Genetic Analysis of a Translational Repressor

描述(由申请人提供)：蛋白质对RNA的特异性识别是各种生物过程的基础。例如，基因表达的许多步骤，包括RNA剪接和蛋白质合成，都依赖于RNA和蛋白质的广泛相互作用。此外，RNA-蛋白质识别在重要的病毒病原体的生命周期中发挥着重要的作用，包括人类免疫缺陷病毒和丙型肝炎病毒，仅举两例。许多与RNA结合的蛋白质都是通过β-折叠来实现这一点的。事实上，我们现在认识到整个类别的相关蛋白质的存在，这些蛋白质已经通过突变适应了结构上同源的β-折叠上惊人多样性的RNA的结合。RNA噬菌体的外壳蛋白为研究这种形式的RNA识别提供了一个不同寻常的实验模型。由于相关病毒使用具有相同三级折叠的外壳蛋白与不同的RNA结合，它们提供了探索氨基酸序列变异如何改变RNA结合位点的特异性的机会。最好理解的例子是MS2外壳蛋白-RNA复合体，它已经被用功能和结构方法进行了深入的研究。这项申请寻求建立在这一知识的基础上，通过努力对具有不同RNA结合特异性的几种进化变异的外壳蛋白进行类似的详细理解。还提出了一些实验，旨在通过故意进化MS2结构框架上的新RNA结合活动来探索外壳蛋白β-折叠的适应性极限。了解外壳蛋白如何适应不同RNA的结合，应该能阐明通常情况下管理β-折叠识别RNA的原则。

DESCRIPTION (provided by applicant): Specific recognition of RNAs by proteins is fundamental to a wide variety of biological processes. For example, a number of steps in gene expression, including RNA-splicing and protein synthesis depend on extensive interactions of RNAs and proteins. Moreover, RNA-protein recognition plays an essential role in the life cycles of important viral pathogens, including the Human Immunodeficiency and Hepatitis C Viruses, to name only two. Many of the proteins that bind RNA do so using a beta-sheet. In fact, we now recognize the existence of whole classes of related proteins that have been adapted by mutation to the binding of an amazing diversity of RNAs on structurally homologous beta-sheets. The coat proteins of the RNA bacteriophages offer an unusually accessible experimental model for studying this form of RNA recognition. Since related viruses use coat proteins with the same tertiary fold to bind different RNAs, they provide the opportunity to explore how variation in amino acid sequence alters the specificity of the RNA-binding site. The best understood example is the MS2 coat protein-RNA complex, which has been intensively studied using both functional and structural approaches. This application seeks to build on that knowledge by working toward a similarly detailed understanding of several evolutionarily variant coat proteins with divergent RNA-binding specificities. Also proposed are experiments designed to probe the limits of adaptability of the coat protein beta-sheet through the deliberate evolution of new RNA-binding activities on the MS2 structural framework. Understanding how coat protein can be adapted to the binding of diverse RNAs should illuminate principles that govern the recognition of RNA by beta-sheets generally.