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

Bacterial transcription and translation are frequently linked through a transcribing RNA polymerase following the leading ribosome, a process termed transcription-translation coupling (TTC). Three distinct TTC structures, the “collided expressome” (TTC-A), the “coupled expressome” (TTC-B) and the “long-range coupled expressome” (TTC-LC) have been reported, but the biological significance of all complexes is still subject to uncertainty. Furthermore, all of the ribosomes in the structures are determined in a static state. The underlying translation dynamics within these TTCs remain elusive. Here, we reconstitute the complete active transcription-translation system, using antibiotics and a series of different lengths of mRNAs to trap intermediate coupling states, and determine Cryo-EM structures showing the snapshots for the dynamic reaction trajectory. The results show five states of TTC-B representing the whole stages of the translation elongation cycle. TTC-B is compatible with ribosome conformational changes during translation elongation and coordinates transcription elongation with translation elongation. The results show two distinct TTC-A in translational pre-translocation and translocation intermediate states in which the RNAP becomes unstable as the translation progresses. The results further show that TTC-A is the crucial state where the ribosomes could exert mechanical force on RNAP, leading to the potential transition between TTC-A and TTC-B within mRNA spacer ranging from 7 to 9 codons and suggesting a ribosome-dependent transcription termination within mRNA spacer shorter than 7 codons. The results further show TTC-LC is also compatible with ribosome conformational changes during translation elongation cycle but with less stability compared with TTC-B and shows the potential transition with TTC-B within mRNA spacer of 13 codons. Our results provide a comprehensive blueprint detailing the dynamic interplay of transcription-translation coupling.