Structural insights into RNA promoters for RNA polymerase II-catalyzed RNA-templated transcription

这个项目将阐明通常参与细胞DNA转录的DNA指导的RNA聚合酶如何也能够识别RNA模板。为了了解转录聚合酶如何识别RNA模板，该项目旨在确定众所周知的RNA模板(马铃薯纺锤体块茎类病毒)中调节元件的分子基础，该元件对招募转录酶至关重要。为了实现这一目标，该项目将使用分子、生化和生物信息学方法的组合。从这个项目中获得的知识可能会促进基于RNA的基础研究技术的发展，以及在生物医学和农业中的应用，从而使社会长期受益。该项目还将作为培养本科生和研究生的工具。特别是，三角洲州立大学的本科生，位于美国最富裕的地区之一，缺乏必要的研究培训设施，每年将参加暑期研究，以获得研究经验，并为STEM相关职业做准备。一个可检验的假说得到已发表的和初步的数据支持，即马铃薯纺锤块茎类病毒招募核糖核酸特异的转录因子(S)，从而提高核糖核酸聚合酶II(POL II)催化的核糖核酸模板转录的效率。RNA启动子区域由最近发现的RNA特异性转录因子TFIIIA-7ZF的结合区定义。本项目将结合引物延伸分析(SHAPE)分析的选择性2‘-羟基酰化分析、功能突变、基于Pol II的体外转录实验、RNA足迹分析等方法，对马铃薯纺锤形块茎类病毒及其近缘基因组中RNA启动子的分子基础/结构进行定位和表征。该项目的结果将有助于更好地了解RNA启动子的性质，并有可能开发RNA启动子用于基础科学及其在农业上的应用。在这个项目中，来自密西西比州两所大学的本科生将获得分子生物学基础研究的经验。这个奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

This project will shed light on how a DNA-directed RNA polymerase, normally involved in transcription of cellular DNA, is also able to recognize RNA templates. To understand how transcription polymerases can recognize RNA templates, this project aims to determine the molecular basis of a regulatory element within a well-known RNA template (potato spindle tuber viroid) that is critical for recruiting the transcription enzymes. To achieve this goal, this project will employ a combination of molecular, biochemical, and bioinformatic approaches. The knowledge gained from this project may facilitate the development of RNA-based technologies for basic research as well as applications in biomedicine and agriculture, thus benefiting society in the long term. This project will also serve as a vehicle for training undergraduate and graduate students. Particularly, undergraduate students from Delta State University, located in one of the least affluent regions in the US and lacking the necessary facility for research training, will participate in summer research annually to gain research experience and to be prepared for STEM-related careers.Supported by published and preliminary data, a testable hypothesis is that potato spindle tuber viroid recruits RNA-specific transcription factor(s), thereby enhancing the efficiency of RNA polymerase II (Pol II)-catalyzed RNA-templated transcription. The RNA promoter region is defined by the binding region of a recently identified RNA-specific transcription factor, TFIIIA-7ZF. This project will map and characterize the molecular basis/structure of RNA promoters within both sense and antisense genomes of potato spindle tuber viroid and its relatives, using a combination of selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) analysis, functional mutagenesis, Pol II-based in vitro transcription assay, RNA footprinting assays, etc. The outcomes from this project will help better understand the nature of RNA promoters and potentially exploit RNA promoters for usage in basic science and its applications in agriculture. While working on this project, undergraduate students from two universities in the state of Mississippi will receive experience in basic research in molecular biology.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.