The NusG paralog RfaH mediates bacterial transcription–translation coupling in genes that contain a DNA sequence element, termed an ops site, required for pausing RNA polymerase (RNAP) and for loading RfaH onto the paused RNAP. Here, we report cryo-electron microscopy structures of transcription–translation complexes (TTCs) containing Escherichia coli RfaH. The results show that RfaH bridges RNAP and the ribosome, with the RfaH N-terminal domain interacting with RNAP and the RfaH C-terminal domain interacting with the ribosome. The results show that the distribution of translational and orientational positions of RNAP relative to the ribosome in RfaH-coupled TTCs is more restricted than in NusG-coupled TTCs because of the more restricted flexibility of the RfaH interdomain linker. The results further suggest that the structural organization of RfaH-coupled TTCs in the ‘loading state’, in which RNAP and RfaH are located at the ops site during formation of the TTC, is the same as the structural organization of RfaH-coupled TTCs in the ‘loaded state’, in which RNAP and RfaH are located at positions downstream of the ops site during function of the TTC. The results define the structural organization of RfaH-containing TTCs and set the stage for analysis of functions of RfaH during translation initiation and transcription–translation coupling.

NusG 旁系同源物 RfaH 介导包含 DNA 序列元件（称为 ops 位点）的基因中的细菌转录-翻译偶联，该序列元件是暂停 RNA 聚合酶 （RNAP） 和将 RfaH 加载到暂停的 RNAP 上所必需的。在这里，我们报告了含有大肠杆菌 RfaH 的转录-翻译复合物 （TTC） 的冷冻电子显微镜结构。结果表明，RfaH 桥接 RNAP 和核糖体，RfaH N 末端结构域与 RNAP 相互作用，RfaH C 末端结构域与核糖体相互作用。结果表明，由于 RfaH 结构域间接头的灵活性更有限，因此 RfaH 偶联 TTC 中 RNAP 相对于核糖体的翻译和取向位置的分布比 NusG 偶联 TTC 更受限制。结果进一步表明，RfaH 偶联的 TTC 在“加载状态”下的结构组织，其中 RNAP 和 RfaH 位于 TTC 形成过程中的操作位点，与“加载状态”下 RfaH 偶联的 TTC 的结构组织相同，其中 RNAP 和 RfaH 位于操作的下游位置在 TTC 运作期间的站点。结果定义了含 RfaH 的 TTC 的结构组织，并为分析翻译起始和转录-翻译耦合期间的 RfaH 功能奠定了基础。