Unraveling the role of iron-sulfur clusters in RNA polymerase

智力价值：这个项目的目的是阐明铁硫（Fe-S）簇的功能作用，最近发现的亚基的古真核RNA聚合酶（RNAP）。RNAP是一种多亚基酶，它复制DNA以产生RNA，这是一个称为转录的基本过程，发生在所有活细胞中，是基因表达的第一步。RNAP的核心亚基在生命的所有三个领域（细菌、酵母和真核生物）中都是保守的。在结构和复杂性上，古细菌的RNAP类似于真核生物的RNAP，而不是不太复杂的细菌的RNAP，尽管古细菌和细菌都是缺乏细胞核的单细胞生物。此外，Fe-S簇只在古细菌和真核生物的RNAP中发现，而在所有细菌的RNAP中都不存在。具体而言，Fe-S簇被发现在一些古细菌的RNAP的RpoD亚基和一些真核RNAP的同源亚基中。RpoD不直接参与RNA合成，但促进RNAP的组装并调节其活性。这些发现提出了有趣的问题，将在该项目中解决：什么是RNAP中的Fe-S簇的目的？为什么只有某些物种含有它们？Fe-S团簇如何调节RNAP的组装或活性以响应环境信号？这些问题将得到解决使用甲烷产生古菌Methanosarcina acetivorans（马）作为模式生物。预测MaRpoD结合两个Fe-S簇。因为M.尽管食乙酸菌仅在没有氧气的情况下生长，但簇可能用于调节响应氧气或氧化应激的RNAP的组装或活性。该项目的具体目标包括：1）确定MaRpoD中Fe-S簇的数量和类型及其氧化还原特性，包括对氧气的敏感性。2)发展M.用能够表达突变体rpoD等位基因的食乙酸菌来测试不能结合Fe-S簇的缺陷型MaRpoD的存在对MaRNAP活性和Ma菌株生存力的影响。这些研究有望揭示Fe-S簇合物是否对MaRNAP的形成和活性至关重要，从而促进其他哺乳动物或真核生物RNAP功能和进化的研究。 更广泛的影响：该项目将有助于对学生进行严格厌氧微生物和厌氧微生物的生物化学、生理学和遗传学方面的教育和培训，这是一个在各级教育中代表性不足的领域。 该项目将资助两名研究生，并为本科生提供研究机会。 来自不同背景的本科生将与阿肯色州大学一起从历史悠久的黑人学院和大学、西班牙裔服务机构和部落学院招募？乔治华盛顿卡佛研究计划。 每年夏天，该项目将为其中一名学生提供支持，在PI的实验室进行研究。PI将继续为阿肯色州高中学生提供暑期研究机会，担任阿肯色州大学数学和科学向上拓展暑期项目的导师。学生将有机会在西北阿肯色州区域科学和工程博览会上展示他们的发现。一个先进的生物学实验室课程的本科生将得到加强，从研究项目整合新的发现，技术和概念。

Intellectual merit:The objective of this project is to elucidate the functional role of the iron-sulfur (Fe-S) clusters, recently discovered in subunits of archeal and eukaryal RNA polymerase (RNAP). RNAP is a multi-subunit enzyme that copies DNA to produce RNA, in a fundamental process called transcription that occurs in all living cells and is the first step in gene expression. The core subunits of RNAP are conserved in all three domains of life (Bacteria, Archaea, and Eukarya). In structure and complexity, archaeal RNAPs resemble those of eukarya, rather than the less complex RNAPs of bacteria, even though both archaea and bacteria are single-celled organisms lacking nuclei. Furthermore, Fe-S clusters are only found in the RNAPs of archaeal and eukaryal species and are absent from all bacterial RNAPs. Specifically, Fe-S clusters are found in the RpoD subunits of the RNAPs of some archaea and in the homologous subunits of some eukaryal RNAPs. RpoD is not directly involved in RNA synthesis but serves to promote the assembly of RNAP and regulate its activity. These findings raise intriguing questions that will be addressed in the project: What is the purpose of the Fe-S clusters in RNAP? Why do only certain species contain them? How do the Fe-S clusters function to regulate the assembly or activity of RNAP in response to environmental signals? These questions will be addressed using the methane-producing archaeon Methanosarcina acetivorans (Ma) as a model organism. MaRpoD is predicted to bind two Fe-S clusters. Because M. acetivorans only grows in the absence of oxygen, it is likely the clusters serve to regulate the assembly or activity of RNAP in response to oxygen or oxidative stress. The specific aims of the project include: 1) determine the number and type of Fe-S clusters in MaRpoD and their redox-properties, including the sensitivity to oxygen. 2) Develop strains of M. acetivorans capable of expressing mutant rpoD alleles to test the affects on MaRNAP activity and Ma strain viability of the presence of defective MaRpoD that are unable to bind Fe-S clusters. These studies are expected to reveal whether the Fe-S clusters are critical for the formation and activity of MaRNAP, and thereby to stimulate studies of the function and evolution of RNAPs in other Archaeal or Eukaryal species. Broader impacts: The project will contribute to the education and training of students in the biochemistry, physiology, and genetics of strictly anaerobic microorganisms and Archaea, a field that is underrepresented at all education levels. The project will support two graduate students and provide research opportunities for undergraduates. Undergraduates from diverse backgrounds will be recruited from Historically Black Colleges and Universities, Hispanic-serving institutions, and Tribal Colleges in conjunction with the University of Arkansas? George Washington Carver research program. Each summer the project will provide support for one of these students to conduct research in the laboratory of the PI. The PI will continue to provide summer research opportunities for Arkansas High School students by serving as a mentor with the Upward Bound Summer Program in Math and Science at the University of Arkansas. Students will be provided the opportunity to present their findings at the Northwest Arkansas Regional Science and Engineering fair. An advanced Prokaryotic Biology laboratory course for undergraduates will be enhanced, integrating new findings, techniques, and concepts from the research project.