Talk Abstracts

Talk on Friday 01:30-01:45pm submitted by Elizaveta Karchuganova

Structure, binding and activity of the human tRNA modification enzyme TRIT1 with tRNA-Sec

Elizaveta Karchuganova (Department of Chemistry, University of Michigan), Daniel J. Corey (Department of Chemistry, University of Michigan), Markos Koutmos (Department of Chemistry, Department of Biophysics, University of Michigan)

Abstract:
tRNA molecules carry a wide array of chemical modifications added post-transcriptionally. The human enzyme TRIT1 (tRNA Isopentenyl Transferase 1) installs an isopentenyl group onto an adenosine at the 37th position of some cytosolic and mitochondrial tRNAs resulting in the formation of N⁶-isopentenyl adenosine 37 (i⁶A37). This modification aids in tRNA folding and maturation process and is especially important for proper recognition of the mRNA codon and overall translational efficiency during protein synthesis. Impairments within the i⁶A37 installation process are known to be linked to such detrimental conditions as encephalopathy, myoclonic epilepsy and different types of cancer. However, how TRIT1 selects its substrates remains unclear: kinetics have been measured only with short anticodon stem-loop fragments, and binding to full-length tRNA has not been quantified. We aim to uncover what governs tRNA recognition, its positioning in the active site of the enzyme, and ultimately, modification. We used Electrophoretic Mobility Shift Assay (EMSA) with fluorescently labeled full-length tRNAs to interrogate binding, and a continuous pyrophosphate detection assay (PPiLight) to track enzymatic activity. We found that full-length tRNA-Sec binds TRIT1 with an apparent affinity at or below the low nanomolar range. Binding does not require catalysis or the isopentenyl moiety donor: the catalytically inactive T32A variant and wild-type TRIT1, with or without a DMAPP analog, bound comparably, and the complex persisted at higher NaCl concentrations. The specificity constant for full-length tRNA-Sec (k_cat/K_M ~0.05 M^-1 min^-1) was comparable to that reported for an anticodon stem-loop fragment. Finally, we have determined the first cryo-EM structure of human TRIT1 in complex with tRNA-Sec. This sets the stage for testing which features of TRIT1 and its tRNA substrates matter for recognition.

References:
1. Yarham, J. W.; Lamichhane, T. N.; Pyle, A.; Mattijssen, S.; Baruffini, E.; Bruni, F.; Donnini, C.; Vassilev, A.; He, L.; Blakely, E. L.; Griffin, H.; Santibanez-Koref, M.; Bindoff, L. A.; Ferrero, I.; Chinnery, P. F.; McFarland, R.; Maraia, R. J.; Taylor, R. W. Defective i6A37 Modification of Mitochondrial and Cytosolic tRNAs Results from Pathogenic Mutations in TRIT1 and Its Substrate tRNA. PLoS Genet. 2014, 10 (6), e1004424. https://doi.org/10.1371/journal.pgen.1004424.
2. Fradejas-Villar, N.; Bohleber, S.; Zhao, W.; Reuter, U.; Kotter, A.; Helm, M.; Knoll, R.; McFarland, R.; Taylor, R. W.; Mo, Y.; Miyauchi, K.; Sakaguchi, Y.; Suzuki, T.; Schweizer, U. The Effect of tRNA[Ser]Sec Isopentenylation on Selenoprotein Expression. Int. J. Mol. Sci. 2021, 22 (21), 11454. https://doi.org/10.3390/ijms222111454.

Keywords: tRNA modification, TRIT1, tRNA-Sec