Skip to Main Content
 

Global Search Box

 
 
 
 

Files

File List


Hyejeong Kim Masters Thesis 2013.pdf (2.4 MB)

ETD Abstract Container

Abstract Header

Characterization of the N-terminal region of tRNA m1G9 methyltransferase (Trm10)

Kim, Hyejeong
http://rave.ohiolink.edu/etdc/view?acc_num=osu1372619914

Abstract Details

2013, Master of Science, Ohio State University, Biochemistry.
tRNA m1G9 methyltransferase, Trm10, catalyzes m1G9 formation in a number of tRNAs in a SAM-dependent manner. Trm10 is highly conserved throughout Eukarya and Archaea, but it does not share any sequence homology to known methyltransferases and its high-resolution structure has not been determined. Although the enzyme exhibits a distinct pattern of tRNA substrate specificity, it is unknown how Trm10 recognizes and interacts with its tRNA substrates on a molecular level. Here, we investigate the role of the N-terminal region in Trm10, particularly in yeast and human variants, mainly using biochemical analyses such as in vitro activity assays, single-turnover kinetics and fluorescence quenching and anisotropy. Based on the observation that the N-terminal region of Trm10 has an abundance of positively-charged residues, we hypothesized that this region might play a role in tRNA binding. The approach used in this thesis to test this postulate was to create N-terminal truncations and substitution variants, and characterize this region using the variant proteins. In particular, the Trm10 variants exhibited distinct features from the wild-type in terms of activity, kinetics and also substrate specificity, suggesting that these residues are important for some aspect of Trm10 function. However, since deletion of a large portion of the N-terminal region still resulted in active enzyme, and individual alterations of positive residues resulted in fully active protein under some conditions, we can conclude that these residues are not absolutely essential for catalysis and do not participate directly in catalysis of methyltransfer in vitro. Two independent measurements of tRNA binding affinity by Trm10 were also developed during this work. Although there were some differences between the assays, there was general agreement that the N-terminal residues investigated here do not play a substantial role in overall tRNA binding and therefore, the role of these residues is more complicated than previously hypothesized. However, the fluorescent binding assays provided the first opportunity to investigate the binding affinity of Trm10 for both substrate and non-substrate tRNAs. These data suggest that there may exist a catalytic step following the initial tRNA binding even that is responsible for substrate specificity. These results are intriguing and will facilitate related studies, particularly once the tertiary structure of Trm10 is elucidated.
Jane Jackman (Advisor)
Venkat Gopalan (Committee Member)
Karin Musier-Forsyth (Committee Member)
64 p.
Biochemistry
Trm10; tRNA m1G9 methyltransferase; N-terminal region of Trm10; Trm10 bindinig affinity

Recommended Citations

Citations

  • Kim, H. (2013). Characterization of the N-terminal region of tRNA m1G9 methyltransferase (Trm10) [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1372619914

    APA Style (7th edition)

  • Kim, Hyejeong. Characterization of the N-terminal region of tRNA m1G9 methyltransferase (Trm10). 2013. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1372619914.

    MLA Style (8th edition)

  • Kim, Hyejeong. "Characterization of the N-terminal region of tRNA m1G9 methyltransferase (Trm10)." Master's thesis, Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1372619914

    Chicago Manual of Style (17th edition)

osu1372619914
529
© 2013, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.