Student Theses and Dissertations

Nathan Harper

Date of Award

2025

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Thesis Advisor

Sebastian Klinge

Abstract

The ribosome is the macromolecular machine responsible for all protein synthesis in the cell and is one of the ancient molecular machines involved in the transfer of genetic information to functional molecules. The ribosome is made up of two conserved ribonucleoprotein complexes: the large subunit, responsible for peptide bond synthesis, and the small subunit, responsible for interrogating the match between mRNA codon and tRNA anti-codon. The assembly of the ribosome is a complex process which is tightly controlled to generate functional subunits which exhibit high translational accuracy and processivity. During ribosome biogenesis, ribosomal proteins and a multitude of additional assembly factors, of which there are over 200 in yeast, orchestrate rRNA processing, folding, and modification. While the vast majority of proteins are made by cytoplasmic ribosomes in eukaryotes, a select few proteins are made by specialized ribosomes in the mitochondria. The existence of these mitoribosomes points to the evolutionary origin of these organelles as proteobacteria which formed a symbiotic relationship with a host archaeal cell. The mitoribosome is responsible for translation of mitochondrially encoded respiratory chain subunits, and therefore assembly of these ribosomes is a key part of cellular energy production. The mitoribosome retains highly conserved regions of bacterial and cytoplasmic ribosomes, but displays many structural and functional differences, most notably significant rRNA deletions and the expansion of the r-protein complement. Little is known about the assembly of the mitoribosome. While some assembly factors involved in the process have been identified, exactly how they work, how biogenesis is controlled, and how the assembly system evolutionarily relates to bacterial and eukaryotic systems, is unknown. To address this, we have used cryo-electron microscopy to determine six high resolution structures of human mitoribosomal small subunits undergoing native assembly (Chapter 2). In structurally characterizing this assembly pathway, we can now understand the role of assembly factors in controlling stepwise folding and modification of key functional centers in the mitochondrial rRNA such as the decoding center. In parallel, we solved structures of three similar mitoribosomal small subunit assembly intermediates from the yeast S. cerevisiae. By comparing these assembly pathways, we can connect ribosome structure with assembly mechanisms to highlight how mature structure and biogenesis are evolutionarily coupled (Chapter 3). Similarities in the human pathway to bacterial small subunit biogenesis spurred us to turn to E. coli to determine whether the conserved assembly factors across these two systems play analogous roles in assembly. In purifying and solving structures of endogenous intermediates from E. coli, we observed surprising aspects of the pathway which have been previously overlooked, including the involvement of a DNA-bending protein (Chapter 4). Together, this work provides the first insights into mitoribosomal small subunit biogenesis, revealing the structure and function of assembly factors, the role of novel players in ribosome assembly pathways, and key principles of small subunit assembly across life.

Comments

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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