Structural basis for co-translational assembly of homo-oligomeric proteins in cis and in trans

The efficient formation of protein complexes is essential for cellular function. Across all kingdoms of life, protein complexes frequently assemble co-translationally, either when one subunit is fully synthesized before interaction with nascent partner subunits (co-post) or when multiple nascent subunits interact co-translationally (co-co), with homomeric proteins particularly enriched among co-co assembling complexes. However, it is unknown whether co-co assembly of homomers occurs on the same mRNA in cis or across neighbouring mRNAs in trans, and how ribosomes spatially organize to enable co-co assembly. Using E. coli homodimeric protein PheA as a proof-of-concept model, we show by ribosome profiling and cryo-EM structural analysis that it employs the co-co assembly route. Co-co assembly of PheA is facilitated by the proximity of polypeptide exit tunnels, but does not rely on fixed ribosomal orientations. Surprisingly, PheA was identified as an exceptional candidate where trans-assembly is potentially the primary mode of PheA co-co assembly, generating large polysomal networks for PheA synthesis. Cis-assembly of PheA was less frequent and occurred mainly between non-adjacent ribosomes on the mRNA due to spatial restraints imposed by the arrangement of directly neighbouring ribosomes in polysomes. These findings reveal fundamental principles of how cells structurally organise fast and efficient co-translational protein complex assembly.