Poster abstracts
Poster number 3 submitted by Aiswarya Krishnamohan
Unexpected mechanistic features of a multifunctional tRNA methyltransferase
Aiswarya Krishnamohan (Ohio State Biochemistry Program, Dept. of Chemistry and Biochemistry, The Ohio State University), Jane Jackman (Dept. of Chemistry and Biochemistry, The Ohio State University)
Abstract:
The tRNA m1G9 methyltransferase (Trm10) family enzymes methylate the guanosine and sometimes adenosine residues at the 9th position (A9/G9) of multiple tRNAs using S-adenosyl methionine (SAM) as the methyl group donor. We and others recently characterized inherited mutations in a human Trm10 homolog (TRMT10A) that are associated with a multisymptomatic disease syndrome characterized by defects in glucose metabolism and cognitive function. Moreover, a strong 5-fluorouracil hypersensitive phenotype is associated with deletion of trm10 in S. cerevisiae. Despite these indications that Trm10 plays important roles in biological systems, the mechanism by which the lack of Trm10 activity negatively impacts cellular function is not known. Moreover, although Trm10 has been classified as a member of the SpoU-TrmD (SPOUT) family of methyltransferases, it does not share many of the key mechanistic features associated with members of this enzyme family, suggesting that Trm10 enzymes exhibit a novel mechanism of catalysis which remains unknown. To address these questions about the mechanism and function of Trm10, a detailed biochemical and kinetic analysis of Trm10 variants with alterations in highly conserved residues was performed, providing evidence for an atypical tRNA methyltransferase mechanism that characterizes this enzyme family.
Using a structure of Trm10 solved in the absence of tRNA as a guide, we investigated the catalytic roles of residues proposed to interact with the SAM methyl donor, a putative general base, and a proposed tRNA binding surface. While our data support the crystallographically-observed SAM binding site, we provide direct evidence contradicting the identification of the proposed general base and tRNA binding surface. To further probe the ability of Trm10 to specifically recognize certain substrate tRNAs, we developed a highly sensitive electromobility shift assay (EMSA), which allowed us to visualize conformational differences between Trm10:tRNA complexes formed with substrate vs. non-substrate RNA that are consistent with the observed differences in enzyme activity. These new and unexpected findings will enable future efforts to fully understand the mechanism of catalysis and substrate recognition by Trm10 so that its complex effects on biology can be understood.
Keywords: tRNA modification, Enzyme mechanism