EM OF POLYSOME FORMATION DURING MEMBRANE PROTEIN SYNTHESIS

膜蛋白合成过程中多聚体形成的电子显微镜

• 批准号: 7602771
• 负责人: James Anthony Swartz
• 金额: $ 0.89万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-13 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7602771
• 关键词: Area; Cells; Computer Retrieval of Information on Scientific Projects Database; Cryoelectron Microscopy; Cytolysis; Docking; Environment; Funding; Grant; Image; Individual; Institution; Knowledge; Membrane; Membrane Proteins; Messenger RNA; Polyribosomes; Procedures; Protein Biosynthesis; Proteins; Reaction; Research; Research Personnel; Resources; Ribosomes; Source; Structure; System; Technology; Translating; United States National Institutes of Health; Vesicle; desire

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have developed a cell-free protein synthesis system which produces high yields of membrane proteins in E.coli inner membrane vesicles. These membrane proteins are inserted into the vesicles co-translationally through docking of the ribosomes to translocon channels embedded in the membranes. We would like to verify that, analogous to soluble protein synthesis, multiple membrane protein-producing ribosomes can translate from a single mRNA and that these ribosomes will each be docked to individual translocons. Ultimately, we would like to use cryoEM to image these polysomes docked to our vesicle membranes. The mechanisms involved in membrane protein synthesis are currently an active area of research. Though polysome formation has been hypothesized for membrane proteins, to the best of our knowledge they have not been imaged directly. Our cell-free synthesis system provides many advantages previously unavailable for polysome studies. First, the reaction occurs in an open environment allowing direct access to the polysome structures without harsh cell lysis procedures that might damage fragile polysomes. Second, the system produces only the single desired target protein, so results are not obscured by polysomes synthesizing unrelated soluble proteins. Thus, we believe cell-free technology provides a more optimal platform for imaging these polysomes.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们已经开发了一种无细胞蛋白质合成系统，该系统在大肠杆菌内膜囊泡中产生高产量的膜蛋白。 这些膜蛋白通过核糖体与嵌入膜中的易位子通道的对接而协同地插入囊泡中。 我们想验证，类似于可溶性蛋白质的合成，多个膜蛋白生产核糖体可以从一个单一的mRNA翻译，这些核糖体将每个对接到个人translocons。 最终，我们想使用cryoEM来成像这些停靠在囊泡膜上的多聚核糖体。 涉及膜蛋白合成的机制是目前的一个活跃的研究领域。 虽然多核糖体的形成已被假设为膜蛋白，据我们所知，他们还没有被直接成像。 我们的无细胞合成系统提供了许多以前无法用于多核糖体研究的优点。 首先，反应发生在开放的环境中，允许直接进入多核糖体结构，而没有可能破坏脆弱的多核糖体的苛刻的细胞裂解程序。 其次，该系统只产生单一的目标蛋白，因此结果不会被合成不相关可溶性蛋白的多核糖体所掩盖。 因此，我们相信无细胞技术为这些多聚核糖体的成像提供了一个更理想的平台。