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Structural and mechanistic study of co-translationally interacting nascent chains

Project description

Decoding co-co assembly

An assembly of components put together at different stages leads to co-co assembly. What is more, co-co assembly is mostly employed for the formation of homo-oligomers and exists in all kingdoms of life. Still, very little is known about the molecular mechanisms that drive it. The EU-funded Co-coAssembly project will study mechanisms of co-co assembly using the dimeric chorismate mutase as a representative top candidate from a high throughput screen for co-co assembling protein complexes in E. coli. Employing special tomography, the study will focus on the three-dimensional arrangement of E. coli ribosomes to assess how the organisation of translational machinery allows co-co assembly.

Objective

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). An ongoing 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.
I propose to study 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, I will analyse 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, I will study the co-translational cascade of folding steps of chorismate mutase by utilizing FRET on in vitro prepared nascent chains. Finally, I plan to explore 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 DnaK and in mutants that synthesize proteins with reduced translation kinetics.

Keywords

Coordinator

RUPRECHT-KARLS-UNIVERSITAET HEIDELBERG
Net EU contribution
€ 174 806,40
Address
SEMINARSTRASSE 2
69117 Heidelberg
Germany

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Region
Baden-Württemberg Karlsruhe Heidelberg, Stadtkreis
Activity type
Higher or Secondary Education Establishments
Links
Total cost
€ 174 806,40