Molecular mechanisms of RNA Polymerase I Transcription Elongation

这项工作旨在填补我们对三种特殊RNA聚合酶之一RNA Pol I如何将核糖体DNA转录成核糖体RNA的理解中的基本空白。这一功能与细胞生长和增殖直接相关。这项研究将对RNA Pol I如何扩展RNA链进行详细的物理描述，这些发现将有助于更广泛的科学界理解为什么高等生物进化出三种RNA聚合酶，Pol I， II和III，而低等生物只需要一种。该项目还将询问亚基在这种多亚基RNA聚合酶中的作用。此外，学生将有机会获得酶学博士水平的专业知识，这是在公共和私营部门寻求。本科生还将获得研究培训，并在STEM领域的未来职业生涯中变得更有见识和竞争力。最后，制定了一项具体计划，以支持在STEM领域招聘和留住女性。由14个亚基RNA Pol I催化的转录延伸是核糖体生物发生所必需的。Pol I合成5.8 S， 18s和25s核糖体rna。尽管它对细胞存活至关重要，但人们对Pol I催化转录延伸的机制知之甚少。这项研究将通过产生Pol i催化的单核苷酸和多核苷酸结合的详细动力学机制来推进转录知识。瞬态动力学技术，特别是化学猝灭流和新的分析方法将用于确定反应中的速率决定步骤以及这些步骤如何被调节。对DNA聚合酶、细菌RNA Pol和真核生物RNA Pol II进行了大量研究；然而，对真核生物Pol I和III知之甚少。该项目的结果将能够比较三种真核RNA聚合酶之间的基本机制差异，并有助于深入了解导致它们专业化的进化压力。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 work seeks to fill a fundamental gap in our understanding of how one of the three specialized RNA polymerases, RNA Pol I, transcribes ribosomal DNA into ribosomal RNA. This function is directly linked to cell growth and proliferation. The research will produce a detailed physical description of how RNA Pol I extends the RNA chain, and the findings will aid the broader scientific community in understanding why higher organisms have evolved three RNA polymerases, Pol I, II, and III, when lower organisms only require one. The project will also interrogate the role of subunits within this multi-subunit RNA polymerase. Additionally, students will have opportunities to gain Ph.D. level expertise in enzymology, which is sought in both the public and private sector. Undergraduate students will also gain research training and become more informed and competitive for future careers in STEM fields. Finally, a specific plan is in place to support recruitment and retention of women in STEM fields.Transcription elongation catalyzed by the fourteen subunit RNA Pol I is essential for ribosome biogenesis. Pol I synthesizes the 5.8 S, 18 S, and 25 S ribosomal RNAs. Despite its essential requirement for cell viability, little is known regarding the mechanism of Pol I catalyzed transcription elongation. This research will advance knowledge of transcription by producing a detailed kinetic mechanism for single and multi-nucleotide incorporation catalyzed by Pol I. Transient state kinetic techniques, specifically chemical quenched-flow and novel analysis methods will be employed to determine rate determining steps in the reaction and how these might be regulated. Substantial research has been done on DNA polymerases, bacterial RNA Pol, and eukaryotic RNA Pol II; however, much less is known about eukaryotic Pol I and III. The results of this project will enable comparison of the fundamental mechanistic differences between the three eukaryotic RNA polymerases, and help provide insights into the evolutionary pressures that led to their specialization.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.