Dynamics of co-transcriptional riboswitch folding through single-molecule microscopy

项目摘要和摘要： 被称为核糖开关的RNA元件对于许多 原核生物。核糖开关通常在mRNA的5‘非翻译区的上游发现，并折叠 共转录，从结构上阻碍信使核糖核酸转录和/或翻译。在特定的情况下 RNA结合配体、核糖开关在二级结构中发生变化：1)不允许翻译 启动，或2)导致改变的转录终止。核糖开关在真核生物中很少发现，因此 提出了一种新的漏洞，可以作为抗菌治疗的靶点。它已经被清楚地证明了 在文献中，新生的RNA结构影响转录聚合酶的转录活性 大量化验，但几乎没有关于转录聚合酶本身如何影响新生RNA的工作 结构作为回报。此外，RNA结构已被证明影响活性部位的构象 聚合酶，但从来没有在核糖开关的背景下。在这项研究中，我们将全面审查 利用切割技术，核糖开关影响RNAP转录的机制，反之亦然 尖端单分子技术。 除了这项申请中提出的新科学之外，这项奖学金将进一步加强我的培训， 让我成为一名独立的科学家。我将参加研讨会、工作坊、辅导和授课 机会，以及职业发展和网络机会提供给我的不同 密歇根大学提供的课程集合(包括由以下机构组织的课程： 文学、科学和艺术，生命科学研究所，化学系， 核糖核酸生物医学，密歇根生命科学奖学金，研究生和博士后办公室 事务和密歇根大学博士后协会)。此外，尼尔斯·沃尔特博士已经 展示了出色的指导技能，他的实验室由一大群不同的博士后组成 我可以找到支持和指导的研究员和研究生。我们的实验室还包括一个大型的 许多有才华的本科生将为我提供许多合作的机会 更年轻的学员。总体而言，这项提案中计划的研究，以及由此给予我的支持 计划，将丰富我和核糖开关RNA生物学领域。

Project Abstract and Summary: RNA elements known as riboswitches are critical for proper genome expression and maintenance in many prokaryotes. Riboswitches are generally found upstream, in the 5’ untranslated region of an mRNA, and fold co-transcriptionally to structurally impede mRNA transcription and/or translation. In the presence of specific RNA binding ligands, riboswitches undergo alterations in secondary structure that either: 1) disallow translation initiation, or 2) result in altered transcription termination. Riboswitches are rarely found in eukaryotes, and thus present a novel vulnerability that can be targeted for anti-bacterial therapies. It has been clearly demonstrated in the literature that nascent RNA structure influences transcriptional activity of the transcribing polymerase in bulk assays, but little work has been done on how the transcribing polymerase itself influences nascent RNA structure in return. Further, RNA structure has been shown to impact active site conformations of the polymerase, but never in the context of the riboswitch. In this study we will comprehensively examine the mechanisms by which riboswitches influence the transcribing RNAP, and vice versa, using cutting edge single-molecule techniques. In addition to the novel science proposed in this application, this fellowship will further enhance my training, and strengthen me as an independent scientist. I will participate in seminars, workshops, mentoring and teach opportunities, as well as professional development and networking opportunities afforded to me by the diverse collection of programs offered at the University of Michigan (including programs organized by: the College of Literature, Sciences, and the Arts, the Life Sciences Institute, the Department of Chemistry, the Center for RNA Biomedicine, the Michigan Life Sciences Fellowship, the Office of Graduate Student and Postdoctoral Affairs, and the University of Michigan Postdoctoral Association). Further, Dr. Nils Walter has already demonstrated excellent mentorship skills, and his lab is comprised of a large, diverse group of postdoctoral fellows and graduate students with whom I can find support and guidance. Our lab also includes a large number of talented undergraduates which will provide me with many opportunities for collaborating with younger mentees. Overall, the research planned in this proposal, and the support granted to me by this program, will enrich both me and the field of riboswitch RNA biology.