Biophysical Chemistry of a DEAD/H Protein

DEAD/H 蛋白的生物物理化学

• 批准号: 6827704
• 负责人: OLKE C UHLENBECK
• 金额: $ 25.6万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6827704
• 关键词: Escherichia coli; RNA binding protein; adenosinetriphosphatase; bacterial proteins; chemical kinetics; crosslink; enzyme activity; helicase; photochemistry; protein engineering; protein structure function; ribosomal RNA; ribosomes; site directed mutagenesis; thermodynamics

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.

描述（由申请人提供）：该项目的长期目标是获得对一大类普遍存在的蛋白质（称为DEXD/H蛋白质）的机制性理解，DEXD/H蛋白质作为重要因子参与许多涉及RNA的细胞过程。DExD/H蛋白被认为作为RNA解旋酶催化大RNA的构象变化，然而，已经提出了其他功能。目的是对纯化的蛋白质进行生物化学和生物物理实验，这将补充许多其他小组的广泛努力，这些小组将分子遗传学和分子生物学方法应用于更复杂的生理环境中的相同蛋白质。本项目最初选择了E.大肠杆菌DbpA作为模型进行详细的研究，因为其特殊的实验易处理性。它不仅在生物化学上表现良好，而且与所有其他DExD/H蛋白相比，它对靶RNA表现出非常紧密的结合和高度特异性，这简化了结构和生化实验。我们已经确定DbpA以独特的方式与RNA相互作用，表明它具有解旋酶活性，并了解如何实现高亲和力和特异性。目前的目标包括（1）机械实验，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)定义DbpA如何与23 S rRNA相互作用的生物化学实验和（3）设计用于确定DbpA作用的细菌核糖体组装途径中的步骤的分子微生物学实验。最后，高通量的RNA结合，ATP酶和RNA解旋酶测定微量滴定板将开发，以测定许多其他DExD/H蛋白，包括18个家族成员参与酵母核糖体组装。