Periodic Reporting for period 1 - Co-coAssembly (Structural and mechanistic study of co-translationally interacting nascent chains)
Période du rapport: 2020-05-01 au 2022-04-30
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.
i) Using cryoelectron microscopy to investigate the organization of polysomes expressing co-co candidate
Work performed:
a) prepared constructs expressing co-translational dimerizing and non-dimerizing co-co assemblying candidate
b) Assessed the amount of gene expression upon overexpression
c) Prepared, measured, and analyzed tomograms of overexpressing E.coli cells
Results:
1) E.coli polysomes undergo small rearrangement of conformation upon expressing co-translationally dimerizing gene
2) This leads to transient configuration of ribosomes where the polypeptide exit tunnels get into close proximity
ii) Utilizing FRET to study the folding cascade of co-co assemblying nascent chain
Work performed:
a) Established a FRET assay on stalled nascent chains using a fluorescent protein and fluorescently modified amino acid incorporated via stop codon suppression
b) Recorded and analyzed multiple samples of co-co assembling nascent chain with different chain length to stimulate different stages of translation
c) Established an enzymatic assay on PheA dimerization domain
Results:
1) Formation of secondary structure of the PheA dimerization domain starts deeply inside the polypeptide exit tunnel
2) Fraction of folded nascent chain increases in the later stages of translation
3) Folding of the dimerized PheA is native like
iii) Investigating the role of chaperones and translation speed on co-co assembly
Work performed:
a) Prepared and analyzed disome-selective ribosome profiling sequencing libraries of cells with variable translation speed
b) Analyzed results from disome-selective ribosome profiling sequencing libraries form wild-type and Trigger Factor-deleted strains
Results:
1) Little to no impact of translation speed variability on the efficiency and onset
2) Only very minor impact of the chaperone trigger factor on assembly efficiency and onset
Dissemination of results:
1) Review in Frontiers in Molecular Biosciences (doi: 10.3389/fmolb.2021.689755)
2) Contribution to recent publication in Cell Reports (doi: 10.1016/j.celrep.2022.111776)
3) Poster presentation at the Complex Life of RNA conference, Heidelberg, October 2022