Functional Role of BYDV 3' RNA Translation Enhancer Element

知识价值：植物病毒引起的疾病影响到世界各地的大量粮食作物，可能对粮食供应和经济状况造成严重后果。大多数病毒“侵占”宿主细胞的蛋白质合成机制来制造病毒蛋白质。大麦黄矮病毒(BYDV)是世界上分布最广、经济意义最大的两种植物病毒之一，危害世界各地的小麦、大麦和燕麦作物。该项目的目标将推进植物病毒的基础科学，同时促进改进有害植物病毒控制的技术，以及利用植物生产高价值蛋白质的改良病毒工程。BYDV含有一种不寻常的RNA结构，即翻译增强子(BTE)，位于mRNA的3‘非翻译区(UTR)，与5’非翻译区茎环相互作用。这种远程相互作用通过一种尚未完全了解的机制促进病毒mRNA的有效翻译。该项目将使用BYDV的3‘非编码区作为模型系统，以阐明涉及3’非编码区元件的远程RNA相互作用的机制。该项目的总体目标是确定使病毒mRNA能够有效地与浓度高得多的细胞RNA竞争以获得核糖体和翻译因子的分子相互作用。病毒蛋白质合成起始复合体组装的三个关键步骤将被研究：1)真核起始因子与病毒3‘-BTE的结合；2)核糖体与病毒3’-BTE和5‘-UTR序列的相互作用；3)病毒3’-BTE和5‘-UTR区之间的相互作用。一个具有不同翻译效率的BYDV mRNA库将被用来确定eIF结合和翻译之间的相关性，核糖体与病毒RNA序列的结合亲和力，以及3‘和5’-UTRs之间的相互作用在每个步骤中的作用。该项目将使用一系列生化和生物物理技术来实现这些目标，包括核糖体脚印分析、直接荧光分析和通过质谱分析分析的RNA结合分析。广泛的影响：研究的意义和所采用的实验方法的范围将为从高中到研究生院的学生在生化分析和定量生物物理测量方面提供极好的培训机会。每个级别的学生将接受适当的分子生物学技术培训，包括克隆蛋白的表达、翻译分析和其他重要的生化技术。该项目中使用的荧光光谱和质谱学将为更多的高级学生提供定量和生物物理培训。亨特学院招收和招收了大量在科学领域未被充分代表的少数族裔学生，并为他们赞助了一个夏季研究项目。将从这个项目中招募学生参与研究。因此，该项目将有助于培训一支多元化的科学技术队伍。高中教师将在夏季在实验室进行研究，使用定量荧光测量来确定平衡和热力学性质，并增强他们在生物系统中应用热力学的内容知识。首席调查员将与曼哈顿社区学院(一家西班牙裔服务机构)的教职员工合作，为他们的教职员工和学生增加研究机会，在适当的时候提供最先进的设备和研究指导。通过参与研究项目，高中教师和社区学院的教职员工将增加他们对当前研究技术的知识，以及向他们的学生传达科学的兴奋的能力。

Intellectual Merit: Plant viruses cause diseases that affect a significant number of food crops world-wide with potentially severe consequences on food supplies and economic conditions. Most viruses 'commandeer' the host cell's protein synthesis machinery to make viral proteins. Barley yellow dwarf virus (BYDV) is one of the two most widespread and economically significant plant viruses, affecting wheat, barley and oat crops worldwide. The aims of this project will advance the basic science of plant viruses, while enabling technologies for the improved control of harmful plant viruses and the engineering of modified viruses for production of high-value proteins using plants. BYDV contains an unusual RNA structure, a translational enhancer (BTE), in the 3' untranslated region (UTR) of the mRNA that interacts with a 5' UTR stem loop. This long-range interaction promotes efficient translation of the viral mRNA by a mechanism that is not yet fully understood. The project will employ the 3' UTR of BYDV as a model system to elucidate mechanisms of long-range RNA interactions involving 3'UTR elements. The overall goal of the project is to identify the molecular interactions that enable viral mRNA to compete efficiently with the much higher concentrations of cellular RNA for access to ribosomes and translation factors. The three critical steps in the assembly of viral protein synthesis initiation complexes will be investigated: 1) The binding of eIFs (eukaryotic initiation factors) to the viral 3'-BTE; 2) the interaction of ribosomes with viral 3'-BTE and 5'-UTR sequences; and 3) the interaction between viral 3'-BTE and 5'-UTR regions. A library of BYDV mRNAs with varying translational efficiency will be used to determine the correlation between eIF binding and translation, the binding affinity of ribosomes for viral RNA sequences, and the role of the interaction between 3' and 5'-UTRs in each of these steps. The project will employ a range of biochemical and biophysical techniques to achieve these aims, including ribosome 'toeprint' analysis, direct fluorescence assays, and RNA binding assays analyzed by mass spectroscopic analysis.Broader Impacts: The significance of the research and the range of experimental approaches employed will provide excellent training opportunities for students, ranging from the high school to the graduate school level, in both biochemical assays and quantitative biophysical measurements. Students at each level will receive appropriate training in molecular biology techniques including expression of cloned proteins, translational assays, and other important biochemical techniques. Fluorescence spectroscopy and mass spectrometry used in this project will provide quantitative and biophysical training for more advanced students. Hunter College recruits and enrolls significant numbers of minority students under-represented in the sciences, and also sponsors a summer research program for them. Students will be recruited from this program to participate in the research. Thus, the project will contribute to training a diverse workforce in science and technology. High school teachers will carry out research in the lab during the summers, using quantitative fluorescence measurements to determine equilibrium and thermodynamic properties and enhance their content knowledge of applications of thermodynamics to biological systems. The Principal Investigator will work with faculty from the Borough of Manhattan Community College (an Hispanic serving institution) to increase research opportunities for their faculty and students, providing access to state-of-the-art equipment and research guidance when appropriate. By participating in the research project, high school teachers and community college faculty will increase their knowledge of current research techniques and their abilities to convey to their students the excitement of science.