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Turk, Martin (2016): Architecture of the 90S pre-ribosome complex from C. thermophilum. Dissertation, LMU München: Fakultät für Chemie und Pharmazie



Assembly of a single ribosome is a complex task, which requires in eukaryotes the concerted action of over 200 non-ribosomal factors and a large number of snoRNAs. The pre-rRNA undergoes co-transcriptional processing, modifications and cleavage events, which separate the biogenesis pathways of the small and large ribosomal subunits at an early stage in the nucleolus. Despite the biochemical characterization of the majority of the involved factors it is not known how these components are structurally arranged in the 90S pre-ribosome, the earliest pre-ribosomal particle in the biogenesis pathway. My work showed the cryo-EM structure of the 90S pre-ribosome from C. thermophilum at 7.3 Å average resolution, revealing a perforated particle comprised of 19 β-propellers located mainly at the periphery and intertwined with dsRNA helices. Most of the electron density could be accounted for and assigned to the 90S sub-complexes UTP-A, UTP-B, Mpp10, Rcl1/Bms1, U3 snoRNP as well as the partially folded pre-18S rRNA in a head-down orientation, thereby providing an overall architecture of the 90S complex. At the foot of the ct90S we identified the UTP-A sub-complex, which binds the first dsRNA helices of the nascent rRNA transcript at the 5’ end of the 5’ETS. The growing transcript then interacts with UTP-B components and hybridizes with the 3’ hinge region of the U3 snoRNP in the body part of the complex. Following the 5’ETS sequence further we encounter the pre-18S rRNA, which will ultimately form the RNA core of the small (40S) ribosomal subunit. With respect to the 90S the pre-40S is oriented upside down, pointing towards the core of the 90S. We find the 5’ domain of the pre-40S to a large extent developed and resembling the mature conformation, albeit requiring a final compaction step. Part of the central domain platform is folded, but shifted with respect to its mature position relative to the 5’ domain. The head is least resembling its mature state and could be partially identified on the basis of the corresponding bound r-proteins at their mature positions and Emg1, known to modify the pre-18S rRNA in that region. The gradual development of the 18S from 5’ domain first and 3’ end last is similar to how ribosomes assemble in bacteria. Taken together the data provide the first structural view into the architecture of 90S pre-ribosome, and will serve as solid foundation for guiding further biochemical experiments as well as structural studies.