Unraveling the role of iron-sulfur clusters in RNA polymerase

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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.