Figure 2.

hTRMT2A domains bind tRNA^Gln cooperatively. (A) Cartoon depicting protein fragments of hTRMT2A used for EMSAs. (B) EMSA with hTRMT2A FL shows high affinity binding to tRNA^Gln in the nanomolar K_D range. (C) EMSAs with the hTRMT2A RBD, Central and Fusion domain display only low affinity binding to tRNA^Gln in the micromolar K_D range. Because the SUMO-tagged Central domain was used, a SUMO-tag control was included. Binding assays were performed with ³²P-labeled tRNA^Gln. For experiments, poly(U) competitor and labeled RNA were pre-incubated with increasing concentrations of hTRMT2A protein for 20 min. Reaction without protein served as control. Free RNA and RNA–protein complex were separated by 4% native PAGE. Each experiment was performed in triplicate.