Cotranslational Folding and Assembly at the Single-Molecule Level
K. Till (TU Delft - BN/Sander Tans Lab)
Sander J. Tans – Promotor (TU Delft - BN/Sander Tans Lab)
ME Aubin-Tam – Promotor (TU Delft - BN/Marie-Eve Aubin-Tam Lab)
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Abstract
The role of ribosome-bound chaperones and nascent chain interactions in protein biogenesis holds many open questions. This thesis provides new insights into the intricate mechanisms governing co-translational folding and complex assembly. By combining in vivo genome-wide screening results from selective ribosome and disome selective profiling, conducted by our collaborators B. Bukau and G. Kramer at Heidelberg University, with our in vitro single-molecule force spectroscopy and correlated confocal imaging results, we not only gained a better understanding of the general prevalence, but also of the mechanistic details of these cellular processes.
We investigated how Trigger Factor (TF), the only ribosome-associated chaperone in bacteria, influences nascent chain folding of a single-domain protein. We show for the first time that TF accelerates folding on the ribosome. This process is regulated by translation, with the emergence of key peptide segments dictating TF’s ability to compact nascent chains and stabilize partial folds. Beyond chaperone-modulated folding, we also explored how ribosome cooperation drives protein complex formation. Using the intermediate filament lamin as a model, we demonstrate that ribosome proximity enables nascent chains to ‘chaperone each other,’ facilitating coiled-coil formation while preventing misfolding. Notably, when early interactions between nascent chains are inhibited or delayed, they become trapped in misfolded states and are no longer assembly-competent. We further examined the role of timing in nascent chain interactions, using the BTB domain as a model to study the challenging formation of intertwined dimers during translation. We identify a translation-driven temporal control mechanism that ensures proper dimerization. This process opens otherwise inaccessible folding-assembly pathways, bypassing unproductive monomeric states.
Taken together, we have managed, through the powerful combination of in vivo and in vitro approaches, to gain new perspectives on how cells ensure faithful protein biogenesis.