Dissecting the regulatory role of a eukaryotic transcription factor in RNA-templated transcription catalyzed by DNA-directed RNA polymerase II

项目摘要 转录是介导遗传信息和基因组之间相互作用的基本过程。 表型，因此对发育，对环境线索的反应和疾病至关重要。转录是 主要由DNA指导的RNA聚合酶（DdRPs）催化，DdRPs在 真核生物已经确定，来自多个层的调节控制DdRP- 在DNA模板上催化转录。有趣的是，一些DdRP可以识别DNA和RNA， 用于转录的模板。这种RNA模板活性调节细菌中的基因表达， 哺乳动物细胞并被病原体利用（即，类病毒和人丁型肝炎病毒）用于繁殖。 然而，人们对DdRP以RNA为模板转录的机制和机制知之甚少。 利用马铃薯纺锤块茎类病毒（PSTVd）的复制作为模型，初步数据表明， Pol II的第二大亚基中的保守甘氨酸对于DNA指导的而不是RNA模板化的 转录，表明该残基对DNA和RNA模板的不同要求。此外，本发明还提供了一种方法， 数据显示，TFIIS是DNA定向转录所必需的通用转录因子， 对于PSTVd RNA模板转录，这表明需要一组专门的因子， DNA与RNA模板的转录。此外，真核转录因子TFIIIA-7ZF， 结合RNA但不结合DNA，已被证明是促进Pol II催化转录的关键因子 在PSTVd RNA模板上。TFIIIA-7ZF结合位点定位于转录起始点附近的区域 位点，并且对于Pol II结合和PSTVd复制是关键的，表明该结合位点作为RNA结合位点起作用。 启动子总之，这些发现激发了一个核心假设，即Pol II招募了一个专门的群体 选择性地识别RNA作为模板并催化转录。用一种新的体外 转录（IVT）测定，该测定基于Pol II催化的PSTVd转录，本项目的目标是 进一步剖析了专门的Pol II机器转录RNA模板的调节机制。到 为了实现这一目标，提出了三个目标，以进一步剖析TFIIIA-7ZF在RNA模板化中的作用。 转录，绘制TFIIIA-7ZF/Pol II结合结构域，并表征RNA的分子基础 启动子预期的结果将提供新的见解的监管机制的基础RNA- Pol II催化的模板化转录，并为更好地描述RNA启动子提供了有价值的知识。 由于该项目旨在揭示RNA聚合酶工作方式的基本原理， 在真核生物中高度保守，这里获得的结果将提供深入的知识 RNA模板转录。这种深入的知识将导致对因素的充分理解， RNA模板转录的调控机制并促进有效转录的开发 治疗由传染性RNA引起的疾病。

Project Summary Transcription is a fundamental process that mediates the interplay between genetic information and phenotypes, thus vital for development, responses to environmental cues, and diseases. Transcription is primarily catalyzed by DNA-directed RNA polymerases (DdRPs), which are highly conserved among eukaryotic organisms. It has been well established that regulation from multiple layers controls DdRP- catalyzed transcription on DNA templates. Interestingly, some DdRPs recognize both DNA and RNA templates for transcription. This RNA-templated activity regulates gene expression in bacteria and mammalian cells and is employed by pathogens (i.e., viroids and human hepatitis delta virus) for propagation. However, the machinery and mechanism for RNA-templated transcription by DdRPs are poorly understood. Using the replication of potato spindle tuber viroid (PSTVd) as a model, preliminary data showed that a highly conserved glycine in the second largest subunit of Pol II is critical for DNA-directed but not RNA-templated transcription, indicating the different requirements of this residue for DNA and RNA templates. In addition, data showed that TFIIS, a general transcription factor essential for DNA-directed transcription, is dispensable for PSTVd RNA-templated transcription, suggesting that a specialized group of factors are required for transcription of DNA versus RNA templates. Furthermore, the eukaryotic transcription factor TFIIIA-7ZF, which binds RNA but not DNA, has been shown as a critical factor in facilitating Pol II-catalyzed transcription on PSTVd RNA templates. The TFIIIA-7ZF binding site maps to a region next to the transcription initiation site and is critical for Pol II binding and PSTVd replication, suggesting that this binding site acts as an RNA promoter. Together, these findings inspire the central hypothesis that Pol II recruits a specialized group of factors to selectively recognize RNA as templates and catalyze transcription. Using a novel in vitro transcription (IVT) assay that is based on Pol II-catalyzed transcription of PSTVd, the goal of this project is to further dissect the regulatory mechanism for specialized Pol II machinery to transcribe RNA templates. To achieve this goal, three aims are proposed to further dissect the role of TFIIIA-7ZF in RNA-templated transcription, map the TFIIIA-7ZF/Pol II binding domains, and characterize the molecular basis of an RNA promoter. The expected outcomes will provide novel insights into the regulatory mechanism underlying RNA- templated transcription catalyzed by Pol II and provide valuable knowledge to better delineate RNA promoters. As this project aims to uncover the basic principles governing the modus operandi of RNA polymerases that are highly conserved across eukaryotic organisms, the results gained here will provide an in-depth knowledge of RNA-templated transcription. Such in-depth knowledge will lead to the full understanding of factors and the regulatory mechanism underlying RNA-templated transcription and facilitate the development of effective treatments for diseases caused by infectious RNAs.