Student Theses and Dissertations

Author

Zhen Chen

Date of Award

2019

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Kapoor Laboratory

Abstract

Cellular proteins are synthesized by ribosomes, which are ~3 MDa macromolecular complexes comprised of four ribosomal RNAs and ~80 ribosomal proteins in yeast. The biogenesis of such complicated ribonucleoprotein complexes is a highly regulated, multistep process requiring a plethora of more than 200 unique assembly factors. Energy-harnessing enzymes, such as ATPases and GTPases, are needed to remodel the precursors of ribosomes at fast time scales. Mdn1 is an essential dynein-like AAA protein (ATPases Associated with various Activities) that releases specific assembly factors from the precursors of 60S subunit of ribosomes. However, Mdn1’s unusually large size (~5000 amino acids in a single polypeptide) and the transient nature of intermediates of ribosome biogenesis have limited our understanding how Mdn1 remodels pre-60S particles. In addition, the limited homology of Mdn1 to other well-studied proteins, including dyneins, has restricted our understanding of its function. Here, I first combined chemical and biochemical approaches to develop and validate ribozinoindoles (Rbins) as the cell-permeable inhibitors of Mdn1, which are the first potent and selective inhibitors of ribosome biogenesis in eukaryotes. These compounds can be further used to dissect the dynamic functions of Mdn1 during the multistep process of ribosome biogenesis. In addition, I solved three cryo-EM structures of both full-length and truncated Mdn1 (resolution up to 4.0 Å) that provided the first pseudo-atomic models for Mdn1 in two distinct nucleotide states. Remarkably, Mdn1’s the C-terminal MIDAS domain (Metal Ion-Dependent Adhesion Site), which interacts with other ribosome assembly factors, docks onto the N-terminal AAA ring in a nucleotide state-specific manner, even though they are separated by more than 2000 aa. These data suggest that conformational changes in the AAA ring can be directly transmitted to the MIDAS domain, thereby driving the selective release of the MIDAS-bound assembly factors from the precursors of 60S subunit of ribosomes. Together, these chemical biology, biochemical and structural studies of Mdn1 reveal how an AAA protein can contribute to the dynamic ribosome biogenesis process in eukaryotes.

Comments

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

Available for download on Friday, December 18, 2020

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