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Date of Award
Master of Science (MS)
Spiegel, P. Clint
Smirnov, Sergey L.
The two billion years of evolution since the divergence of prokaryotes and eukaryotes has left earth with very few molecules conserved across these two domains of life. One such molecule, the ribosome, is an enormous ribonucleoprotein responsible for translation, the process of converting the information contained within an organism’s genetic code into functional proteins. Translation is facilitated by a number of other proteins, termed translation factors, required to catalyze the synthesis of these proteins. A large number of antibiotics prescribed today target either the ribosome or translation factors, and with increasing antibiotic resistance being found in infectious bacteria there is a greater need for understanding the ribosome and its associated molecules. While knowledge of the ribosome has increased considerably over the past two decades there are still many unknowns surrounding its transition states, how it interacts with translation factors, and even the role some of these translation factors play in the cell. A more thorough understanding of the interactions between ribosomes and translation factors will lead to the generation of more diverse and possibly more potent antibiotics for the treatment of bacterial infections.
The goals of this work were threefold. First, based on the work of previous Spiegel lab graduate student Justin Walter, we aimed to further characterize the role of the L12 ribosomal protein in activation of several translation factors that v utilize the hydrolysis of guanosine 5’-triphosphate (GTP), called GTPases, to exert their function. Walter had shown that removal of L12 led to decreased GTP hydrolysis by these proteins, but was unable to ensure L12 had been completely removed from the ribosomes. Here it is shown that when L12 is completely absent from functional prokaryotic ribosomes the GTPase activity of three translation factors, elongation factor G (EF-G), release factor 3 (RF3), and initiation factor 2 (IF2) all unequivocally cease, showing no activity beyond that of uncatalyzed GTP hydrolysis. A fourth translational GTPase, leader peptidase A (LepA), exhibited a different response, with activity dropping by effectively 50% upon the removal of L12. Reconstitution of these depleted ribosomes with externally purified L12 caused an unambiguous return to full activity for all investigated GTPases.
A second ambition of this work was to analyze the role of the L12 ribosomal protein in binding of translation factors. GTPase binding assays through ultracentrifugation demonstrated that absolute removal of L12 led to a nearly complete abrogation of binding between 70S ribosomes and EF-G, IF2, and RF3. LepA exhibited diminished binding in the presence of L12 deficient ribosomes, but maintained a level significantly above baseline. To further assess the effect of L12 depletion on binding, BioLayer Interferometry was utilized to quantitatively measure the binding affinity between EF-G and 70S or 70SΔL12. EF-G and 70S interactions fell within previously established KD values, averaging ~160 nM. Preincubation of EF-G with 70SΔL12 maintained this affinity, suggesting that little vi to no EF-G associates with depleted ribosomes, while preincubation of EF-G with intact 70S ribosomes caused a > 10,000 fold increase in the KD, indicating EF-G has a strong association with 70S ribosomes when L12 is present.
The final objective herein was to determine the roles of domains 4 and 5 and subdomain G’ of EF-G in the hydrolysis of GTP. EF-GΔ5 and EF-GΔ4,5 were previously produced in the Spiegel lab. Here it is shown that EF-GΔ4 and EF-GΔG’ are both expressed in the soluble fraction of E. coli cells and are readily isolated. The GTPase activity of each mutant relative to full length EF-G was calculated. EF-GΔ4 and EF-GΔ4,5 exhibited an activity of roughly 65% of wild type EF-G, suggesting the loss of the 4 domain confers the same disadvantage as the loss of the 4 and 5 domains. Meanwhile, EF-GΔ5 maintained 85% activity, showing the loss of the 5 domain is less detrimental to GTPase activity than either the Δ4 or Δ4,5 mutants. EF-GΔG’ confers a loss of around 90% activity compared to EF-G, suggestive of a crucial role of this domain in EF-G activity or binding, despite being absent in other homologous translational GTPases.
Western Washington University
Subject – LCSH
GTPase-activating protein; GTPase-activating protein--Inhibitors; Ribosomes--Structure; Guanosine triphosphatase; Catalysis; Genetic translation
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Carlson, Markus A., "Structural Requirements for Ribosome-Dependent GTPase Activity and Binding" (2015). WWU Graduate School Collection. 421.