管氧气扮演着关键的角色 在许多生物系统中，氧气形成的分子机制 人们对细胞的感知知之甚少。我们的长期目标是找出 细胞如何感知和反应环境中氧气的变化。这个 大肠杆菌转录因子FNR提供了一种理想的系统，在其中 来研究这个问题，因为它在全球范围内调节基因表达 作为对缺氧的反应。在厌氧条件下，FNR 刺激厌氧呼吸酶的转录，它还 抑制至少两种需氧呼吸复合体的合成。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 缺氧引起的生理信号和效应器 在好氧细胞中将FNR从不活跃的形式转化为 厌氧条件下的活跃转录。因此，最终的 我们实验的目标是从生物化学的角度定义FNR的活性 受缺氧的影响。这一目标的最终实现 将需要确定FNR的效应器，开发一种体外 系统来监控FNR特异的DNA相互作用，并确定是否 FNR的构象通过效应器结合而改变。我已选择FNR* 在存在的情况下激活FNR靶操纵子转录的突变体 氧气。这些Fnr*突变体提供了一个独特的机会来开发一种 研究FNR-DNA相互作用的体外系统，因为它们绕过了需要 对于先前分析过野生型的未知效应器 蛋白质很难。我将确认这些FnR*突变体的表型 与它们与靶序列结合的独特能力有关 体内存在氧气。我将纯化FNR*蛋白以确定是否 它们包含任何结合的假体基团并识别效应器。在……里面 此外，我将开发一个体外系统来确定效应器 结合改变野生型FNR的构象。基因分析将开始 追踪所提出的信号转导途径中的步骤 缺氧诱导的生理信号对效应器结合和 FNR激活。最后，对FNR突变体的分析将使我们能够 确定这种蛋白质允许其对氧气做出反应的区域 剥夺。这种跨学科的方法将使我能够证明 缺氧是如何调节FNR活性的。

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.