Student Theses and Dissertations

Date of Award

2024

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Thesis Advisor

Sebastian Klinge

Keywords

mitochondrial ribosome, small subunit (mtSSU), assembly intermediates, cryo-electron microscopy (cryo-EM), assembly factors, evolutionary comparison

Abstract

Mitochondrial ribosomes (mitoribosomes) synthesize proteins encoded within the mitochondrial genome that are assembled into oxidative phosphorylation complexes. As such, the biogenesis of mitoribosomes is essential for ATP production and cellular metabolism. Despite significant progress in elucidating the structures of mature mitoribosomes, a comprehensive understanding of their assembly pathways remains elusive. This thesis presents the first structural characterization of assembly intermediates of the mitochondrial small subunit (mtSSU) in eukaryotes. To gain mechanistic insight into the process of SSU assembly in the mitochondria, I set out to enrich and characterize intermediates of SSU assembly in the mitochondria of S. cerevisiae. By employing a combination of endogenous tagging, affinity purification, mass spectrometry, and cryo-electron microscopy, three distinct assembly intermediates (States 1-3) were successfully visualized at resolutions ranging from 3.2 to 3.8 Å, revealing the stepwise progression of mtSSU biogenesis. These structures unveiled the roles of previously uncharacterized assembly factors Rsm22 and Ccm1. Initial biochemical analysis of additional intermediates suggests the role of factors Mtg3 and Rmd9 at earlier stages of assembly (Chapter 2). Parallel work in H. sapiens was completed by Nathan Harper allowing the visualization of six assembly intermediates at resolutions ranging from 2.4 Å to 3.0 Å. These structures provide insight into the functions of assembly factors NOA1, TFB1M, RBFA, ERAL1, METTL17, and MCAT and allow visualization of the early stages of mtSSU assembly in humans for the first time (Chapter 3). Comparative analysis of assembly pathways in these 2 key species (Chapter 4) reveal conserved and unique features of mtSSU assembly in both species. By comparing conserved mechanisms of assembly within the mitochondria of these 2 species to their bacterial homologs, we shed light into the evolution of ribosome assembly in the mitochondria. This thesis significantly advances our understanding of mtSSU assembly mechanisms in yeast and humans and lays the foundation for understanding the evolutionary trajectory of SSU assembly across different species.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

License and Reuse Information

Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.

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