来自大肠杆菌的翻译起始复合物的冷冻电镜结构。

The cryo-EM structure of a translation initiation complex from Escherichia coli.

作者信息

Allen Gregory S, Zavialov Andrey, Gursky Richard, Ehrenberg Måns, Frank Joachim

机构信息

Howard Hughes Medical Institute, Health Research, Inc. at the Wadsworth Center, Albany, New York 12201, USA.

出版信息

Cell. 2005 Jun 3;121(5):703-12. doi: 10.1016/j.cell.2005.03.023.

DOI:10.1016/j.cell.2005.03.023

PMID:15935757

Abstract

The 70S ribosome and its complement of factors required for initiation of translation in E. coli were purified separately and reassembled in vitro with GDPNP, producing a stable initiation complex (IC) stalled after 70S assembly. We have obtained a cryo-EM reconstruction of the IC showing IF2GDPNP at the intersubunit cleft of the 70S ribosome. IF2GDPNP contacts the 30S and 50S subunits as well as fMet-tRNA(fMet). IF2 here adopts a conformation radically different from that seen in the recent crystal structure of IF2. The C-terminal domain of IF2 binds to the single-stranded portion of fMet-tRNA(fMet), thereby forcing the tRNA into a novel orientation at the P site. The GTP binding domain of IF2 binds to the GTPase-associated center of the 50S subunit in a manner similar to EF-G and EF-Tu. Additionally, we present evidence for the localization of IF1, IF3, one C-terminal domain of L7/L12, and the N-terminal domain of IF2 in the initiation complex.

摘要

70S核糖体及其在大肠杆菌中启动翻译所需的因子补充物被分别纯化，并在体外与GDPNP重新组装，产生了一个在70S组装后停滞的稳定起始复合物（IC）。我们获得了IC的冷冻电镜重建图像，显示IF2GDPNP位于70S核糖体的亚基间裂隙处。IF2GDPNP与30S和50S亚基以及fMet-tRNA(fMet)接触。这里的IF2采用了一种与最近IF2晶体结构中所见截然不同的构象。IF2的C末端结构域与fMet-tRNA(fMet)的单链部分结合，从而迫使tRNA在P位点形成一种新的取向。IF2的GTP结合结构域以类似于EF-G和EF-Tu的方式与50S亚基的GTPase相关中心结合。此外，我们提供了关于IF1、IF3、L7/L12的一个C末端结构域以及IF2的N末端结构域在起始复合物中定位的证据。