REGULATION OF GENE EXPRESSION BY OXYGEN

氧气对基因表达的调节

• 批准号: 2900752
• 负责人: PATRICIA J KILEY
• 金额: $ 22.52万
• 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 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2900752
• 关键词: DNA directed RNA polymerase; Escherichia coli; bacterial genetics; cellular respiration; conformation; gene expression; hypoxia; iron compounds; microorganism culture; microorganism metabolism; nucleic acid sequence; oxygen; oxygen tension; protein purification; transcription factor

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.

该项目的长期目标是了解生物化学和 使细胞能够感知和适应氧气变化的分子事件 in their其environment环境.因为氧气对许多生物的生存至关重要 生物，这个问题具有广泛的生物学意义。解决 针对这个问题，我们正在研究大肠杆菌的转录因子 FNR在氧气反应中全面调节基因表达 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活性受氧可用性调节的观察结果， 为我们提供了一个机会， 氧气我们的数据表明，该蛋白质含有一个[4Fe-4S]簇 看起来像是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。以确定如何 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暴露于氧气后的损失。为了验证 这种Fe-S簇对氧不稳定性与生理学有关， 将比较体外和体内FNR失活率， WT FNR和一系列具有增加的活性的FNR* 突变蛋白 在氧气的存在下。如果鉴定出FNR* 突变替换 改变了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在体内。为了确定氧气或氧自由基 超氧化物在灭活FNR方面更有效， 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 是至关重要的，因为这样的结果将表明 氧化应激信号系统和氧气信号系统之间的联系 剥夺此外，Fe-S簇合物作为纳米晶的潜在用途也被认为是可能的。 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中可以提供的多功能。 我们实验的另一个目标是确定FNR如何激活 转录在其靶启动子，并确定是否既σ 7 O FNR依赖性需要RNA聚合酶的α亚基和α亚基 转录激活明确FNR的这一功能， 生理重要性，因为这种蛋白质是一个全球调节许多 厌氧诱导基因此外，使用共活化剂，如 在一些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，如硝酸盐和cAMP的变化， 缺乏氧气我们的实验应该提供必要的 阐明FNR在这些更复杂的相互作用的基础 发起人。