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Characterization of Prolyl-tRNA Synthetase, YbaK, and ProXp-ala Editing Mechanism

Chen, Lin
http://orcid.org/0000-0002-3682-9270
http://rave.ohiolink.edu/etdc/view?acc_num=osu148053070268849

Abstract Details

2016, Doctor of Philosophy, Ohio State University, Chemistry.
Aminoacyl-tRNA synthetases (ARSs) attach cognate amino acids onto corresponding tRNAs to produce aminoacyl-tRNAs, which are delivered to the ribosomes. High accuracy of ARSs ensures the fidelity of protein synthesis and accumulation of errors introduced by ARSs could result in protein misfolding and aggregation, which has detrimental effects on cell growth. ARSs have developed proofreading/editing activity to correct mistakes throughout evolution. Previous studies have reported a triple-sieve editing mechanism of Escherichia coli prolyl-tRNA synthetase (ProRS), in which the first “coarse” sieve is the active site of ProRS rejecting larger amino acids, the second “fine” sieve is an editing domain, known as the insertion domain (INS) that specifically deacylates Ala-tRNAPro, and the third “fine” sieve is YbaK, a free-standing editing protein that clears Cys-tRNAPro in trans. In some bacteria, a distinct triple-sieve mechanism has been discovered, which relies on trans-editing factors exclusively. In this case, INS is absent in ProRS and instead ProXp-ala, another free-standing protein, hydrolyzes Ala-tRNAPro. YbaK and ProXp-ala belong to the INS superfamily with sequence homology and structural similarities; however, YbaK lacks inherent tRNA specificity while ProXp-ala recognizes acceptor stem elements. It was proposed that YbaK finds its substrate Cys-tRNAPro by forming a ternary complex with ProRS and tRNAPro, which has been difficult to characterize due to its transient nature. Azetidine-2- carboxylic acid (Aze), a proline analog, can also be misacylated by ProRS. It has toxic effects on growth of some plants, like Arabidopsis thaliana, but it is present in the roots of Beta vulgaris. Whether plant species that are resistant to Aze are inherently more specific or possess editing functions that prevent mistranslation has not been studied. ProXp-ala is also found to be distributed in higher eukaryotes, such as humans, but little is known about human ProXp-ala function in vitro or in the cell. In this work, we have characterized the ternary complex of ProRS/tRNAPro/YbaK and determined the stoichiometry as 2:1:1 using native mass spectrometry coupled with ion mobility. Competition binding studies were performed to confirm the specific binding of tRNAPro by ProRS and ProXp-ala in contrast to the lack of tRNA specificity of YbaK. Additionally, A. thaliana ProRS was shown to mischarge Aze onto tRNAPro and fails to deacylate the mischarged species. Although attempts to purify active B. vulgaris ProRS were unsuccessful, we hypothesize that its lack of Aze misacylation activity or pre- transfer editing towards Aze confers Aze resistance. A B. vulgaris ProRS encoding transgene transferred into wild-type A. thaliana, however, did not confer Aze-resistance. This could be due to the presence of wild-type A. thaliana ProRS or alternatively, due to the inadvertent inclusion of an N-terminal extension found only in the chloroplast form of the B. vulgaris enzyme. Finally, human ProXp-ala localization was monitored in vivo. We found that C-terminal tagged ProXp-ala-mCitrine is distributed throughout HeLa cells, whereas N-terminal tagged mCitrine-ProXp-ala is localized in the cytoplasm. To further characterize its function in the cell, future studies will focus on identifying cellular RNAs that interact specifically with human ProXp-ala.
Karin Musier-Forsyth (Advisor)
Thomas Magliery (Committee Member)
Vicki Wysocki (Committee Member)
139 p.
Biochemistry; Chemistry

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Citations

  • Chen, L. (2016). Characterization of Prolyl-tRNA Synthetase, YbaK, and ProXp-ala Editing Mechanism [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu148053070268849

    APA Style (7th edition)

  • Chen, Lin. Characterization of Prolyl-tRNA Synthetase, YbaK, and ProXp-ala Editing Mechanism. 2016. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu148053070268849.

    MLA Style (8th edition)

  • Chen, Lin. "Characterization of Prolyl-tRNA Synthetase, YbaK, and ProXp-ala Editing Mechanism." Doctoral dissertation, Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu148053070268849

    Chicago Manual of Style (17th edition)

osu148053070268849
118
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This open access ETD is published by The Ohio State University and OhioLINK.