Nascent Proteins Interact with Key Regions of the Outer Surface of the Ribosome

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While in vitro protein folding is reasonably well understood, very little is known about how nascent chains achieve their native state in the complex cellular environment. The earliest stages of protein folding as nascent chains emerge out of the ribosome are still poorly characterized. Proteins and rRNA located within the ribosomal exit tunnel and outer surface, for instance, may interact with nascent proteins and affect their folding. Previous studies in the Cavagnero group showed that ribosome-bound nascent chains (RNCs) derived from the intrinsically disordered protein PIR and its highly negatively charged variants exhibit a significant spatial bias, consistent with the presence of non-covalent interactions between RNCs and the ribosomal surface. In this work, we probe RNC-ribosomal-protein contacts by a combination of chemical crosslinking and Western blotting, and show direct evidence for well-defined interactions between RNCs of PIR and specific ribosomal proteins. Surprisingly, the interacting ribosomal proteins are clustered only on one side of the ribosomal surface, relative to the exit tunnel. The observed interactions are particularly strong when about 15 amino acids have emerged outside of the ribosomal exit tunnel, and get weaker as the chain gets longer. This result suggests that the interactions are targeted towards the prevention of co-translational aggregation. This work corroborates an emerging view according to which the ribosome plays a fundamental role in protein biogenesis that extends well beyond the mere catalysis of peptide bond formation.