Mechanism for the selection of undamaged physiological substrates by the ATP-dependent protease Lon

该项目将确定降解各种蛋白质的细胞蛋白酶Lon如何调节核糖核酸（RNA）遗传物质的转录。由于长链介导的蛋白质降解和RNA转录在细菌和人类中是保守的，从这项工作中产生的发现将提供对维持生命重要的普遍过程的工作原理的见解。PI假设Lon和生理结合伙伴之间对同一细胞蛋白的竞争性相互作用决定了该细胞蛋白的命运，并最终决定了其功能。该项目将通过确定纯化的大肠杆菌Lon蛋白酶（ELon）如何影响构成大肠杆菌RNA聚合酶（ERNAP）、lambda N蛋白、NusA蛋白和含有nutL序列的核酸的RNA转录复合体的组装来验证这一假设。此外，拟议的研究将确定RNA转录过程的组装在多大程度上保护lambda N蛋白免受ELon降解。这个项目将把研究与教育结合起来。这个项目更广泛的影响在于向俄亥俄州克利夫兰的贫困儿童推广科学素养。参与该项目的受训者将学习向公众传授和传播科学概念，为他们从事STEM教育事业做好准备。培养本科生和博士后从事有机合成、分子克隆、蛋白质纯化和标记、电子显微镜和酶学等方面的研究。学员将学习向公众传授和传播科学概念。这种经历将为未来的STEM教育工作者提供培训基础。该项目将确定大肠杆菌Lon蛋白酶（Elon）是否参与RNA转录的调控。该项目将测试Lon通过选择性地降解抗终止RNA转录所需的lambda N蛋白来破坏RNA转录的假设。将使用酶动力学、诱变和电子显微镜成像方法的组合来评估大肠杆菌Lon蛋白酶（ELon）对构成大肠杆菌RNA聚合酶（ERNAP）、lambda N蛋白、NusA蛋白和含有nutL序列的核酸的RNA转录复合体的动力学组装的影响。RNA转录对长链介导的蛋白水解lambda N蛋白的影响也将被评估。由于Lon存在于大多数生物体中，因此有关Lon的生理学相关机制发现可以转化为了解Lon同系物在真核生物中的作用。为阐明Lon的机制而开发的定量方法将有利于采用相同的方法来研究其他atp依赖性蛋白酶，以揭示该酶家族之间的机制差异。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

This project will determine how the cellular protease Lon, which degrades various proteins, regulates the transcription of a genetic material known as ribonucleic acid (RNA). Since Lon-mediated protein degradation and RNA transcription are conserved in bacteria and humans, findings generated from this work will provide insights into the workings of a universal process that is important for sustaining life. The PI hypothesizes that the competing interaction between Lon and the physiological binding partner(s) for the same cellular protein dictates this cellular protein's fate and, ultimately, function. The project will test this hypothesis by determining how purified Escherichia coli Lon protease (ELon) affects the assembly of the RNA transcription complex constituting the Escherichia coli RNA polymerase (ERNAP), the lambda N protein, the NusA protein, and the nucleic acid containing the nutL sequence. In addition, the proposed research will determine the extent to which the assembly of the RNA transcription process protects the lambda N protein from degradation by ELon. This project will integrate research into education. The broader impact of this project lies in promoting science literacy to underprivileged children in Cleveland, Ohio. Trainees participating in this project will learn to teach and communicate scientific concepts to the general public, which will prepare them for an education career in STEM. Undergraduate students and post-doctoral fellows will be trained to conduct research in organic synthesis, molecular cloning, protein purification and labeling, electron microscopy, and enzymology. The trainees will learn to teach and communicate scientific concepts to the general public. This kind of experience will provide a training ground for future educators in STEM.This project will determine if the Escherichia coli Lon protease (Elon) participates in the regulation of RNA transcription. The project will test the hypothesis that Lon disrupts RNA transcription by selectively degrading the lambda N protein, which is needed for antitermination RNA transcription. A combination of enzyme kinetics, mutagenesis, and electron microscopy imaging approaches will be used to evaluate the impact of Escherichia coli Lon protease (ELon) on the kinetic assembly of the RNA transcription complex constituting the Escherichia coli RNA polymerase (ERNAP), the lambda N protein, the NusA protein, and the nucleic acid containing the nutL sequence. The effect of RNA transcription on Lon-mediated proteolysis of the lambda N protein will also be evaluated. Since Lon is found in most organisms, physiologically relevant mechanistic findings on ELon could be transformed to understand the workings Lon homologs in eukaryotes. The quantitative approach developed for elucidating the mechanism of Lon would benefit the adaptation of the same approach to study other ATP-dependent proteases to reveal mechanistic differences among this family of enzymes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.