Cotranslational Folding and Assembly at the Single-Molecule Level

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 as-
sembly. By combining in vivo genome-wide screening results from selective ri-
bosome 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 for-
mation. 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.