dmwilson_phd_bio_2020 (1).pdf (30.66 MB)
Assembly of Functional Centers in the Ribosomal 60S Subunit
thesisposted on 2021-04-14, 18:12 authored by Daniel WilsonDaniel Wilson
Ribosomes are evolutionarily ancient nanomachines that are composed of ribosomal RNA (rRNA) and ribosomal proteins (r-proteins), and function to translate the genetic code in the form of mRNA to the functional units of a gene, proteins. Carrying out this task with speed and accuracy requires the rRNA to be folded and positioned precisely so that it can catalyze the reactions necessary to produce proteins. Therefore, the many complex steps involved in assembling ribosomes can be thought of as being centered around one goal; make sure that the rRNA matures properly. R-proteins and assembly factors are proteins that evolved to ensure that the rRNA matures and stabilizes into functional centers, which carry out the various tasks of translation. For example, the small (40S) subunit of the ribosome contains the decoding center, which translates the genetic code in mRNA, while the large (60S) subunit contains the peptidyl transferase center (PTC), which synthesizes proteins. Answering questions concerning how these functional centers form and mature in eukaryotic cells has only become possible in recent years, thanks to advancements in cryo-electron microscopy (cryo-EM), a technique that enables researchers to visualize immature ribosomes in their native states. My work discussed in this dissertation focuses on how the nascent polypeptide exit tunnel (NPET), another functional center of the 60S subunit, is assembled in the baker’s yeast, Saccharomyces cerevisiae. This functional center acts as a passageway for newly synthesized protein chains to thread out of the large subunit and into the cytoplasm of the cell, where the protein then begins to fold. It is also the target of many antibiotics, including erythromycin. Far from being a passive tunnel, the NPET actively interacts with proteins as they are being synthesized. These interactions are crucial in guiding early folding stages of proteins as well as situational control of gene expression. By mutating r-protein L4 and assembly factors Nog1, Rei1, and Reh1 found inside the NPET and studying the mutant immature ribosomes using biochemistry and cryo-EM, my work revealed that a particular rRNA helix in the NPET (H74) must mature properly for the large subunit to complete maturation. These studies were the first to use cryo-EM to study how a mutation in a functional center affects ribosome assembly. The model I have built from these data has laid a foundation for future exploration of how functional centers are constructed during ribosome assembly.
- Biological Sciences
- Doctor of Philosophy (PhD)