Translational Enhancement by tRNA Mimics

tRNA 模拟物的翻译增强

• 批准号: 1411836
• 负责人: Anne Simon
• 金额: $ 74.88万
• 依托单位: University of Maryland, College Park
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1411836&HistoricalAwards=false
• 关键词: Translational; Enhancement; tRNA; Mimics

This project addresses the question of how viruses, which depend on the infected host cells for making virus proteins, use the host cell protein-producing machinery, the ribosome, in a manner that is highly efficient. The research will exploit and extend the novel discovery that the genetic material of a virus contains specific structures that can bind to the ribosomes and that thereby allow them to be quickly and efficiently reused. Key outcomes of this project will be broadly applicable in many fields, including in biotechnology to enhance the efficiency of protein production. The outcomes may also lead to novel approaches to control a variety of viruses that infect plants and animals. This project will provide educational and research training opportunities for high school students, college undergraduates, graduate students and postdoctoral researchers. The outcomes of the research will be presented at scientific conferences, taught in introductory courses to biology students, and used to introduce undergraduates and high school students to the life cycle of viruses. The principal investigator will work with Advanced Placement students at DuVal High School (98% under-represented minority students) to prepare them for college, serve on the board of the National Academy of Sciences partnership with the entertainment industry and will also serve as a scientific consultant for programming by the Discovery and National Geographic Channels.The plant virus "Pea enation mosaic umbravirus" has a bipartite positive-strand RNA genome containing two 3' proximal T-shaped structures (kl-TSS and 3'TSS). These structures are critical for translation of the uncapped viral RNAs into proteins. 3'TSS is an example of the long-known, better-studied tRNA-like structures found in viral RNAs, but the existence of the kl-TSS was unsuspected and came to light during previous research. The newly found, internal kl-TSS, with its three-way branched secondary structure, binds to 40S, 60S and 80S ribosomes and can simultaneously engage in a long-distance interaction with 5H2, a coding region hairpin located near the 5'Untranslated Region (UTR). The 3'TSS is also likely to connect with 5H2, and the whole system will be exploited to learn how different tRNA-mimics interact with ribosomes to enhance translation. Specifically, a novel "pick-up and delivery" hypothesis will be evaluated, i.e., the idea that multiple TSS may be connected via RNA:RNA interactions with the ends of virus-encoded Open Reading Frames (ORF), to gather ribosomal subunits downstream of an ORF at the end of translation and relay them back to the 5'UTR for another round of translation. In addition, it will be tested whether the kl-TSS can enhance translation of capped mRNAs produced from expression constructs. These experiments will inform our understanding of how this novel and possibly widespread use of tRNA mimicry operates to enhance translation and to control plant virus life cycles. The project will also lead to the development of expression cassettes with enhanced translation of encoded proteins with potentially extensive agricultural as well as laboratory applications.

该项目解决了依赖于受感染的宿主细胞来制造病毒蛋白的病毒如何以高效的方式使用宿主细胞蛋白质生产机器-核糖体的问题。这项研究将利用和扩展新的发现，即病毒的遗传物质含有可以与核糖体结合的特定结构，从而使它们能够快速有效地重复使用。该项目的主要成果将广泛适用 在许多领域，包括生物技术，以提高蛋白质生产的效率。这些结果还可能带来控制感染植物和动物的各种病毒的新方法。该项目将为高中生、大学本科生、研究生和博士后研究人员提供教育和研究培训机会。研究成果将在科学会议上发表，在生物学学生的入门课程中教授，并用于向本科生和高中生介绍病毒的生命周期。首席研究员将与杜瓦尔高中的大学先修课程学生一起工作（98%的少数民族学生）为他们上大学做准备，担任美国国家科学院与娱乐业合作伙伴关系的董事会成员，并将担任探索频道和国家地理频道节目的科学顾问。植物病毒“豌豆enation mosaic umbravirus”具有双向阳性-链RNA基因组，含有两个3'近端T形结构（kl-TSS和3' TSS）。这些结构对于将未加帽的病毒RNA翻译成蛋白质至关重要。3 'TSS是在病毒RNA中发现的长期已知的、更好研究的tRNA样结构的一个例子，但是kl-TSS的存在在以前的研究中被怀疑并曝光。新发现的内部kl-TSS具有三向分支二级结构，可与40 S、60 S和80 S核糖体结合，并可同时与位于5 '非翻译区（UTR）附近的编码区发夹5 H2进行长距离相互作用。3 'TSS也可能与5 H2连接，整个系统将被用来了解不同的tRNA模拟物如何与核糖体相互作用以增强翻译。具体而言，将评估一种新的“拾取和递送”假设，即，多个TSS可以通过RNA：RNA相互作用与病毒编码的开放阅读框架（ORF）的末端连接，以在翻译结束时收集ORF下游的核糖体亚基并将它们传递回5 'UTR进行另一轮翻译。此外，将测试kl-TSS是否可以增强由表达构建体产生的加帽mRNA的翻译。这些实验将使我们了解这种新的、可能广泛使用的tRNA模拟是如何增强翻译和控制植物病毒生命周期的。该项目还将导致开发表达盒，增强编码蛋白质的翻译，具有潜在的广泛农业和实验室应用。