In-cell architecture of an actively transcribing-translating expressome
In-cell architecture of an actively transcribing-translating expressome
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DOI：
10.1126/science.abb3758
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发表时间：
2020-07-31
期刊：
影响因子：
56.9
通讯作者：
Rappsilber, Juri
中科院分区：
文献类型：
--
作者：
O'Reilly, Francis J.;Xue, Liang;Rappsilber, Juri
在细胞内进行的结构生物学研究可以捕获分子机器在其原生环境中的作用。在这项工作中，我们开发了一个整合的细胞内结构的方法，使用基因组减少的人类病原体肺炎支原体。我们结合了全细胞交联质谱，细胞冷冻电子断层扫描，和综合建模，以确定在亚纳米分辨率的转录和翻译表达体的细胞内结构。表达体包含RNA聚合酶（RNAP）、核糖体和转录延伸因子NusG和NusA。我们在NusG结合的延伸RNAP和核糖体之间的界面上定位了NusA，并提出它可以介导转录-翻译偶联。翻译抑制使表达体解离，而转录抑制使表达体停滞并重新排列，因此，活性表达体结构需要细胞内翻译和转录延伸。
Structural biology studies performed inside cells can capture molecular machines in action within their native context. In this work, we developed an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae. We combined whole-cell cross-linking mass spectrometry, cellular cryo-electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at subnanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpointed NusA at the interface between a NusG-bound elongating RNAP and the ribosome and propose that it can mediate transcription-translation coupling. Translation inhibition dissociated the expressome, whereas transcription inhibition stalled and rearranged it. Thus, the active expressome architecture requires both translation and transcription elongation within the cell.