Molecular drivers of cotranslational protein assembly in bacteria

Protein complexes are pivotal to all major cellular processes. Translation-coupled complex assembly is ubiquitous in eukaryotes, but its molecular basis and prevalence in bacteria remain poorly understood. Here, we combine ribosome profiling and single-molecule optical tweezers to establish principles governing cotranslational assembly of four structurally diverse complexes in Escherichia coli. We reveal how heterotetramers form cotranslationally via hierarchical assembly pathways, with initial heterodimer formation priming the assembly of higher-order oligomers through a topology-dependent organization of homomeric and heteromeric interfaces. Co-post assembly, defined as interaction between nascent and fully synthesized subunits, initiates when dimerization domains are completely exposed at the ribosome. The assembly order is predominantly unidirectional and matches the operon gene order, with upstream-encoded subunits being synthesized first, and then engaging downstream nascent partner subunits. The co-post assembly efficiency depends both on the native operon structure and the ribosome-associated chaperone trigger factor, which binds emerging oligomerization domains and whose dissociation marks the onset of partner subunit engagement. The handover from chaperone engagement to cotranslational assembly is mediated by nascent chain folding. Finally, we show that co-post assembly provides a fitness advantage. Together, these findings reveal how genome organization and chaperone-assisted folding coordinate cotranslational assembly to promote efficient complex biogenesis and cellular fitness in bacteria.