Unraveling the role of iron-sulfur clusters in RNA polymerase

揭示铁硫簇在 RNA 聚合酶中的作用

• 批准号: 1121292
• 负责人: Daniel Lessner
• 金额: $ 61.38万
• 依托单位: University of Arkansas
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1121292&HistoricalAwards=false
• 关键词: Unraveling; role; iron; sulfur; clusters

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.

智力价值：这个项目的目标是阐明最近在弓形和真核细胞核糖核酸聚合酶亚单位中发现的铁-硫簇(Fe-S)的功能作用。RNAP是一种多亚单位酶，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的核心亚单位在生命的所有三个领域(细菌、古生菌和真核生物)都是保守的。在结构和复杂性上，古生菌的RNAPs类似于真核细菌的RNAPs，而不是细菌的不那么复杂的RNAPs，尽管古生菌和细菌都是没有核的单细胞有机体。此外，Fe-S簇只在古生物和真核生物的RNAP中存在，而在所有细菌的RNAP中都不存在。具体地说，在一些古生物RNAP的RPOD亚基和一些真核生物的RNAP的同源亚基中发现了Fe-S簇。RPOD不直接参与RNA的合成，但促进RNAP的组装并调节其活性。这些发现提出了将在该项目中解决的耐人寻味的问题：RNAP中的Fe-S星系团的目的是什么？为什么只有某些物种含有它们？铁-S簇如何调节RNAP的组装或活性以响应环境信号？这些问题将以产甲烷的古甲烷嗜乙酸杆菌(MA)作为模式生物来解决。预计MaRpoD将结合两个Fe-S团簇。因为食醋分枝杆菌只能在没有氧气的情况下生长，所以这些簇很可能用于调节RNAP的组装或活性，以响应氧气或氧化应激。该项目的具体目标包括：1)确定MARPOD中铁-S团簇的数量和类型以及它们的氧化还原性质，包括对氧的敏感性。2)建立能够表达突变rpoD等位基因的食醋分枝杆菌菌株，以检测不能与Fe-S簇结合的MaRpoD缺陷对MaRNAP活性和Ma菌株活力的影响。这些研究有望揭示Fe-S簇是否对MaRNAP的形成和活性起关键作用，从而促进对RNAP在其他古生物或真核生物中功能和进化的研究。更广泛的影响：该项目将有助于在严格意义上的厌氧微生物和古生物的生物化学、生理学和遗传学方面对学生进行教育和培训，这是一个在所有教育级别都未得到充分代表的领域。该项目将支持两名研究生，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大学一起从历史上的黑人学院和大学、为西班牙裔服务的机构和部落学院招收？乔治·华盛顿·卡弗的研究项目。每年夏天，该项目将为其中一名学生提供支持，让他们在国际和平研究所的实验室进行研究。PI将继续为阿肯色州高中生提供暑期研究机会，担任阿肯色州大学数学和科学向上跳跃暑期计划的导师。学生将有机会在阿肯色州西北部地区科学与工程博览会上展示他们的发现。将加强为本科生开设的一门高级原核生物实验室课程，整合研究项目中的新发现、新技术和新概念。