Contribution of Nascent Polypeptides of increasing Length to the Apparent Stability of the Bacterial Ribosome

Abstract

The role of the ribosome in the cell extends well beyond catalysis of peptide-bond formation. For instance, the ribosome is emerging as a key player in cotranslational protein folding. On the other hand, whether and how the ribosome assists nascent-protein folding, and how the nascent protein may in turn affect the ribosome, is poorly understood. Recent results suggest that the ribosome facilitatesprotein folding by directly interacting with nascent chains. Yet, whether interactions between the ribosome and the nascent chain have any influence on the ribosome, and whether these interactions promote proper protein folding is currently unknown. Via sucrose-gradient assays, we found that empty ribosomes are more sensitive to urea denaturation than ribosomal complexes carrying either aminoacyl-tRNA or nascent-chains. Further, by taking advantage of tryptophan fluorescence emission, we probed the effect of nascent chains of widely variable sequence and length on the apparent stability of ribosomal proteins (rProteins). We found that there is no statistically significant difference between the contribution of different nascent chains, suggesting that nascent-chain-to-rProtein interactions are either weak or thermodynamically compensated. Hence, there is no difference between the sensitivity to urea of ribosomal initiation complexes carrying aminoacyl tRNA and ribosomal complexes harboring peptidyl tRNA. in all, the above findings lead us to conclude that aminoacyl tRNA, likely in conjunction with mRNA, increases the apparent stability of the ribosome, while the nascent polypeptide does not. Finally, our data led us to propose a multi-step model for the disassembly of ribosome-nascent-chain complexes.