Summary 🇬🇧
Ribosomal protein biosynthesis (translation) is a crucial process in all domains of life. This work aims to investigate the process of translation in the mammalian system by means of single particle cryogenic electron microscopy (cryo-EM). The focus of this thesis lies on the following two aspects of mammalian translation:
1) Translocation by the mammalian cytosolic 80S ribosome. Translocation moves the tRNA2•mRNA module directionally through the ribosome during the elongation phase of translation and is associated with large scale conformational changes within both the ribosome and the bound tRNAs. It is catalyzed by the GTPase eEF2 (EF-G in bacteria). Although knowledge on translocation, especially in the bacterial system, has accumulated in the past years, the detailed mechanisms are not fully understood. In particular, the role of GTP hydrolysis is controversial and structural knowledge on translocation in the mammalian system has been missing.
In this work, three high-resolution structures of in vitro reconstituted authentic intermediates of translocation by the mammalian 80S ribosome are presented. They are trapped by the non-hydrolysable GTP analog GMPPNP and contain, in contrast to similar experiments in the bacterial system, the translocase eEF2 and a complete tRNA•mRNA module. Single-molecule imaging, carried out in collaboration with Prof. Scott Blanchard and colleagues, revealed that GTP hydrolysis principally facilitates rate-limiting, late steps of translocation, consistent with the presented cryo-EM structures. Comparison with the bacterial system showed that distinctions between bacterial and mammalian translocation mechanisms originate from differential dissociation rates of deacylated tRNA from the E site.
Further, a cryo-EM structure of a mammalian 80S ribosome containing a complete tRNA2•mRNA module and eEF2•GDP is presented, which stems from a sample prepared by in vitro translocating a PRE complex using eEF2•GTP. In contrast to the GMPPNP-stalled translocation intermediates, this structure gives insight into the interaction of unstalled eEF2 with the 80S ribosome.
2) The influence of serum on the energy landscape of mammalian translation and on the structure of ribosomal protein eS6. Serum treatment of cells intervenes with many signaling pathways, but it is not known if the energy landscape of translation is altered upon its influence. Serum deprivation and restimulation can be used as a model system to diminish and enhance phosphorylation of ribosomal protein eS6, which is a eukaryote-specific protein on the small ribosomal subunit. The phosphorylation of the C-terminus of eS6 has been investigated since a long time, however, its mechanistic role has not been elucidated yet. In particular, hardly anything is known on possible structural impacts of eS6 phosphorylation.
The presented work reveals that serum deprivation and restimulation do not have an impact on the energy landscape of translation for the ex vivo derived cytosolic fraction of polysomes. However, the observation of different yields of cell lysate from serum deprived and restimulated cells led to the proposition of a new hypothesis that suggests cellular redistribution of ribosomes. The phosphorylation of ribosomal protein eS6, which strongly correlates with serum treatment, does not lead to observable structural changes in the small ribosomal subunit.
Finally, the structural analysis and in silico sorting of the obtained translation intermediates led to the identification of two previously not observed substates of the 80S rotated PRE ribosome and to the unprecedented visualization of two distinct, native inititation complexes.
