A semi-quantitative pull-down assay to study tRNA substrate specificity of modification enzymes
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Updated: Oct 20, 2025
An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity
Published on: October 8, 2018
Lu Han1, Erin Marcus2, Eric M Phizicky2
1Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester School of Medicine, Rochester, NY, United States; Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, United States; Department of Genetics, The Blavatnik Institute, Harvard Medical School, Boston, MA, United States.
Abstract:
A tRNA interacts with numerous proteins throughout its biogenesis and during translation, and a significant portion of these interacting proteins are involved in post-transcriptional modifications. While some of the modifying enzymes use relatively simple recognition elements for substrate recognition, many enzymes selectively modify a specific subset of tRNA species without obvious recognition rules. In this chapter we describe a semi-quantitative pull-down assay to study tRNA substrate specificity of modification enzymes, by using the yeast Saccharomyces cerevisiae m3C32 methyltransferase Trm140 as an example. We also discuss some overall considerations for a successful pull-down experiment, with a focus on practical applications of the dissociation constant KD between the protein and the tRNA and the off-rate.
