A central question in biology is how macromolecular machines function cooperatively. In bacteria, transcription and translation occur in the same cellular compartment, and can be physically and functionally coupled^1,2,3,4. Although high-resolution structures of the ribosome–RNA polymerase (RNAP) complex have provided initial mechanistic insights into the coupling process^5,6,7,8,9,10, we lack knowledge of how these structural snapshots are placed along a dynamic reaction trajectory. Here we reconstitute a complete and active transcription–translation system and develop multi-colour single-molecule fluorescence microscopy experiments to directly and simultaneously track transcription elongation, translation elongation and the physical and functional coupling between the ribosome and the RNAP in real time. Our data show that physical coupling between ribosome and RNAP can occur over hundreds of nucleotides of intervening mRNA by mRNA looping, a process facilitated by NusG. We detect active transcription elongation during mRNA looping and show that NusA-paused RNAPs can be activated by the ribosome by long-range physical coupling. Conversely, the ribosome slows down while colliding with the RNAP. We hereby provide an alternative explanation for how the ribosome can efficiently rescue RNAP from frequent pausing without requiring collisions by a closely trailing ribosome. Overall, our dynamic data mechanistically highlight an example of how two central macromolecular machineries, the ribosome and RNAP, can physically and functionally cooperate to optimize gene expression.

生物学的一个核心问题是大分子机器如何协同工作。在细菌中，转录和翻译发生在同一个细胞区室中，并且可以在物理和功能上耦合^1,2,3,4。尽管核糖体-RNA 聚合酶 （RNAP） 复合物的高分辨率结构为偶联过程提供了初步的机制见解^5,6,7,8,9,10，但我们缺乏这些结构快照如何沿动态反应轨迹放置的知识。在这里，我们重建了一个完整而活跃的转录-翻译系统，并开发了多色单分子荧光显微镜实验，以直接和同时实时跟踪转录延伸、翻译延伸以及核糖体和 RNAP 之间的物理和功能耦合。我们的数据表明，核糖体和 RNAP 之间的物理偶联可以通过 mRNA 环发生在中间 mRNA 的数百个核苷酸上，这一过程由 NusG 促进。我们检测 mRNA 环过程中的主动转录延伸，并表明 NusA 暂停的 RNAP 可以通过长程物理偶联被核糖体激活。相反，核糖体在与 RNAP 碰撞时减慢速度。我们特此提供另一种解释，说明核糖体如何有效地将 RNAP 从频繁的暂停中拯救出来，而不需要紧密跟随的核糖体发生碰撞。总体而言，我们的动态数据在机制上突出了两个中心大分子机制（核糖体和 RNAP）如何在物理和功能上合作以优化基因表达的一个例子。