Student Theses and Dissertations

Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Klinge Laboratory


Ribosomes carry out one of the most fundamental functions of life - the translation of genetic information into functional proteins. The pivotal role of the ribosome in the cell is reflected in its immensely complicated and energy-consuming assembly pathway. The maturation of a eukaryotic ribosome involves more than 200 non-ribosomal factors and the activity of all three RNA polymerases. In yeast, ribosome biogenesis starts with the transcription of the 35S pre-ribosomal RNA in the nucleolus. This large RNA molecule contains three of the four ribosomal RNAs separated by several internal and external transcribed spacer regions. The 5’ external transcribed spacer (5’ETS) is the first RNA domain of the 35S pre-rRNA being transcribed. As it emerges from the RNA polymerase it is bound by UtpA, a 660 kDa complex consisting of 7 essential subunits in yeast. 9 By binding to the nascent pre-rRNA, UtpA triggers the association of multiple other proteins and complexes, which leads to the formation of the ~2 MDa 5’ ETS particle. As transcription continues through the ensuing small subunit rRNA gene more ribosome biogenesis factors as well as ribosomal proteins are recruited and the 5’ ETS particle evolves into the small subunit processome. The small subunit processome, a giant particle, unique and essential to eukaryotes, coordinates the cleavage of the 35S pre-rRNA to separate the maturation of the small and large ribosomal subunit. So far, a functional understanding of the initial events in ribosome biogenesis has been impeded by a lack of structural and biochemical data about the protein complexes facilitating this process and the pre-ribosomal particles they form. To gain mechanistic insights into these earliest steps we set out to delineate the role of UtpA as first building block, vital structural component and organizer of the 5’ ETS particle and the small subunit processome. By using protein-protein and RNA-protein cross-linking techniques combined with negative stain electron microscopy and biochemical assays we were able to define the composite RNA binding site of UtpA and characterize its molecular architecture in the absence of high-resolution structural data (Chapter II). Subsequent structure determination of the small subunit processome by cryoelectron microscopy has not only provided the first fully assigned atomic model of UtpA but visualized how ribosome biogenesis factors keep the ribosomal RNA domains in spatially separated compartments of this large particle (Chapter III). In the small subunit processome, the 5’ ETS particle forms the base onto which the segregated ribosomal RNA domains are folded. To investigate whether the 5’ ETS particle serves as a structural mold for the maturing rRNA domains during earlier assembly stages, we solved the cryo-EM structures of the 5’ ETS particle in intermediates preceding the formation of the small subunit processome (Chapter IV). Combined with the in vivo analysis of artificial pre-rRNA fragments, the architecture of the 5’ ETS particle shows that the initial steps of ribosome assembly are governed by the functional independence of all rRNA domains and the 5’ ETS particle. Completion of ribosomal gene transcription then leads to a conformational change in the 5’ ETS particle and small subunit processome formation. In summary, our work provides structural snapshots and biochemical information on more than 50 ribosome assembly factors during different stages of the initiating steps in eukaryotic ribosome biogenesis. These data form the basis for a three-dimensional model of these essential events in the eukaryotic cell.


A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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