Functional Role of BYDV 3' RNA Translation Enhancer Element

知识价值：植物病毒引起的疾病影响全世界大量粮食作物，可能对粮食供应和经济状况造成严重后果。大多数病毒“征用”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）是两种分布最广、经济意义最大的植物病毒之一，影响世界各地的小麦、大麦和燕麦作物。该项目的目标是推进植物病毒的基础科学，同时使技术能够改进对有害植物病毒的控制，并使修饰病毒的工程用于利用植物生产高价值蛋白质。BYDV含有一个不寻常的RNA结构，翻译增强子（BTE），位于mRNA的3‘非翻译区（UTR），与5’ UTR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了病毒mRNA的有效翻译。该项目将采用BYDV的3'UTR作为模型系统来阐明涉及3'UTR元件的远程RNA相互作用机制。该项目的总体目标是确定分子相互作用，使病毒mRNA能够有效地与更高浓度的细胞RNA竞争，以获得核糖体和翻译因子。研究了病毒蛋白合成起始复合物组装的三个关键步骤：1)真核起始因子（eIFs）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与5′-UTR区域的相互作用。一个具有不同翻译效率的BYDV mrna文库将用于确定eIF结合与翻译之间的相关性，核糖体对病毒RNA序列的结合亲和力，以及3‘和5’- utr之间相互作用在这些步骤中的作用。该项目将采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“足印”分析、直接荧光分析和质谱分析的RNA结合分析。更广泛的影响：研究的意义和所采用的实验方法的范围将为学生提供从高中到研究生水平的优秀培训机会，包括生化分析和定量生物物理测量。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。本项目中使用的荧光光谱和质谱将为更高级的学生提供定量和生物物理训练。亨特学院招收了大量在科学领域代表性不足的少数族裔学生，并为他们赞助了一个暑期研究项目。将从该项目中招募学生参与研究。因此，该项目将有助于培训科学和技术方面的多样化劳动力。高中教师将在暑假期间在实验室进行研究，使用定量荧光测量来确定平衡和热力学性质，并增强热力学在生物系统中的应用的内容知识。首席研究员将与曼哈顿社区学院（一所西班牙裔服务机构）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。通过参与这项研究项目，高中教师和社区大学教师将增加他们对当前研究技术的了解，并提高他们向学生传达科学兴奋的能力。

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.