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Master's thesis final 7-22-16.pdf (4.06 MB)

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Variants of Human Lysyl-tRNA Synthetase: In vitro Activity and Relevance to Human Disease

McVey, Chase A
http://rave.ohiolink.edu/etdc/view?acc_num=osu1469113725

Abstract Details

2016, Master of Science, Ohio State University, Chemistry.
Aminoacyl-tRNA synthetases are an ancient family of enzymes that maintain the fidelity and efficiency of translating the cell’s genetic code. Specifically, they are responsible for the correct attachment of amino acids to their cognate tRNA molecules for use in ribosomal peptide synthesis. Erroneous placement of amino acids onto tRNAs, known as mischarging, can lead to incorrectly incorporated amino acids, misfolding, or degradation of protein products. This study examines several single nucleotide mutations in the human KARS gene which codes for lysyl-tRNA synthetase (LysRS). These mutations have been detected in patients and associated with severe neurodegenerative and sensory disabilities. By purification and in vitro steady-state aminoacylation experiments, it was revealed that the mutant LysRS proteins exhibit varying levels of reduced enzymatic activity. The decrease in activity may be due to disrupting LysRS dimerization in solution, altering contacts between the synthetase and the tRNA, or directly affecting active site residues. This could potentially lead to a build-up of uncharged tRNA in the cell. In response, protein synthesis could be restricted by a host of translational control pathways. The severe associated phenotypes may also be due to the modulation of LysRS’s alternative non-translational functions. Many synthetases have evolved appended domains and secondary functions that lie outside their canonical function in protein synthesis. LysRS is one such synthetase, having an N-terminally appended domain, the eukaryotic extension, in addition to its anticodon-binding and aminoacylation domains. The “bifunctional” KARS gene’s mRNA undergoes alternative splicing to produce both mitochondrial and cytoplasmic LysRS, as well as additional splice variant proteins. These splice variants often do not contain the canonical aminoacylation domain required for its role in protein synthesis but retain the appended domains. This study also examines the properties of one such protein: spliced protein N9, spLysRSN9, or simply N9. The N9 protein was expressed and purified from E. coli, characterized for oligomeric state and fold, and tested for binding of tRNA and aminoacylation. It was shown that N9 is a fully folded dimeric protein in solution that could bind tRNAs in a similar manner to LysRS, but is catalytically inactive. While previously detected in Jurkat T-cell lysate, the reason behind the cellular production of these splice variants of LysRS remains unclear.
Karin Musier-Forsyth, PhD (Advisor)
Jane Jackman, PhD (Committee Member)
98 p.
Biochemistry; Chemistry
Human Lysyl-tRNA Synthetase, In vitro Activity, Human Disease

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Citations

  • McVey, C. A. (2016). Variants of Human Lysyl-tRNA Synthetase: In vitro Activity and Relevance to Human Disease [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1469113725

    APA Style (7th edition)

  • McVey, Chase. Variants of Human Lysyl-tRNA Synthetase: In vitro Activity and Relevance to Human Disease. 2016. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1469113725.

    MLA Style (8th edition)

  • McVey, Chase. "Variants of Human Lysyl-tRNA Synthetase: In vitro Activity and Relevance to Human Disease." Master's thesis, Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1469113725

    Chicago Manual of Style (17th edition)

osu1469113725
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