Date of Award
2024
Degree Type
Dissertation
Degree Name
Doctor of Philosophy in Biological and Environmental Sciences
Specialization
Cell and Molecular Biology
Department
Cell & Molecular Biology
First Advisor
Steven T. Gregory
Abstract
The ribosome is the instrument by which all biological life on earth translates a message of ribonucleic acid (mRNA) into proteins. The modern-day ribosome has evolved into a highly complex and exceedingly efficient macromolecular machine responsible for protein synthesis. The two primary functional activities of the ribosome, decoding and peptide bond formation, have been attributed to rRNA. While three rRNA bases have precisely-defined roles in decoding, no direct role for any RNA or protein residues within the active site has been established in catalysis of peptide bond formation.
This dissertation investigates the intramolecular RNA and intermolecular protein-RNA interactions within the ribosomal peptidyl transferase center (PTC) - the catalytic site of peptide bond formation - to broaden our understanding of their structural and functional implications for the mechanism of protein synthesis. Utilizing the extreme thermophile Thermus thermophilus as a model organism, I employ a variety of molecular techniques to generate a collection of 23S rRNA mutations and r-protein mutations within the active site of the bacterial ribosome. I also examine the impacts of these ribosomal mutations by characterizing the growth and fitness phenotypes compared to wild-type T. thermophilus. I also conduct directed evolution of the acquired rRNA mutations to assess the fitness landscapes and sequence alternatives of the PTC, providing insights into possible evolution pathways in the primordial active site. Further, I explore the innate robustness of the PTC applying the functional characterization of mutants and examining the structural implications of rRNA and r-protein mutations within the active site. Finally, I address one of the fields greatest quandaries by directly probing the necessity for r-protein-rRNA interactions within the PTC and demonstrate conclusively that no individual r-protein component is required for catalytic activity by the PTC. The work described in this dissertation provides my efforts towards defining the boundaries of the modern-day PTC.
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.
Recommended Citation
Killeavy, Erin, "DEFINING THE PEPTIDYL TRANSFERASE ACTIVE SITE OF THE RIBOSOME" (2024). Open Access Dissertations. Paper 1636.
https://digitalcommons.uri.edu/oa_diss/1636