The internally Cy5 labeled tRNAAlaUGC (0.1 μM) was incubated with endogenous yeast Elp123 subcomplex or recombinant wild-type or mutant forms of Elongator (0.1 to 0.9 μM) for 10 min on ice. The reaction was carried out in the buffer condition containing 20 mM Hepes (pH 7.5), 100 mM NaCl, 5 mM MgCl2, and 3 mM DTT. Next, samples were supplemented with 30% glycerol and resolved on a tris-boric acid EDTA–based 6% native polyacrylamide gel electrophoresis (PAGE) gels containing 15% sucrose using EMSA. Gels were run with 120 V for 1 hour and 40 min in 1× tris-glycine/DTT buffer at 4°C. Individual cofactors (1 mM), such as acetyl-CoA, SAM, and ATP, were added as indicated in figures. To quantify the tRNA binding affinity of Elp123 and Elongator, microscale thermophoresis assays were performed. The reactions (20 μl) were carried out as described above and applied to premium capillaries (NanoTemper). The interactions were determined using the MO.Control software (NanoTemper), and dissociation constants (Kd’s) were calculated based on triplicate measurement using the MO.Affinity software (NanoTemper).

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