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Biochemical characterization of catalytic mechanism and substrate recognition by the atypical SPOUT tRNA methyltransferase, Trm10

Krishnamohan, Aiswarya Lakshmi
http://rave.ohiolink.edu/etdc/view?acc_num=osu1512039838462506

Abstract Details

2017, Doctor of Philosophy, Ohio State University, Biochemistry Program, Ohio State.
The tRNA m1G9 methyltransferase (Trm10) is a member of the SpoU-TrmD (SPOUT) superfamily of methyltransferases. Trm10 homologs are widely conserved throughout Eukarya and Archaea. Despite possessing the trefoil knot characteristic of SPOUT enzymes, Trm10 does not share the same quaternary structure or key sequences with other members of the SPOUT family. Further, Trm10 displays a unique property where specificity of the target purine varies among different homologs ranging from obligate m1G9 or m1A9 methyltransferases to bifunctional enzymes that catalyze both m1G9 and m1A9, an expansion of substrate specificity not observed in other tRNA methyltransferases. These observations suggest that Trm10 enzymes may possess a distinct active site and a novel mechanism of catalysis. In this thesis, I describe detailed biochemical characterization of m1G9-specific Saccharomyces cerevisiae Trm10 and m1G9/m1A9-specific Thermococcus kodakarensis Trm10, providing evidence for an atypical tRNA methyltransferase mechanism that characterizes this enzyme family. The results argue against a previously proposed general base mechanism and suggest that the rate-determining step most likely involves a conformational change that positions the substrate nucleotide in the active site aided by a highly conserved network of active site residues. I also describe analysis of the Trm10: tRNA binding mode that likely occurs through a non-traditional 1:1 complex. Using RNA structure probing experiments, I provide evidence for structural effects imposed by Trm10 upon binding to substrate tRNA, possibly to gain access to R9 that is buried in the tRNA core and identify residues on Trm10 that are involved in these effects. These findings provide critical insight into the apparently unique active site of Trm10 enzymes that has the ability to catalyze different nucleotides.
Jane Jackman (Advisor)
Juan Alfonzo (Committee Member)
Venkat Gopalan (Committee Member)
Kotaro Nakanishi (Committee Member)
154 p.
Biochemistry
tRNA modification; RNA biology; enzyme mechanism; substrate recognition; Trm10

Recommended Citations

Citations

  • Krishnamohan, A. L. (2017). Biochemical characterization of catalytic mechanism and substrate recognition by the atypical SPOUT tRNA methyltransferase, Trm10 [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1512039838462506

    APA Style (7th edition)

  • Krishnamohan, Aiswarya. Biochemical characterization of catalytic mechanism and substrate recognition by the atypical SPOUT tRNA methyltransferase, Trm10. 2017. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1512039838462506.

    MLA Style (8th edition)

  • Krishnamohan, Aiswarya. "Biochemical characterization of catalytic mechanism and substrate recognition by the atypical SPOUT tRNA methyltransferase, Trm10." Doctoral dissertation, Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1512039838462506

    Chicago Manual of Style (17th edition)

osu1512039838462506
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© 2017, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.