STRUCTURE/FUNCTION OF SRP RNA

这项研究的目的是推进分子生物学的发展。 了解蛋白质跨膜转运的基本过程 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着核心作用。 的SRP RNA是SRP的重要组成部分， 具有重要功能的遗传学保守位点。 的目标 我们的研究是为了了解这种结构和功能的作用， 人体系统中的RNA 我们将定义人类SRP的这些区域， 参与SRP介导蛋白的特定方面的RNA 分泌和SRP组装的方法， 诱变，定点交联，结构探测，和 与克隆的SRP组分的系统发育比较。的主要议题 研究内容包括：（1）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 完全由重组成分重组而成 通过定点突变来测试 分泌蛋白 SRP循环中的单个事件，如信号 承认，翻译逮捕，并释放逮捕，将是 鉴定 大SRP结构域中的一个RNA开关， 将表征蛋白质SRP 54的结合。(2)SRP大会。 纯化的重组SRP组分将用于详细测定 SRP组装途径中的事件。 SRP的相互作用 将通过系统的定点突变研究具有SRP RNA的蛋白质。 诱变，RNA-蛋白质邻域将通过使用 位点定向光化学交联，随后鉴定 交联的核苷酸和氨基酸。 这些数据 实验将被视为集体，以完善一个三维 SRP RNA的模型。(3)的结构域的生物物理表征 与信号肽密切相关的蛋白质SRP 54， 与SRP RNA相互作用。这些研究将侧重于具体的 最保守的RNA-蛋白质相互作用的关键特征 区域，并将定义其与整体的关系。 蛋白质转运过程。

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.