In-cell architecture of an active transcription-translation complex

Structural biology performed inside cells can capture molecular machines in action within their native context. Here we develop an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae. We combine whole-cell crosslinking mass spectrometry, cellular cryo-electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at sub-nanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpoint NusA at the interface between the ribosome and a NusG-bound elongating RNAP, and propose it could mediate transcription-translation coupling. Transcription inhibition stalls and rearranges the expressome, whereas translation inhibition dissociates it, demonstrating that the elongating expressome architecture requires active translation and transcription within the cell.