Biophysical Chemistry of a DEAD/H Protein

描述(申请人提供)：该项目的长期目标是获得对一大类普遍存在的蛋白质的机械性理解，称为DEXD/H蛋白质，它们作为基本因子参与许多涉及RNA的细胞过程。DExD/H蛋白被认为是催化大RNA构象变化的RNA解旋酶，然而，其他功能也被提出。其目的是对纯化的蛋白质进行生化和生物物理实验，这将补充许多其他小组的广泛努力，将分子遗传学和分子生物学方法应用于在更复杂的生理环境中的相同蛋白质。本项目最初选择E.coliDbpA作为详细研究的模型，因为它具有特殊的实验可操作性。它不仅在生化上表现良好，而且与所有其他DExD/H蛋白相比，它对目标RNA表现出非常紧密的结合和高度的特异性，这简化了结构和生化实验。我们已经确定DbpA以一种独特的方式与RNA相互作用，表明它具有解旋酶活性，并理解了高亲和力和特异性是如何实现的。目前的目标包括(1)机制实验，以了解DbpA如何作为解旋酶，以及它是否被设计为只打开几个碱基对。(2)确定DbpA如何与23S rRNA相互作用的生化实验；(3)旨在确定DbpA作用于细菌核糖体组装途径的步骤的分子微生物学实验。最后，将发展高通量的微量平板RNA结合、ATPase和RNA解旋酶分析方法，以检测许多其他DExD/H蛋白，包括参与酵母核糖体组装的18个家族成员。

DESCRIPTION (provided by applicant): The long term goal of this project is to obtain a mechanistic understanding of a large, ubiquitous class of proteins, termed DEXD/H proteins, which participate as essential factors in many cellular processes involving RNA. DExD/H proteins are believed to act as RNA helicases to catalyze conformational changes in large RNAs, however, other functions have been proposed. The intent is to perform biochemical and biophysical experiments on purified proteins that will complement extensive efforts by many other groups applying molecular genetic and molecular biological methods to the same proteins in their more complex physiological setting. This project initially chose E. coli DbpA as a model for detailed study, because of its exceptional experimental tractability. Not only is it biochemically well-behaved, but, in contrast with all other DExD/H proteins, it shows very tight binding and high specificity for its target RNA, which simplifies structural and biochemical experiments. We have established that DbpA interacts with RNA in a unique manner, shown that it has helicase activity and understood how the high affinity and specificity is achieved. Current aims include (1) mechanistic experiments to understand how DbpA acts as a helicase and whether it is designed to only open a few base pairs. (2) biochemical experiments defining how DbpA interacts with 23S rRNA and (3) molecular microbiological experiments designed to determine the step in the bacterial ribosome assembly pathway where DbpA acts. Finally, high throughput RNA binding, ATPase and RNA helicase assays in microtiter plates will be developed, in order to assay many other DExD/H proteins, including the 18 family members involved in yeast ribosome assembly.