Structural and functional studies of the ribosomes : a closer look at expansion segments and translational dynamics in eukaryotes

Abstract: The ribosome is the basic machinery for protein synthesis in all living cells. In the last decade, there has been an explosion of structural information on the ribosome. The eukaryotic ribosome is larger than the prokaryotic counterpart and contains several extra features, even though they have similar core structures. The major size and shape differences are due to added ribosomal proteins and the eukaryote specific expansion segments in ribosomal RNA. In this thesis, the secondary structure of several expansion segments was determined in an effort to explore the structural, and possibly functional, differences between prokaryotic and eukaryotic ribosomes. An experimental modeling method was chosen, since the exclusive use of phylogenetic covariation analysis results in inconclusive structures, due to the variability of the segments. A wide range of chemical and enzymatic modification reagents was used to probe the ribosomal RNA. The experimental data was combined with phylogenetic alignments, and applied in computer modeling based on energy minimization. Secondary structure models are presented for ES15 and ES39 in 28S rRNA from mouse, rat and rabbit, in addition to wheat in the latter case. Models are also proposed for V4 in 18S rRNA from mouse and wheat. Hopefully, these models can in time be fit into the three-dimensional ribosome structures and give us an indication of the role that expansion segments play in eukaryotic protein synthesis.Experimental probing of the rRNA and analysis of the three-dimensional models of the ribosome has made it clear that the ribosome exhibits inherent dynamic characteristics during translation. The flexibility is especially evident during the elongation cycle. Disruption of the ability of the ribosome to progress through different conformations during elongation destroys the ribosome's capacity to add new amino acids to the growing peptide chain. For instance, the function of elongation factors eEF2 and eEF1 is affected by the ribosome inactivating proteins ricin and -sarcin, respectively. Native ribosomes were treated with these inhibitors, which resulted in modification patterns that only partially overlap. This indicates that the different effects of the two inhibitors in ribosomal function are reflected in different effects on the conformation of ribosomal RNA. This is especially true at the GTPase activating center of the ribosome, i.e. the interaction site of the elongation factors.