Date of Award

Fall 9-2016

Embargo Period


Degree Type


Degree Name

Doctor of Philosophy (PhD)


Biological Sciences


John L. Woolford, Jr.


Ribosome assembly is a dynamic process that involves the concurrent folding and processing of pre-rRNA upon association of ribosomal proteins (r-proteins), and the binding, function and release of assembly factors. These remodeling events exert their function as timers and switches for the progression of ribosome assembly. Using combined approaches of molecular genetics, proteomics and structural biology, we were able to address several major questions currently faced by those who study ribosome assembly in eukaryotes. The crystal structure of mature yeast ribosomes revealed the exact locations of r-proteins, especially how they interact with rRNA. However, this snapshot of the final product did not provide information on the timing of association of r-proteins with pre-ribosomes and the roles for their rRNA interactions in assembly. In Chapter 2, I describe experiments that establish the hierarchy of ribosome assembly by analyzing the depletion phenotypes of eight r-proteins from the large subunit, as representatives of experiments conducted on 32 large subunit r-proteins. Our results indicate that the solvent interface of the pre-ribosome is stabilized first, followed by the polypeptide exit tunnel, and finally, the central protuberance and subunit-interface. Besides r-proteins, about 75 assembly factors were identified to mediate assembly of 60S ribosomal subunits. In order to dissect the functions of interactions between assembly factors and rRNA or r-proteins, we needed highresolution structural data showing the exact locations of assembly factors in v pre-ribosomes. In Chapter 3, I analyze the near atomic resolution cryo-EM structures of Nog2-associated pre-ribosomes. Besides revealing the locations of about 30 assembly factors, these structures enabled visualization of the three major remodeling events that occur during late nuclear stages of ribosome assembly. One striking feature revealed by the crystal structure of mature ribosomes is that many r-proteins contain eukaryote-specific extensions. Although the functions of these extensions were not clear, their intrinsic properties, such as structural flexibility and ability to reach long distances in ribosomes, suggested their involvement in the sequential binding of r-proteins to pre-ribosomes. Previous ribosome reconstitution studies pointed to sequential binding of r-proteins, where upon initial contact, r-proteins and rRNA form encounter complexes that are stabilized later, as assembly proceeds. In Chapter 4, I establish that portions of r-proteins are stabilized at different stages of assembly, based on the rRNA domain with which they interact. In addition, through the clues provided by the cryo-EM structures, I suggest a communication paradigm mediated by the eukaryote-specific N-terminal extension of L8 during late nuclear stages of assembly. Finally, we and others identified a drastic remodeling event, an ~180° rotation of the central protuberance, that happens shortly before nuclear export of pre-ribosomes. The mechanism of this rotation was not known. In Chapter 5, I provide multiple testable hypotheses on the mechanism of rotation of the central protuberance, based on interactions that are broken or established upon rotation.

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