Description du projet
Décoder le co-assemblage
Un assemblage de composants réunis à différentes étapes aboutit au co-assemblage. Le co-assemblage est principalement employé pour la formation d’homo-oligomères et existe dans tous les règnes de la nature. Nous savons pourtant très peu de choses sur les mécanismes moléculaires qui l’entraînent. Le projet Co-coAssembly, financé par l’UE, étudiera les mécanismes du co-assemblage à l’aide de la chorismate mutase dimérique comme principale représentante issue d’un criblage à haut débit des complexes protéiques en co-assemblage d’E. coli. À l’aide d’une tomographie spéciale, l’étude sera axée sur l’agencement en trois dimensions des ribosomes d’E. coli pour déterminer la manière dont l’organisation de la machinerie translationnelle permet le co-assemblage.
Objectif
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.
Champ scientifique
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Programme(s)
Régime de financement
MSCA-IF - Marie Skłodowska-Curie Individual Fellowships (IF)Coordinateur
69117 Heidelberg
Allemagne