Functional Analysis of TEV Internal Ribosome Entry Site

对于那些缺乏帽状结构的mrna，与经典的翻译模型发生了重大偏差。这些mrna具有一个结构化的非编码5' RNA区域，称为IRES（内部核糖体进入位点）。IRES也开始在细胞RNA中被发现。一般来说，它们是在整体翻译受到损害的情况下发现的。病毒mRNA的5'非翻译区（UTR）足以赋予其帽独立翻译，然而IRES翻译（包括病毒翻译）的机制尚不清楚。本项目将以烟草蚀刻病毒（TEV）的5′UTR为模型系统，阐明IRES的功能机制。利用化学探针和诱变技术对tev5′UTR二级结构进行了定位。将审查启动过程的三个相互关联的阶段。它们是1)eIF结合，2)解旋酶活性和3)核糖体结合活性。每个这些活动将测量一个选定的具有不同翻译活性的TEV结构库。eIF结合将通过直接荧光法监测。与解旋酶活性相关的碱基配对变化将通过在RNA和核磁共振测量中加入特定位置的荧光标记来阐明。核糖体结合将通过直接荧光法和蔗糖梯度分析来测量。这里描述的实验将阐明分子相互作用，使病毒IRES有效地与更高浓度的细胞RNA竞争翻译。植物病毒性疾病影响世界范围内大量粮食作物，对经济状况和粮食供应都可能产生严重影响。这项研究的信息不仅有可能用于控制病毒，而且有可能用于开发生产有益营养的所需蛋白质、抗体或具有经济用途的系统。TEV是最简单的IRES元素之一，因此它可以作为一个模型系统来增加我们对更复杂的病毒和细胞IRES功能的理解。这些实验将为从高中到研究生的学生在生化和定量测量方面提供良好的训练。过去参与研究的许多学生都来自代表性不足的少数群体，这种情况未来可能会继续下去。这些学生将为科学和技术领域的多元化和训练有素的劳动力做出贡献。

A major deviation from the classical model of translation occurs for those mRNAs that lack a cap structure. These mRNAs possess a structured, non-coding region of 5' RNA known as an IRES (internal ribosome entry site). IRES are also beginning to be identified in cellular RNA. Generally, they are found under conditions where overall translation is compromised. The 5' untranslated region (UTR) of viral mRNA is sufficient to confer its cap-independent translation, however the mechanism of IRES translation (including viral translation) is not well understood. This project will elucidate the mechanisms of IRES function by using the 5' UTR of tobacco etch virus (TEV) as a model system. The TEV 5' UTR secondary structure has been mapped by chemical probing and mutagenesis. Three inter-related phases of the initiation process will be examined. These are 1) eIF binding, 2) helicase activity and 3) ribosome binding activity. Each of these activities will be measured for a select library of TEV constructs with differing translational activity. The eIF binding will be monitored by direct fluorescence assays. Base pairing changes associated with helicase activity will be elucidated by using fluorescent labels incorporated into specific locations in the RNA and nmr measurements. Ribosome binding will be measured by direct fluorescence methods and sucrose gradient analysis. The experiments described here will elucidate the molecular interactions that allow viral IRES to compete effectively with the much higher concentrations of cellular RNA for translation. Plant viral diseases affect a significant number of food crops world-wide and can have severe impact both on economic conditions and food supply. There is potential to use information from this research not only to control viruses but also to develop systems to produce desired proteins which are nutritionally beneficial, antibodies or have economic uses. TEV is one of the simplest IRES elements, so it serves as a model system to increase our understanding of more complex viral and cellular IRES function. The experiments will provide excellent training for students from high school through graduate school in both biochemical and quantitative measurements. Many of the students involved in research in the past have been from underrepresented minority groups and this is likely to continue in the future. These students will contribute to a diverse and well trained workforce in science and technology.