Date of Award
Doctor of Philosophy (PhD)
The ribosome is a complex macromolecule responsible for the synthesis of all proteins in the cell. In yeast, it is made of four ribosomal RNAs and 79 proteins, asymmetrically divided in a small and large subunit. In a growing yeast cell, more than 2000 ribosomes are assembled every minute. The ribosome is assembled through a highly complex process involving more than 200 trans-acting factors. Ribosome assembly begins in the nucleolus where RNA polymerase I transcribes a long polycistronic RNA, the 35S preribosomal RNA which contains the sequences for three of the four ribosomal RNAs, as well as spacer sequences which are transcribed and removed during assembly. Ribosome assembly factors bind co-transcriptionally to the nascent chains of preribosomal RNA and coordinate its correct folding, modification and cleavage. While most ribosome assembly factors have been identified, the function of numerous factors is still unknown. The timing of their involvement in ribosome assembly has not been characterized, limiting our understanding of their function. To further our knowledge of this essential cellular process, we set out to characterize the order of assembly of ribosomal assembly factors on the first half of the nascent preribosomal RNA, which forms the earliest precursors of the small subunit (Chapter II). Moreover, using state-of-the-art cryo-electron microscopy we determined the structure at near-atomic resolution of the earliest yet intermediate of small subunit assembly, the small subuit processome (Chapter III). The combined insights from the structure of this early intermediate and co-transcriptional assembly of pre-ribosomal complexes has redefined our understanding of early ribosome assembly. The cell invests more than 70 factors into the early events of small subunit assembly, with a combined molecular weight of more than 5 megadalton, four times the size of the mature small subunit. This impressive number of factors form a structural blueprint for the spatial segregation and individual maturation of the domains of the 18S. Ribosome assembly factors perform a multitude of functions within this platform, including specific modification of the ribosomal RNA and the concerted coordination of important RNA elements. We have also determined a new mechanism by which cells regulate ribosome biogenesis in response to nutritional depletion. We finally set out characterize the timing of recruitment of assembly factors to the second half of the 35S pre-ribosomal RNA, which leads to the formation of the earliest large subunit precursors (Chapter IV). This has allowed us to complete a new model for the co-transcriptional assembly of pre-ribosomal complexes. Our work has not only provided new insights into the role of more than 100 factors of early ribosome biogenesis but will serve as a platform for the further characterization of individual factors, as well as the regulation of ribosome assembly.
Chaker-Margot, Malik, "Making Ribosomes: Biochemical and Structural Studies of Early Ribosome Biogenesis in Yeast" (2018). Student Theses and Dissertations. 472.