STRUCTURE/FUNCTION OF SRP RNA

这项研究建议的目的是促进对 蛋白质通过生物膜运输的基本过程。我们的 特别关注的是信号识别粒子(SRP)的作用 在分泌过程中起着中心作用。所有SRP的一个主要成分是RNA 分子，其区域是普遍保守的。我们的目标是 了解这种RNA在人类体内的结构和功能作用 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 通过结合使用定点突变、定点突变、定点突变 定向交联，并与克隆的系统发育进行比较 SRP组件。这项建议拟研究的主要课题包括： (1)信号识别和蛋白质的分子机制 易位。用突变的SRP RNA重组的SRP将用于 在无细胞条件下测试分泌蛋白的易位能力 翻译-转位系统。受影响的各个步骤 在SRP周期中，例如信号识别、翻译停止和 释放逮捕的人，将得到确认。(2)SRP组件。突变体 SRP RNA将通过系统的定点突变构建，或者 随机诱变后选择，用于研究SRP的相互作用 与SRP RNA结合的蛋白质。上的绑定位置的信息 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)。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居。(3) RNA四核苷酸环相互作用的生物物理特征 与SRP19蛋白结合。这些研究将集中在特定的特征 SRP保守区的关键蛋白质-RNA相互作用 确定其与蛋白质转运的整个过程的关系。

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.