REGULATION OF GENE EXPRESSION BY OXYGEN

该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。由于氧气对于许多生物的生存至关重要 有机体，这个问题具有广泛的生物学意义。致地址 这个问题，我们正在研究大肠杆菌的转录因子 FNR 全局调节基因表达以响应氧气 剥夺。本提案中的实验将回答两个核心问题 关于 FNR 如何调节其靶基因转录的问题 在厌氧条件下。 FNR 活性受氧气利用率调节的观察结果表明 为我们提供了剖析细胞传感机制的机会 氧气。我们的数据表明该蛋白质含有 [4Fe-4S] 簇 它似乎充当氧气传感器。拟议的实验将 证明氧气是否直接控制 FNR 的活性 导致该 Fe-S 簇的氧化降解。确定如何 Fe-S簇影响FNR活性， 二聚化和 DNA 结合将与 Fe-S 簇的速率进行比较 FNR 暴露于氧气后损失。为了测试这个想法 该 Fe-S 簇对氧的不稳定性与生理相关， 将比较体外和体内 FNR 失活率 WT FNR 和一系列活性增强的 FNR* 突变蛋白 在氧气存在下。如果发现 FNR* 突变体取代 改变 Fe-S 团簇的稳定性，我们可以得出结论： Fe-S 簇的氧依赖性失活调节活性 FNR 体内。确定氧或氧自由基 超氧化物更能有效地灭活FNR，降低 将测定体内和体外超氧化物水平对 FNR 功能的影响。 确定 FNR [4Fe-4S] 簇是否对超氧化物敏感 至关重要，因为这样的结果将表明存在链接 氧化应激信号系统和氧气信号系统之间的关系 剥夺。此外，Fe-S 团簇作为 FNR 中的氧气或超氧化物传感器不断增加 这些金属中心在生物学中可以提供多种功能。 我们实验的另一个目标是确定 FNR 如何激活 在其目标启动子处转录并定义 sigma7O 是否 FNR 依赖性需要 RNA 聚合酶的 α 亚基和 α 亚基 转录激活。定义FNR这个函数是很有用的 生理重要性，因为这种蛋白质是许多细胞的全局调节剂 厌氧诱导基因。此外，还可以使用辅助激活剂，例如 一些 FNR 依赖性启动子上的 NarL 和 CAP 允许大肠杆菌整合 其他环境信号，例如硝酸盐和 cAMP 的变化 没有氧气。我们的实验应该提供必要的 为阐明 FNR 在这些更复杂的相互作用中的相互作用奠定了基础 发起人。

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.