EM OF POLYSOME FORMATION DURING MEMBRANE PROTEIN SYNTHESIS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们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小泡中产生高产率的膜蛋白。这些膜蛋白通过核糖体与嵌入在膜中的转运子通道的对接，以共翻译的方式插入到小泡中。我们想要验证的是，类似于可溶性蛋白质的合成，多个产生膜蛋白的核糖体可以从单个mRNA翻译而来，并且这些核糖体都将与单独的转座子对接。最终，我们希望使用低温电子显微镜对这些连接到我们的囊泡膜上的多聚体进行成像。 膜蛋白合成的机制是目前研究的热点。尽管多聚体的形成被假设为膜蛋白，但就我们所知，它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不适用于多聚体研究的优点。首先，反应发生在开放的环境中，允许直接进入多聚体结构，而不需要苛刻的细胞裂解程序，否则可能会损坏脆弱的多聚体。其次，该系统只产生单一的所需目标蛋白，因此结果不会被合成无关的可溶性蛋白的多聚体所掩盖。因此，我们认为无细胞技术为这些多聚体提供了一个更理想的成像平台。

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.