Structure and function of SRP RNA

描述(由申请人提供)： 这项研究计划的目的是促进对蛋白质通过生物膜运输的基本过程的分子理解。我们特别关注信号识别颗粒(SRP)，因为它在蛋白质靶向和分泌中发挥核心作用。SRP RNA是每个SRP的重要组成部分，包含几个具有重要功能的保守特征。我们研究的目标是了解这种RNA在人类系统中的结构和功能作用。我们将结合定点突变、定点交联、结构探测、比较分析、生化和生物物理研究，确定人类SRP RNA中参与SRP介导的蛋白质分泌和SRP组装的区域。需要研究的主要课题包括：(1)SRP RNA参与SRP组装、信号识别和蛋白质转位的机制。将重组组分重组成复合体，以研究SRP的形成和分泌蛋白的转位能力。改变的复合体对SRP循环中个别事件的影响，如信号肽识别、翻译停滞和释放停滞，将被识别。(2)SRP68和SRP72蛋白在人SRP中的结构和功能作用。SRP RNA与SRP68和SRP72的相互作用将通过系统的定点突变来研究。RNA-蛋白质邻域将通过使用定点光化学交联法确定，然后鉴定交联的核苷酸和氨基酸。这些实验的数据将被集中查看，并用于生成人类SRP的三维分子模型。(3)SRP54的M结构域与SRP RNA螺旋8结合的生物物理特征，以及重组信号融合蛋白衍生物(SFP)的信号和含有改变的信号肽的操作。这些研究将集中于两个关键相互作用的具体特征，并确定它们与SRP功能的动态关系。

DESCRIPTION (provided by applicant): 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 targeting and secretion. The SRP RNA is an essential component of every SRP and contains several conserved features 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 SRP-mediated protein secretion and SRP assembly by using a combination of site-directed mutagenesis, site-directed cross-linking, structure probing, comparative analyses, biochemical, and biophysical studies. The major topics to be investigated are: (1) Mechanisms that involve SRP RNA in SRP assembly, signal recognition, and protein translocation. Complexes will be reconstituted from recombinant components to study the formation of the SRP and translocation competence of secretory proteins. The effects of altered complexes on individual events in the SRP-cycle, such as signal peptide recognition, translation arrest, and release of the arrest, will be identified. (2) Structural and functional role of proteins SRP68 and SRP72 within the human SRP. The interactions of SRP RNA with SRP68 and SRP72 will be studied by systematic site-directed mutagenesis. 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 and used to generate a three-dimensional molecular model of human SRP. (3) Biophysical characterization of the M-domain of SRP54 which binds to the SRP RNA helix 8 and the signal as well as of recombinant signal fusion protein derivatives (SFPs) manipulated to contain altered signal peptides. These studies will focus on the specific features of two critical interactions and define their dynamic relationship to SRP function.