STRUCTURE/FUNCTION OF SRP RNA

SRP RNA 的结构/功能

• 批准号: 2186553
• 负责人: CHRISTIAN W ZWIEB
• 金额: $ 11.76万
• 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 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2186553
• 关键词: SDS polyacrylamide gel electrophoresis; biological signal transduction; cell free system; crosslink; dogs; membrane transport proteins; molecular cloning; mutant; nucleic acid structure; protein structure function; protein transport; ribonucleoproteins; site directed mutagenesis

The objective of this research proposal is to advance the 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 secretion. A major component of all SRPs is an RNA molecule, regions of which are universally conserved. Our goal 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 SRP assembly by using a combination of site-directed mutagenesis, site- directed cross-linking, and phylogenetic comparisons with cloned SRPcomponents. The major topics to be investigated in this proposal are: (1) The molecular mechanisms involved in signal recognition and protein translocation. SRPs reconstituted with mutant SRP RNAs will be used to test the translocation competence of secretory proteins in a cell-free translation-translocation system. The individual steps which are affected in the SRP-cycle, such as signal recognition, translation arrest, and release of the arrest, will be identified. (2) SRP assembly. Mutants of SRP RNA will be constructed by systematic site-directed mutagenesis, or selected after random mutagenesis, to study the interactions of the SRP proteins with the SRP RNA. Information about the binding sites on the proteins will be obtained by testing the ability of mutant polypeptides to interact with the SRP RNA or with partially assembled ribonucleoprotein particles (RNPs). Photochemical cross-linking with modified nucleotides introduced into the SRP RNA at predetermined positions will be used to identify neighborhoods at the single nucleotide and amino acid level. (3) Biophysical characterization of an RNA tetranucleotide loop interaction with protein SRP19. These studies will focus on the specific features of a critical protein-RNA interaction in a conserved region of the SRP and define its relationship to the overall process of protein translocation.

本研究计划的目的是增进对 蛋白质跨生物膜转运的基本过程。我们的 特别关注的是信号识别粒子 (SRP)，因为它发挥作用 分泌中起核心作用。所有 SRP 的主要成分是 RNA 分子，其区域普遍保守。我们的目标是 了解该 RNA 在人体中的结构和功能作用 系统。我们将定义人类 SRP RNA 的那些区域 参与 SRP 介导的蛋白质分泌和 SRP 的特定方面 通过使用定点诱变、定点诱变的组合进行组装 定向交联以及与克隆的系统发育比较 SRP 组件。该提案要研究的主要主题是： (1)信号识别和蛋白质相关的分子机制 易位。用突变 SRP RNA 重组的 SRP 将用于 测试无细胞中分泌蛋白的易位能力 翻译易位系统。受影响的各个步骤 在 SRP 周期中，例如信号识别、翻译抑制和 逮捕后释放，身份将被确定。 (2)SRP组装。突变体 SRP RNA 将通过系统定点诱变构建，或 随机诱变后选择，以研究 SRP 的相互作用 带有 SRP RNA 的蛋白质。有关结合位点的信息 蛋白质将通过测试突变多肽的能力来获得 与 SRP RNA 或部分组装的核糖核蛋白相互作用 粒子（RNP）。与修饰核苷酸的光化学交联 在预定位置引入 SRP RNA 将用于 在单核苷酸和氨基酸水平上识别邻域。 (3) RNA 四核苷酸环相互作用的生物物理表征 与蛋白质SRP19。这些研究将集中于特定特征 SRP 保守区域中关键的蛋白质-RNA 相互作用 定义其与蛋白质易位整个过程的关系。