The eukaryotic ribosome in focus : a chemical footprinting analysis of ribosome-ligand interactions in in vivo assembled complexes

Abstract: Protein synthesis is an essential biological process that occurs on ribosomes. The process requires an involvement of numerous components like mRNA, tRNA and translation factors. The mammalian 80S ribosome, the focus of this thesis, is composed of four rRNAs and approximately 80 proteins distributed in two subunits the 40S and 60S subunit. The rRNA itself plays an essential role in subunit association, tRNA selection, peptide bond formation, mRNA and translation factor binding to the ribosome. Furthermore, the rRNA undergoes structural changes during different phases of translation.During protein synthesis the ribosome usually interacts with more than one component at a time. Therefore, to understand the mechanism of translation in more detail, it is important to determine the interplay between the different translational components as well as to identify the functional sites within the ribosome that interact with the various ligands. However, the large number of components involved makes it difficult to identify the specific ligand binding site for each factor. Furthermore, the identification of various ligand binding sites is even more complicated as the ribosome is a dynamic particle that changes conformation during the protein synthesis. The flexibility of the eukaryotic rRNA was investigated by using ribosomal inactivating proteins (RIPs) ?-sarcin and ricin that modify neighbouring sites in the ?-sarcin/ricin-loop. Structural changes were seen in the active site, suggesting that the two RIPs locked the ribosome in two different conformations. Foot-prints from mRNA, tRNA and the nascent peptide were also identified in the active site of in vivo assembled translational active eukaryotic ribosomal complexes. Many of the footprints overlap with previously identified binding sites on the prokaryotic ribosome whereas some are unique. Thus, these studies show that the mRNA and tRNA binding sites are similar on eukaryotic and prokaryotic ribosomes as well as in in vivo and in vitro assembled complexes.During the course of this study it was observed that the secondary structure of a localized region in 18S rRNA in the 40S subunit deviated from the proposed secondary structure models. Similar modification patterns were observed in several different eukaryotic ribosomal complexes assembled in vivo. The data differ from modification experiments performed in prokaryotes and may be caused by differences in modification conditions.