Nascent-Chain Interaction Networks and Their Effect on the Bacterial Ribosome

In order to become bioactive, proteins need to be biosynthesized and protected from aggregation during translation. The ribosome and molecular chaperones contribute to both of these tasks. While it is known that some ribosomal proteins (r-proteins) interact with ribosome-bound nascent chains (RNCs), specific interaction networks and their role within the ribosomal machinery remain poorly characterized and understood. Here, we find that RNCs of variable sequence and length (beyond the 1st C-terminal reside) do not modify the apparent stability of the peptidyl-transferase center (PTC) and r-proteins. Thus, RNC/r-protein interaction networks close to the PTC have no effect on the apparent stability of ribosome-RNC complexes. Further, fluorescence anisotropy decay, chemical-crosslinking and Western blots show that RNCs of the foldable protein apoHmp1-140 have an N-terminal compact region (63-94 residues) and interact specifically with r-protein L23 but not with L24 or L29, at the ribosomal-tunnel exit. Longer RNCs bear a similar compact region and interact either with L23 alone or with L23 and another unidentified r-protein, or with molecular chaperones. The apparent strength of RNC/r-protein interactions does not depend on RNC sequence. Taken together, our findings show that RNCs encoding foldable protein sequences establish an expanding specific interaction network as they get longer, including L23, another r-protein and chaperones. Interestingly, the ribosome alone (i.e., in the absence of chaperones) provides indiscriminate support to RNCs bearing up to ca. 190 residues, regardless of nascent-chain sequence and foldability. In all, this study highlights the unbiased features of the ribosome as a powerful nascent-protein interactor. Significance StatementThe presence of interactions between nascent chains bearing a foldable amino-acid sequence (with no signal or arrest tags) and specific ribosomal proteins has never been experimentally demonstrated, up to now. Here, we identify the ribosomal protein L23 as a specific nascent-chain interacting partner. We show that L23 establishes noncovalent contacts with nascent chains of the multi-domain foldable model protein apoHmp, which lacks signal/arrest sequences. Interactions with another ribosomal protein and with the trigger-factor and Hsp70 chaperones were also detected. Interestingly, ribosomal-protein/nascent-chain complexes have similar apparent stability, in the case of nascent chains of variable sequence and degree of foldability. These findings are significant because they advance our knowledge on ribosome-mediated nascent-protein interaction networks and suggest avenues to prevent undesirable aggregation.