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Examining tRNA binding properties of hKRS domains

Horne, Daniel J.
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1337715480

Abstract Details

2012, MS, University of Cincinnati, Arts and Sciences: Chemistry.
Human immunodeficiency virus type 1 (HIV-1) initiates replication of its RNA genome by priming from the 3¿¿¿¿ end of a host cell tRNA complementary to the primer binding site on the viral genome. The virus packages its primer, human lysyl tRNA during virus assembly incorporating human lysyl-tRNA synthetase (hKRS) into virions using viral Gag and Gag-Pol proteins. Here, we report our attempts to study the tRNA binding properties, and in particular KD and stoichiometry of binding of the domains of Human lysyl-tRNA synthetase (hKRS) by UV/Vis and circular dichroism (CD). hKRS consists of three domains, which can function independently, a tandem domain, or as the full length enzyme. Cognate lysyl tRNA and non-cognate phenylalanine tRNA were used for an attempt to determine KD and stoichiometry upon binding to hKRS. We were able to obtain some conclusive results pertaining to KD and stoichiometry for the domains of hKRS. For example, significant binding has been observed between yFtRNA and 2D domain as determined both by CD and UV/Vis. With regards to stoichiometry, a yFtRNA:rrACB C_S titration was performed and a 1:1 stoichiometric ratio was estimated by CD. Also, a KD value of 18 uM for the yFtRNA:2D native complex was obtained by UV/Vis. Finally, an online computerized algorithm named DICHROWEB was used to show that that the secondary structure of various truncated variants of the ACB domain were less disordered in solution than the full length ACB. With respect to all constructs, each domain has at least 50% random coil on average and the trend of greatest to least random coil goes as follows modnhKRS>2D protein >rrACBcs> ghKF_ACB> flhKRS>catalytic>GED_ACB. However, most of our attempts in determining KD and stoichiometry were unsuccessful. Upon further investigation of the various domains of hKRS', we were not able to determine qualitative binding between yFtRNA and modnhKRS, ACB, or flhKRS as well as hktRNA to 2D domain by CD because of improper procedures and analysis. In fact, only 4 out 15 CD titrations were reproduced for qualitative binding studies and stoichiometry of binding. In the future, increasing the concentrations of the components for the target complexes should be done to increase signal to background ratio. This should help us obtain more reproducible results in the future.
Pearl Tsang, PhD (Committee Chair)
Apryll Stalcup, PhD (Committee Member)
70 p.
Chemistry
Human immunodeficiency virus

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Citations

  • Horne, D. J. (2012). Examining tRNA binding properties of hKRS domains [Master's thesis, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1337715480

    APA Style (7th edition)

  • Horne, Daniel. Examining tRNA binding properties of hKRS domains. 2012. University of Cincinnati, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1337715480.

    MLA Style (8th edition)

  • Horne, Daniel. "Examining tRNA binding properties of hKRS domains." Master's thesis, University of Cincinnati, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1337715480

    Chicago Manual of Style (17th edition)

ucin1337715480
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© 2012, all rights reserved.
This open access ETD is published by University of Cincinnati and OhioLINK.