In-cell cryo-electron tomography of Mycoplasma pneumoniae reveals that bacterial translation is regulated through multilayered coordination mechanisms including a preferred initiation pathway where IF2 associates after initiator tRNA recruitment, physiologically relevant transcription-translation collisions during elongation, a threading-based reinitiation mechanism enabled by persistent RNAP-ribosome coupling, and reinitiation-dependent control of large subunit membrane attachment—a mechanism conserved from bacteria to mammalian cells that is structurally visualized here through resolution of the long-hypothesized transertion supercomplex.

Translation is a central process in gene expression. Regulation of this process is complex, depends on factors that include cell state and the subcellular environment, and is subject to modulation via crosstalk to processes like transcription or translocation. Here, we used cryo-electron tomography of native and antibiotic-perturbed Mycoplasma pneumoniae cells to resolve 140 maps that recapitulate bacterial translation during the initiation, elongation, termination, and recycling phases. We visualized multiple transcription-translation complexes, allowing us to propose a threading-based translation reinitiation mechanism, and to provide structural evidence for a long-hypothesized supercomplex that coordinates transcription, translation, and membrane attachment. We further resolved abundant membrane-associated large ribosomal subunits, and suggest that dissociation from membranes depends on the conditional initiation of new translation, consistent with a conserved mechanism in mammalian cells. This work visualizes the multilayered control of bacterial translation and demonstrates the power of in-cell structural biology to investigate regulatory circuits in gene expression.