STRUCTURE/FUNCTION OF SRP RNA

SRP RNA 的结构/功能

• 批准号: 2634718
• 负责人: CHRISTIAN W ZWIEB
• 金额: $ 16.07万
• 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/2634718
• 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.

这项研究的目的是促进对以下问题的理解： 蛋白质跨生物膜运输的基本过程。我们 特别关注的是信号识别颗粒（SRP），因为它发挥 在分泌中起重要作用。所有SRPs的主要成分是RNA 分子，其区域普遍保守。我们的目标是 了解这种RNA在人类中的结构和功能作用， 系统我们将定义人SRP RNA的那些区域， 参与SRP介导的蛋白质分泌的特定方面， 通过使用定点诱变、定点突变和定点突变的组合进行组装， 定向交联，并与克隆的 SRP组件。本建议书拟研究的主要课题为： (1)信号识别和蛋白质合成的分子机制 易位用突变体SRP RNA重构的SRP将用于 测试分泌蛋白在无细胞培养基中的易位能力， 易位系受影响的各个步骤 在SRP循环中，如信号识别、翻译停滞和 释放的逮捕，将被确定。(2)SRP组件。突变体 SRP RNA将通过系统性定点诱变构建，或 随机诱变后选择，以研究SRP的相互作用 蛋白质与SRPRNA。上的结合位点的信息。 蛋白质将通过测试突变多肽 与SRP RNA或部分组装的核糖核蛋白相互作用 颗粒（RNP）。用修饰的核苷酸进行光化学交联 在预定位置引入SRP RNA的RNA将用于 在单核苷酸和氨基酸水平上识别邻近区域。（三） RNA四核苷酸环相互作用的生物物理特性 蛋白质SRP 19这些研究将侧重于以下方面的具体特点： 在SRP的保守区域中的关键蛋白质-RNA相互作用， 定义其与蛋白质移位的整个过程的关系。