Descripción del proyecto
Descodificación del cocoensamblaje
El ensamblaje de los componentes juntos en diferentes etapas resulta en el cocoensamblaje. Además, el cocoensamblaje se emplea principalmente en la formación de homooligómeros y existe en todos los reinos de la vida. Sin embargo, se sabe muy poco sobre los mecanismos moleculares que lo impulsan. El proyecto Co-coAssembly, financiado con fondos europeos, estudiará los mecanismos del cocoensamblaje usando la enzima dimérica corismato mutasa como la principal candidata representativa de un cribado de alto rendimiento de los complejos enzimáticos de cocoensamblaje en «E. coli». El estudio, que utilizará una tomografía especial, se centrará en la disposición tridimensional de los ribosomas de «E. coli» para evaluar cómo la maquinaria traslacional permite el cocoensamblaje.
Objetivo
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.
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MSCA-IF - Marie Skłodowska-Curie Individual Fellowships (IF)Coordinador
69117 Heidelberg
Alemania