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

剖析真核转录因子在 DNA 指导的 RNA 聚合酶 II 催化的 RNA 模板转录中的调节作用

基本信息

批准号：
10047065
负责人：
Ying Wang
金额：
$ 41.09万
依托单位：
MISSISSIPPI STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2023-07-31
项目状态：
已结题

项目摘要

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.

项目摘要转录是一个基本过程，介导遗传信息和表型，因此对发展，对环境线索和疾病的反应至关重要。转录是主要由DNA定向的RNA聚合酶（DDRP）催化，在真核生物。已经有很好的确定，来自多层的调节控制DDRP- DNA模板上的催化转录。有趣的是，一些DDRP识别DNA和RNA 转录模板。这种RNA模拟活性调节细菌中的基因表达哺乳动物细胞由病原体（即病毒和人类肝炎三角洲病毒）使用。但是，DDRP的RNA-启用转录的机制和机制知之甚少。将马铃薯纺锤块茎病毒（PSTVD）的复制作为模型，初步数据表明高度 POL II的第二大亚基中保守的甘氨酸对于DNA定向但不是RNA-甘氨酸至关重要转录，表明该残基对DNA和RNA模板的不同要求。此外，数据表明，TFII是DNA指导转录必不可少的一般转录因子，是可分配的对于PSTVD RNA模拟转录，这表明需要一组专业的因素 DNA与RNA模板的转录。此外，真核转录因子tfiiia-7zf，结合RNA而不是DNA的哪个已显示为促进pol II催化转录的关键因素在PSTVD RNA模板上。 TFIIIA-7ZF结合位点映射到转录启动旁边的区域位点，对于Pol II结合和PSTVD复制至关重要，表明该结合位点充当RNA 发起人。这些发现共同启发了中心假设，即Pol II招募了一个专业团体选择性识别RNA作为模板并催化转录的因素。使用小说体外基于PSTARY催化转录的转录（IVT）测定，该项目的目的是进一步剖析了专门的POL II机械的调节机制以转录RNA模板。到实现这一目标，提出了三个目标，以进一步剖析TFIIIA-7ZF在RNA-Temped的作用转录，绘制TFIIIA-7ZF/POL II结合域，并表征RNA的分子基础发起人。预期的结果将为RNA-的调节机制提供新的见解。由POL II催化的模板转录，并为更好地描绘RNA启动子提供了宝贵的知识。由于该项目旨在揭示有关RNA聚合酶作案操作的基本原则，在真核生物中是高度保守的，这里获得的结果将提供深入的知识 RNA模拟转录。这种深入的知识将导致对因素和 RNA图转录的基础调节机制，并促进有效的发展由感染性RNA引起的疾病治疗。