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Steiner Dissertation _Final.pdf (2.34 MB)

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Regulation of Translational Quality Control by Phenylalanyl-tRNA Synthetase

Steiner, Rebecca Elizabeth
http://rave.ohiolink.edu/etdc/view?acc_num=osu1561468654993642

Abstract Details

2019, Doctor of Philosophy, Ohio State University, Biochemistry Program, Ohio State.
Aminoacyl tRNA synthetases (aaRSs) are critical enzymes in translation, being required to accurately make functional proteins. aaRSs are responsible for correctly aminoacylating a tRNA with the cognate amino acid through a two-step reaction mechanism. It is important that translational accuracy is maintained through proofreading activity in order to prevent mistranslation because translational errors have been implicated in disease progression and inappropriate stress response activation. This research aimed to examine how aaRSs function during stress. Translational quality control is important for Escherichia coli and Saccharomyces cerevisiae to appropriately respond to nutrient stress. In the absence of phenylalanyl-tRNA synthetase (PheRS) proofreading, mischarged tRNAPhe accumulates, increasing the pool of aminoacylated tRNA (aa-tRNA). Stopping the accumulation of uncharged tRNA stops the activation of the stringent response and general amino acid control (GAAC) pathway in E. coli and S. cerevisiae, respectively. Therefore, in S. cerevisiae we hypothesized that PheRS proofreading activity could also play a role in the target of rapamycin (TOR) pathway, which has been shown to have cross-talk with GAAC. We found that disrupting PheRS proofreading disrupts translational quality control and perturbs the regulation of multiple stress response networks. Further we demonstrated that PheRS proofreading impacts the nitrogen source dependent growth, and consequently, proofreading is required for proper regulation of GATA-mediated response to nitrogen stress. Previously, it was demonstrated that oxidation of threonyl-tRNA synthetase inhibits proofreading activity and Ser-tRNAThr accumulates, increasing the possibility for mistranslation. Oxidative stress also alters the amino acid pools, through oxidation of phenylalanine into tyrosine isomers, increasing the presence of cytotoxic non-cognate amino acids. How PheRS responds to this change in the amino acid pools during oxidative stress was not understood. We discovered that oxidation of PheRS causes a conformational change to the enzyme, generating a hyper-accurate PheRS and maintaining aminoacylation activity. Positive regulation of PheRS proofreading during oxidative stress protects the cell from adverse growth conditions and has potential to help maintain the stringent response in Salmonella. Defects in mitochondrial translation have been implicated in multiple diseases. Mutations across all domains of mitochondrial-PheRS have been found in 21 patients with a wide range of disease phenotypes. In our case study, we analyzed a patient who has abnormalities in brain scans and presents with pure spastic paraplegia. Upon exosome sequencing we discovered one chromosome has a nonsense mutation that truncates PheRS, deleting a portion of the catalytic domain. The other chromosome has a single nucleotide polymorphism (SNP) in the active site domain of mitochondrial PheRS, P136H. This mutation reduces the catalytic efficiency of PheRS by 10-fold, potentially being the underlying cause of the disease phenotype. Taken together these findings show that maintenance and regulation of PheRS catalytic and proofreading activities are important both for normal growth of cells and for the cell’s ability to appropriately respond to stress.
Michael Ibba (Advisor)
134 p.
Biochemistry
Oxidative Stress, PheRS, translation, proofreading, biochemistry, kinetics

Recommended Citations

Citations

  • Steiner, R. E. (2019). Regulation of Translational Quality Control by Phenylalanyl-tRNA Synthetase [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1561468654993642

    APA Style (7th edition)

  • Steiner, Rebecca. Regulation of Translational Quality Control by Phenylalanyl-tRNA Synthetase. 2019. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1561468654993642.

    MLA Style (8th edition)

  • Steiner, Rebecca. "Regulation of Translational Quality Control by Phenylalanyl-tRNA Synthetase." Doctoral dissertation, Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1561468654993642

    Chicago Manual of Style (17th edition)

osu1561468654993642
326
© 2019, some rights reserved.Creative Commons License Regulation of Translational Quality Control by Phenylalanyl-tRNA Synthetase by Rebecca Elizabeth Steiner is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by The Ohio State University and OhioLINK.