BIOPHYSICAL CHEMISTRY OF A DEAD/H PROTEIN

描述：(摘自申请者的摘要)这个项目的目标是 了解大肠杆菌DbpA的功能，DbpA是 DEAD/H家族蛋白参与多种细胞通路，包括 核糖核酸。DEAD/H蛋白将ATP的水解与RNA结合结合在一起，并 建议对RNA的二级和三级结构进行修饰。E.Coli DbpA及其基因 枯草杆菌同源菌株YxiN被选择用于详细的结构和机理 研究是因为，与几乎所有其他DEAD/H蛋白不同，它们紧密结合并 具体到23S rRNA的离散区域。均衡与速率 ATPase反应最小动力学方案中各步骤的常数 将决心建立一个结构-功能研究的框架。这个 DbpA在重组rRNA中作为解旋酶的可能性将是 检查过了。RNA修饰、光交联和蛋白质工程 实验将测试DbpA的不同结构域如何与其同源基因相互作用 RNA，以及蛋白质与RNA的接触在催化循环中是否发生变化。 最后，将使用DbpA中断菌株来搜索 DbpA在大肠杆菌细胞中的作用机制。

DESCRIPTION: (From the Applicant's Abstract) The goal of this project is to obtain an understanding of the function of E. coli DbpA, a member of the large DEAD/H family of proteins that participate in many cellular pathways involving RNA. DEAD/H proteins couple the hydrolysis of ATP with RNA binding and are proposed to modify RNA secondary and tertiary structure. E. Coli DbpA and its B. subtilis homologue YxiN were chosen for detailed structural and mechanistic studies because, unlike nearly all other DEAD/H proteins, they bind tightly and specifically to a discrete region of 23S rRNA. The equilibrium and rate constants for the steps in the minimal kinetic scheme of the ATPase reaction will be determined to establish a framework for structure-function studies. The possibility that DbpA acts as a helicase in restructuring rRNA will be examined. RNA modification, photocrosslinking, and protein engineering experiments will test how different domains of DbpA interact with its cognate RNA, and whether the protein-RNA contacts change during the catalytic cycle. Finally, a DbpA disruption strain of E. coli will be used to search for the mechanism of action of DbpA in E. coli cells.