Real-time tracking of transcription-translation coupling

In prokaryotic cells, transcription and translation occur in the same compartment, enabling a functional interaction between both gene expression machineries. Several studies, including recent high-resolution structures, demonstrate a physical link that establishes coupling between both machineries either mediated by direct interactions between the RNAP and the ribosomeor by factor-mediated interactions via the transcription factors NusA and NusG. In contrast, recent work challenges the notion that transcription and translation are generally coupled. Here, to understand what promotes coupling and how it is regulated, we reconstituted in vitro the complete active transcription-translation coupling system and developed multicolor single-molecule fluorescence microscopy assays to track in real-time how single RNA molecules are actively transcribed (Duss et al., Cell, 2019) and simultaneously translated by the ribosome. Furthermore, by site-specifically labeling the ribosome and the RNAP, we can detect the direct physical interaction between both machineries. Our data shows that physical coupling between RNAP and the ribosome can be transient and depends on the mRNA length between both machineries. We also detect different coupled states while the polymerase moves away from the ribosome, likely representing the different states found by cryoEM (Wang & Ebright, Science, 2020; Webster & Weixlbaumer, Science, 2020; O’Reilly & Rappsilber, Science, 2020). RNAP and the ribosome can also remain physically coupled even if the transcription machinery moves more than 100 nucleotides away from the ribosome suggesting that physical and functional coupling can be unrelated. Overall, by tracking the conformation and interaction dynamics of different macromolecular machines over time, our methods allow us to investigate how different macromolecular machines cooperate to fine-tune gene expression.