Almost all fundamental biological processes involve protein complexes and therefore, efficient folding and assembly of homo- and hetero-oligomers is critical for cellular functionality and integrity. Recent studies have shown that many protein complexes assemble co-translationally by one fully-synthesized subunit engaging another subunit that is still in nascent state (co-post assembly). A recent study in the Bukau lab now revealed that assembly can also occur by interaction of two partner nascent chains (co-co assembly). Co-co assembly is mostly employed for the formation of homo-oligomers and exists in all kingdoms of life.
Despite initial evidence of its existence, very little is known about the molecular mechanisms driving co-co assembly. This includes information on whether co-co assembly requires co-localization of two polysomes or can happen on one polysome. Furthermore, it is currently unclear whether co-co interactions require preceding nascent chain folding steps and to what extent co-translationally acting chaperones coordinate the process and the impact of translation speed on co-co assembly.
In this project, we investigated how cellular translation machinery adapts to expression of proteins which assemble while being synthesized (i.e. co-translational assembly). This fundamental research broadens our view on cellular organization and homeostasis providing an expansion to the textbook knowledge of protein synthesis and provides possibility of wider applications of the results in the industry.
The overall goal of this project is to obtain structural and mechanistic information on a co-translational protein complex assembly mechanism that involves the interaction of two nascent polypeptides in bacteria. More specifically, we studied mechanisms of co-co assembly using the dimeric chorismate mutase (PheA) as a representative top candidate from a high throughput screen for co-co assembling protein complexes in E. coli. Employing cryo-electron tomography, we analyzed the three dimensional arrangement of E. coli ribosomes in the context of a polysome to assess how organization of translational machinery allows co-co assembly. Moreover, we investigated the co-translational cascade of folding steps of chorismate mutase by utilizing FRET on in vitro prepared nascent chains. Finally, we explored the impact of co-translationally acting chaperones and translation kinetics on co-co assembly, by performing disome-selective profiling analysis in chaperone mutant cells lacking Trigger Factor and in mutants that synthesize proteins with reduced translation kinetics.
