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 在含有 DNA 序列元件（称为ops位点）的基因中介导细菌转录-翻译偶联，需要暂停 RNA 聚合酶 (RNAP) 并将 RfaH 加载到暂停的 RNAP 上。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，其中RfaH N端结构域与RNAP相互作用，RfaH C端结构域与核糖体相互作用。结果表明，由于 RfaH 域间连接子的灵活性更受限制，RfaH 偶联 TTC 中 RNAP 相对于核糖体的平移和定向位置分布比 NusG 偶联 TTC 更受限制。结果进一步表明，“加载状态”下的 RfaH 偶联 TTC 的结构组织，其中 RNAP 和 RfaH 在 TTC 形成过程中位于ops位点，与“加载状态”下 RfaH 偶联 TTC 的结构组织相同。 “加载状态”，其中 RNAP 和 RfaH 在 TTC 功能期间位于ops位点下游的位置。结果定义了含有 RfaH 的 TTC 的结构组织，并为分析 RfaH 在翻译起始和转录-翻译耦合过程中的功能奠定了基础。