REGULATION OF GENE EXPRESSION BY OXYGEN

这个项目的长期目标是了解生物化学和 允许细胞感知和适应氧气变化的分子事件 在他们的环境中。因为氧气对许多生物的生存是必不可少的 对于生物体来说，这个问题具有广泛的生物学意义。致信地址 这个问题，我们正在研究大肠杆菌转录因子 FNR是一种全局调节基因表达以响应氧气的基因 剥夺。这项提案中的实验将回答两个核心问题 关于FNR如何调控其靶基因转录的问题 在厌氧条件下。 观察到FNR活性受氧气供应的调节 为我们提供了剖析细胞感知机制的机会 氧气。我们的数据表明，这种蛋白质含有一个[4Fe-4S]簇 它看起来像是氧气传感器。拟议中的实验将 证明氧气是否直接控制FNR的活性 导致这个铁-S团簇的氧化降解。要确定如何 铁-S团簇影响FNR活性， 二聚化和DNA结合将与Fe-S团簇的速率进行比较 FNR暴露在氧气中后的损失。来检验这样一种想法 这种铁-S团簇对氧的不稳定性与生理相关， 将比较FNR在体外和体内的失活率 WT FNR和一系列活性增强的FNR*突变蛋白 在氧气存在的情况下。如果确定了FnR*突变替换 改变了Fe-S团簇的稳定性，我们可以得出结论 铁-S团簇的氧依赖失活调节活性 FNR在体内的表达。以确定氧或氧自由基 超氧阴离子更有效地钝化FNR，降低 将检测体内和体外超氧化物歧化水平对FNR功能的影响。 确定FNR[4Fe-4S]团簇是否对超氧化物敏感 具有根本的重要性，因为这样的结果将表明 氧化应激信号系统与氧信号系统之间的关系 剥夺。此外，铁-S团簇的潜在用途 FNR中的氧或超氧化物传感器增加了越来越多的 这些金属中心在生物学上可以提供的多种功能。 我们实验的另一个目标是确定FNR是如何激活的 转录在其目标启动子上，并定义这两个sigma7O 和RNA聚合酶的α亚基是FNR依赖所必需的 转录激活。明确FNR的这一职能具有重大意义 生理上的重要性，因为这种蛋白质是许多 厌氧诱导基因。此外，使用共同激活剂，如 某些FNR依赖启动子上的NarL和CAP允许大肠杆菌整合 其他环境信号，如硝酸盐和cAMP的变化 氧气的缺乏。我们的实验应该提供必要的 为阐明FNR在这些更复杂的环境中的相互作用奠定基础 推动者。

The long term goal of this project is to understand the biochemical and molecular events that allow cells to sense and adapt to changes in oxygen in their environment. Since oxygen is essential for the viability of many organisms, this problem has broad biological significance. To address this problem, we are studying the Escherichia coli transcription factor FNR that globally regulates gene expression in response to oxygen deprivation. The experiments in this proposal will answer two central questions regarding how FNR regulates transcription of its target genes under anaerobic conditions. The observation that FNR activity is regulated by oxygen availability has provided us the opportunity to dissect a cellular sensing mechanism for oxygen. Our data indicate that this protein contains a [4Fe-4S] cluster which appears to act as an oxygen sensor. The proposed experiments will demonstrate whether oxygen directly controls the activity of FNR by causing the oxidative degradation of this Fe-S cluster. To determine how the Fe-S cluster affects FNR activity, the rate of decrease in dimerization and DNA binding will be compared to the rate of Fe-S cluster loss following exposure of FNR to oxygen. To test the idea that instability of this Fe-S cluster to oxygen is physiologically relevant, the rates of FNR inactivation in vitro and in vivo will be compared for WT FNR and a series of FNR* mutant proteins which have increased activity in the presence of oxygen. If FNR* mutant substitutions are identified that alter the stability of the Fe-S cluster, we would conclude that oxygen dependent inactivation of the Fe-S cluster regulates the activity of FNR in vivo. To determine whether oxygen or the oxygen radical superoxide is more effective in inactivating FNR, the effect of lowering superoxide levels on FNR function in vivo and in vitro will be assayed. Determining whether the FNR [4Fe-4S] cluster is sensitive to superoxide is of fundamental importance because such a result would indicate a link between the signaling systems for oxidative stress and those of oxygen deprivation. Furthermore, the potential use of an Fe-S cluster as an oxygen or superoxide sensor in FNR adds to a growing list of the versatile functions that these metal centers can provide in biology. Another goal of our experiments is to determine how FNR activates transcription at its target promoters and define whether both the sigma7O and alpha-subunit of RNA polymerase are required for FNR-dependent transcription activation. Defining this function of FNR is of great physiological importance since this protein is a global regulator of many anaerobically induced genes. In addition, the use of co-activators like NarL and CAP at some FNR-dependent promoters allows E.coli to integrate additional environmental signals such as changes in nitrate and cAMP in the absence of oxygen. Our experiments should provide the necessary foundation for elucidating the interactions of FNR at these more complex promoters.