Date of Award
Master of Science (MS)
Robert L. McFeeters
William N. Setzer
Antibiotics--Biotechnology., Genetic transcription., Hydrolases., Transfer RNA.
With more and more antibiotic-resistant infections occurring worldwide each year, it is imperative that a new class of antibiotics be developed in order for modern medical treatments to continue to advance. To do so effectively, new antibacterial targets must be found and analyzed for their potential as novel antibiotic drugs. Peptidyl-tRNA hydrolase I (Pth1) is potentially one such target. During protein translation, approximately 10% of the time the ribosome stalls. When this happens, and rescue mechanisms fail, the ribosome is disassembled. This leaves the nascent peptide attached to the P-site tRNA, creating peptidyl-tRNA. If peptidyl-tRNA is not recycled, the cell will run out of tRNA for protein translation, which leads to non-viability. Pth1 is employed to recycle peptidyl-tRNA from the cell so that protein translation can continue. As this is the only mechanism most bacterial species possess to perform this action, inhibition of this enzyme would lead to the accumulation of peptidyl-tRNA within the bacterial cell and eventual cell death. Coupled with not being essential in eukaryotes, Pth1 is a promising target for novel antibiotic development. In order to further advance development of Pth1 inhibitors, a high-resolution crystal structure of a bound complex is needed. Currently, apo structures, lower resolution data for partial substrates, and a small angle neutron scattering structure provides general information about the enzyme and enzyme-substrate complex, but not detailed binding interaction information, like an amino acid-specific peptidyl-tRNA in complex with Pth1 would. To assist in the production of a high-resolution crystal structure of the substrate bound complex, methods must be developed to procure amino acid-specific tRNA molecules in order to produce peptidyl-tRNA with selective peptide chains for further study and crystallization. It was the goal of this study to generate tRNAAla as a first step for specific peptide peptidyl-tRNA and then aminoacylate it. Both in vivo and in vitro methods were pursued, allowing for comparison of the methods in terms of efficiency of the reactions and overall yield. The in vitro production of tRNAAla involved a plasmid DNA purification protocol, restriction enzyme digest, and in vitro transcription reaction, while the in vivo production method involved induction of bacterial cultures and subsequent purification through precipitation reactions. Alanyl-tRNA synthetase (ARS875) was also produced in vivo, allowing production of aminoacyl-tRNA. Results of this study find that the in vivo methods reliably and efficiently produce large yields of both tRNAAla and ARS875. This method of production can now be used to produce selective peptidyl-tRNA for further binding studies, as well as production of a high-resolution crystal structure of the enzyme-substrate complex.
Byler, Brandie, "A comparative analysis of tRNA[superscript Ala] production" (2019). Theses. 284.