Poster abstracts
Poster number 149 submitted by Matthew Yacoub
Characterizing Structural and Thermodynamic Properties of a Human Homolog of TRMT1
Matthew Yacoub (Department of Chemistry, University of Michigan), Catherine Wilhelm (Department of Chemistry, University of Michigan), Shayna Brotzman (Department of Chemistry, University of Michigan), Daniel Corey (Department of Chemistry, University of Michigan), Markos Koutmos (Department of Chemistry, University of Michigan)
Abstract:
Post-transcriptional modifications of transfer RNA (tRNA) affect the stability, three-dimensional structure, and folding of these dynamic biomacromolecules. Such modifications exert downstream effects on critical biological processes such as translation and aminoacylation. TRMT1, a tRNA methyltransferase, modifies guanosine at position 26 of its tRNA substrates to form both monomethylated (m2G26) and dimethylated (m22G26) products. Mutations in human TRMT1 (hTRMT1) have been linked to numerous diseases, including autosomal-recessive intellectual disability, mitochondrial encephalomyopathies, and cancer. Homologs of this enzyme found in Saccharomyces cerevisiae, Pyrococcus horikoshii, and Aquifex aeolicus have previously been kinetically, thermodynamically, and structurally characterized; however, the same characterization has yet to be performed for hTRMT1. This is likely due to the complexity of the human homolog, as it is 300 amino acids longer than its non-human counterparts, conferring instability and intrinsically disordered regions (as predicted by AlphaFold) that make it more difficult to express, purify, and handle. We successfully purified hTRMT1 using affinity and size exclusion chromatography. Next, we aim to determine the structural and thermodynamic properties of the resulting protein samples by setting up crystallization screening trays and negative stains for characterization by X-ray crystallography and cryo-EM, respectively. We further plan to conduct binding and catalytic activity assays in order to characterize the enzyme’s affinity for and ability to methylate its tRNA substrates. These studies will lay the groundwork for future investigations into the mechanisms of pathogenesis caused by patient mutations in hTRMT1.
References:
Machnicka et al. RNA Biol. (2014) 11(12), 1619-1629.
Zhang et al. Hum. Mutat. (2020) 41(3), 600-607.
Ihsanawati et al. J. Mol. Biol. (2008) 383, 871-884.
Awai et al. J. Biol. Chem. (2011) 286(40), 35236-35246.
Keywords: hTRMT1, Modification, Methyltransferase