STRUCTURE/FUNCTION OF SRP RNA

这项研究计划的目的是推动分子生物学的发展 对蛋白质跨膜转运的基本过程的理解 生物膜。我们特别关注的是信号识别 颗粒(SRP)，因为它在蛋白质分泌中起核心作用。SRP RNA是SRP的基本组件，它包含几个 系统发育保护的具有重要功能的地点。的目标是 我们的研究是为了了解这一结构和功能的作用 人体系统中的RNA。我们将定义人类SRP的这些区域 参与SRP介导的蛋白质的特定方面的RNA 结合使用定点定向的SRP和SRP组装的分泌 突变、定点交联、结构探测和 与克隆的SRP组分的系统发育比较。主要议题 有待研究的是：(1)涉及SRP的分子机制 RNA在信号识别和蛋白质转运中的作用。人类SRP将是 完全由将被改变的重组成分重组而成 通过定点突变检测细胞的易位能力 分泌蛋白质。SRP周期中的个别事件，如信号 承认、翻译逮捕和释放逮捕，将是 已确认身份。大型SRP域中的RNA交换机，它控制 将对SRP54蛋白的结合进行表征。(2)SRP的组装。 纯化的重组SRP组分将用于详细确定 SRP组装途径中的事件。SRP的相互作用 含有SRP RNA的蛋白质将通过系统的定点定向研究 突变和RNA-蛋白质邻域将通过使用 定点光化学交联后的鉴定 交联型核苷酸和氨基酸。来自这些的数据 实验将被集体观看，以提炼三维 SRP RNA的模型。(3)领域的生物物理特征 与信号肽紧密结合的SRP54蛋白和 与SRP RNA相互作用。这些研究将集中在具体的 最保守分子中关键的RNA-蛋白质相互作用的特征 SRP RNA区域，并将定义其与整体 蛋白质转位的过程。

The objective of this research proposal is to advance the molecular understanding of the basic process of protein transport across biological membranes. Our particular focus is on the signal recognition particle (SRP) as it plays a central role in protein secretion. The SRP RNA is an essential component of the SRP and contains several phylogenetically conserved sites of functional importance. The goal of our research is to understand the structural and functional role of this RNA in the human system. We will define those regions of the human SRP RNA which are involved in particular aspects of SRP-mediated protein secretion and of SRP assembly by using a combination of site-directed mutagenesis, site-directed cross-linking, structure probing, and phylogenetic comparisons with cloned SRP-components. The major topics to be investigated are: (1) The molecular mechanisms that involve SRP RNA in signal recognition and protein translocation. Human SRP will be reconstituted entirely from recombinant components that will be altered by site-directed mutagenesis to test the translocation competence of secretory proteins. Individual events in the SRP-cycle, such as signal recognition, translation arrest, and release of the arrest, will be identified. An RNA switch in the large SRP domain which controls the binding of protein SRP54 will be characterized. (2) Assembly of the SRP. Purified recombinant SRP components will be used to determine in detail the events in the SRP assembly pathway. The interactions of the SRP proteins with the SRP RNA will be studied by systematic site-directed mutagenesis, and RNA-protein neighborhoods will be determined by using site-directed photochemical cross-linking followed by the identification of the cross-linked nucleotides and amino acids. The data from these experiments will be viewed collectively to refine a three-dimensional model of the SRP RNA. (3) Biophysical characterization of the domain of protein SRP54 that associates closely with the signal peptide and interacts with SRP RNA. These studies will focus on the specific features of a critical RNA-protein interaction in the most conserved region of the SRP RNA and will define its relationship to the overall process of protein translocation.