Structure and function of SRP RNA

SRP RNA的结构和功能

• 批准号: 6742530
• 负责人: CHRISTIAN W ZWIEB
• 金额: $ 20.92万
• 依托单位: UNIVERSITY OF TEXAS HLTH CTR AT TYLER
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6742530
• 关键词: SDS polyacrylamide gel electrophoresis; biological signal transduction; conformation; crosslink; genetic translation; intermolecular interaction; matrix assisted laser desorption ionization; molecular assembly /self assembly; nucleic acid structure; polymerase chain reaction; protein protein interaction; protein signal sequence; protein structure function; protein transport; ribonucleoproteins; ribosomal RNA; secretion; secretory protein; site directed mutagenesis

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.

描述（由申请人提供）： 这项研究的目的是推进蛋白质跨生物膜转运的基本过程的分子理解。我们特别关注的是信号识别颗粒（SRP），因为它在蛋白质靶向和分泌中起着核心作用。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。我们将通过使用定点诱变、定点交联、结构探测、比较分析、生物化学和生物物理学研究的组合来定义参与SRP介导的蛋白分泌和SRP组装的人SRP RNA的那些区域。研究的主要内容有：（1）SRP RNA参与SRP组装、信号识别和蛋白质转运的机制。复合物将从重组组分重构，以研究SRP的形成和分泌蛋白的易位能力。将确定改变的复合物对SRP循环中的单个事件的影响，例如信号肽识别、翻译停滞和停滞的释放。(2)蛋白质SRP 68和SRP 72在人类SRP中的结构和功能作用。将通过系统的定点突变研究SRP RNA与SRP 68和SRP 72的相互作用。RNA-蛋白质邻域将通过使用位点定向光化学交联，然后鉴定交联的核苷酸和氨基酸来确定。这些实验的数据将被集体观察，并用于生成人类SRP的三维分子模型。(3)与SRP RNA螺旋8和信号结合的SRP 54的M-结构域的生物物理表征以及被操纵以含有改变的信号肽的重组信号融合蛋白衍生物（SFP）的生物物理表征。这些研究将集中在两个关键的相互作用的具体特点，并确定其动态关系的SRP功能。