Structures of transcription-translation coupling complexes at each stage of the translation cycle

In the bacterium Escherichia coli, efficient expression of protein-coding genes is achieved through physical coupling of RNA polymerase (RNAP) synthesizing an mRNA and the first ribosome translating the mRNA, with transcription elongation factors NusG and NusA bridging RNAP and ribosome in a transcription-translation complex (TTC). Recent structural studies suggest that the flexibility in NusG and NusA accommodates the changes in ribosome conformation that occur during the translation cycle, but details have been unclear. Here, we report cryo-EM structures of TTCs at each stage of the translation cycle as an actively translating ribosome approaches to a halted transcription complex. We observe sequential conversion of a loosely coupled TTC (TTC-LC) to a tightly coupled TTC (TTC-B) to a collided TTC (TTC-A) as the ribosome approaches the transcription complex. We show that flexibility of the NusG linker and NusA "pantograph" enable TTC-LC and TTC-B to accommodate ribosome conformation at each stage of the translation cycle. We show that, in TTC-A, steric clash between ribosome and RNAP upon ribosome 30S head swiveling inhibits 30S head swiveling, consistent with translation slowdown, and applies mechanical force to the trailing edge of RNAP, causing transcription termination. Our results define the structures and properties of loosely coupled, tightly coupled, and collided TTCs at each stage of the translation cycle.