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Paul Kelly Dissertation Final.pdf (6.85 MB)

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Characterizing the Function of Alanyl-tRNA Synthetase Activity in Microbial Translation

Kelly, Paul Michael
http://rave.ohiolink.edu/etdc/view?acc_num=osu1586259003806337

Abstract Details

2020, Doctor of Philosophy, Ohio State University, Molecular, Cellular and Developmental Biology.
Across all domains of life, mechanisms have evolved to ensure faithful translation of the genetic code. One integral step in the transition from a nucleic acid encoded-genome to functional proteins is the aminoacylation of tRNA molecules. To perform this activity, aminoacyl-tRNA synthetases (aaRSs) activate free amino acids in the cell forming an aminoacyl-adenylate before transferring the amino acid on to its cognate tRNA. These newly formed aminoacyl-tRNA (aa-tRNA) can then be used by the ribosome during mRNA decoding. In Escherichia coli, there are twenty aaRSs encoded in the genome, each of which correspond to one of the twenty proteinogenic amino acids used in translation. Given the shared chemicophysical properties of many amino acids, aaRSs have evolved mechanisms to prevent erroneous aa-tRNA formation with non-cognate amino acid substrates. Of particular interest is the post-transfer proofreading activity of alanyl-tRNA synthetase (AlaRS) which prevents the accumulation of Ser-tRNAAla and Gly-tRNAAla in the cell. Upon mis-activation of the non-cognate serine or glycine, AlaRS will transfer the amino acid to the 3’ end of tRNAAla where it will translocate into a distinct editing active site in the enzyme. Recognition of the non-cognate substrate will lead to hydrolysis of the amino acid from the tRNA leaving both substrates available for subsequent rounds of aminoacylation. It has been previously shown that disruption of the cytosolic and mitochondrial AlaRS editing domains in mice leads to neurodegeneration and embryonic lethality respectively. Here we show that perturbation of the E. coli AlaRS editing domain causes gross perturbation to the E. coli proteome causing a variety of pleiotropic phenotypes including growth defects, motility impairment, and antibiotic sensitivity. Furthermore, we have identified second-site suppressors within the AlaRS editing domain that alleviate these defects. This work highlights the importance of AlaRS fidelity in E. coli and characterizes novel elements within this aaRS editing domain. Given the essentiality for maintaining protein synthesis, aaRSs have been promising targets for therapeutic discovery. Leishmania major is a eukaryotic parasite that infects upwards of a million individuals a year. Current therapeutic options remain limited and have adverse effects on humans. While antibiotics are able to target prokaryotic cellular components specifically, developing therapies against eukaryotic pathogens is more challenging due to conservation between potential pathogen drug targets and their host counterparts. Previous studies have identified anti-fungal therapies that target pathogen aaRS with some degree of specificity, indicating that these essential enzymes could be developed as drug targets for a variety of infections. Using bioinformatic approaches, we have identified several aaRSs, including AlaRS in L. major that appear to be potential targets for anti-leishmanial therapies. Preliminary biochemical analysis have supported these in silico predictions and led to the identification of novel aaRS inhibitors in vitro.
Michael Ibba, Ph.D. (Advisor)
Kurt Fredrick, Ph.D. (Committee Member)
Jane Jackman, Ph.D. (Committee Member)
Natividad Ruiz, Ph.D. (Committee Member)
181 p.
Microbiology; Molecular Biology
Mistranslation; Aminoacyl-tRNA Synthetases; AlaRS; tRNA

Recommended Citations

Citations

  • Kelly, P. M. (2020). Characterizing the Function of Alanyl-tRNA Synthetase Activity in Microbial Translation [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1586259003806337

    APA Style (7th edition)

  • Kelly, Paul. Characterizing the Function of Alanyl-tRNA Synthetase Activity in Microbial Translation. 2020. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1586259003806337.

    MLA Style (8th edition)

  • Kelly, Paul. "Characterizing the Function of Alanyl-tRNA Synthetase Activity in Microbial Translation." Doctoral dissertation, Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1586259003806337

    Chicago Manual of Style (17th edition)

osu1586259003806337
234
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