Generation of ribosome nascent chain complexes for structural and functional studies
Generation of ribosome nascent chain complexes for structural and functional studies
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DOI：
10.1016/j.jsb.2007.01.005
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发表时间：
2007-06-01
影响因子：
3
通讯作者：
Ban, Nenad
中科院分区：
文献类型：
--
作者：
Schaffitzel, Christiane;Ban, Nenad
核糖体新生链复合物（RNCs）的制备是研究核糖体共翻译折叠、靶向和转位的基础。在这里，我们提出了我们的方法，用于生成同质和稳定的RNCs，包括体外翻译和亲和纯化。将与核糖体隧道紧密相互作用的SecM阻滞序列融合到新生多肽链上显著增强了RNC的稳定性。我们已经能够通过工程化具有更高亲和力的标签来增加亲和纯化步骤的产率。用这种方法产生的RNC已成功地用于获得与信号识别颗粒和易位子的复合物的3D冷冻电子显微镜重建。所建立的程序是高效的，如果按比例放大，可以产生足够用于结晶实验的毫克量的RNC。(c)2007年爱思唯尔公司All rights reserved.
Biochemical and structural studies of co-translational folding, targeting and translocation depend on an efficient methodology to prepare ribosome nascent chain complexes (RNCs). Here we present our approach for the generation of homogenous and stable RNCs involving in vitro translation and affinity purification. Fusing the SecM arrest sequence, which tightly interacts with the ribosomal tunnel, to the nascent polypeptide chain significantly enhanced the stability of the RNCs. We have been able to increase the yield of the affinity purification step by engineering a tag with higher affinity. The RNCs generated with this approach have been successfully used to obtain 3D cryo-electron microscopic reconstructions of complexes with the signal recognition particle and the translocon. The established procedure is highly efficient and if scaled up could yield milligram amounts of RNCs sufficient for crystallization experiments. (c) 2007 Elsevier Inc. All rights reserved.