Effect Of Nascent Proteins On The Stability Of The Ribosome

Recent studies showed that many nascent proteins begin folding during translation, sometimes within the vestibule of the ribosomal exit tunnel. On the other hand, how and whether the ribosome assists protein folding, and how protein folding may in turn affect the ribosome, is poorly understood. We recently detected interactions between foldable and intrinsically disordered nascent proteins (not bearing an N-terminal signal sequence) and ribosomal proteins by dynamic fluorescence depolarization in the frequency domain and by chemical cross-linking. These results suggest that the ribosome facilitates in vivo protein folding by directly interacting with nascent chains. Whether interactions between the ribosome and the nascent chain have any thermodynamic influence on the ribosome, and whether these interactions promote proper protein folding however, is not known. Via sucrose-gradient assays, we found that empty ribosomes are less stable than ribosomes carrying tRNA, or ribosomes harboring nascent-chains. In addition, by taking advantage of tryptophan fluorescence emission, we probed the effect of several nascent chains of variable sequence and length on the stability of ribosomal proteins. We found that there is no statistically significant difference between the contribution of different nascent-chain constructs, suggesting that nascent-to-ribosomal protein interactions are either weak or thermodynamically compensated. Hence, there is no apparent difference between the overall stability of ribosomes carrying a tRNA and ribosomes harboring a nascent-chain. In all, the above findings lead us to conclude that tRNA stabilizes the ribosome, while nascent proteins do not. Therefore, intriguingly, the interplay between the ribosome and nascent chains does not critically rely on standard-state net free-energy changes. Finally, our data led us to propose a multi-step model for the disassembly of ribosome-nascent-chain complexes.