Nascent-chain interaction networks and their effect on the bacterial ribosome.

Abstract

In order to become bioactive, proteins need to be biosynthesized and protected from aggregation during translation. Remarkably, the ribosome contributes to both of these tasks. Some ribosomal proteins (r-proteins) are known to noncovalently interact with ribosome-bound nascent chains (RNCs). Yet, specific interaction networks and their role within the ribosomal machinery remain poorly characterized. Here, we found that empty ribosomes are more sensitive to urea denaturation than ribosomes harboring nascent protein chains, including aminoacyl or short peptidyl-tRNA (snc-tRNA). This result suggests that snc-tRNAs play a ribosome-stabilizing role. On the other hand, RNCs of variable sequence and length do not significantly modify the apparent stability of the peptidyl-transferase center (PTC) and r-proteins, suggesting that RNC/r-protein interaction networks close to the PTC have a negligible effect on the apparent ribosome stability. Fluorescence anisotropy decay, chemical-crosslinking and Western blots show that 140-residue-long RNCs of the foldable protein apoHmp have an N-terminal compact region (63-94 residues) and interact with r-protein L23 but not with L24 or L29. Longer RNCs bear a similar compact region and interact either with L23 or with L23 and another unidentified r-protein or with molecular chaperones. Interestingly, the apparent stability of RNC/r-protein complexes does not depend on RNC sequence or length. Taken together, our findings show that RNCs establish preferential interactions with the L23 ribosomal protein whose strength does not depend on RNC amino-acid sequence and length. As the nascent chain elongates, the interaction network extends to additional r-proteins and chaperones. It follows that the ribosome provides uniform, indiscriminate support to nascent proteins regardless of their sequence and foldability. This result highlights the mode of action of this unbiased molecular machine for cellular health.