L 3h serine

The histidylation assay mixture contained 50 mM Tris-HCl (pH 7.7), 100 mM KCl, 10 mM MgCl₂, 8 mM DTT, 2.5 mM ATP, 20 μM L-[¹⁴C] -histidine (PerkinElmer, 320 mCi/mmol) and 5 μM annealed tRNA^His(GUG). 50 nM HisRS or mutants were added to initiate the reactions. All the activity assays were performed with 5′-GMP tRNAs due to their higher activities as the substrates than that of the 5′-GTP tRNAs. The reaction was carried out at 60°C, and aliquots were removed at the designated time points, spotted onto 5% trichloroacetic acid (TCA)-soaked filter pads, and washed twice with 5% cold TCA as well as 95% ethanol. The filter pads were dried and the radioactivity was measured by scintillation counting.
The aminoacylation assays for hSerRS/hGlyRS were carried out under similar conditions to those of HisRS assays. hSerRS assays contained 50 mM Tris-HCl (pH 7.7), 100 mM KCl, 10 mM MgCl₂, 8 mM DTT, 2.5 mM ATP, 20 μM cold serine, 1 μM L-[³H]-serine (PerkinElmer, 22 Ci/mmol), 10 μM annealed tRNA^Ser(UGA), whereas that of hGlyRS contained 150 mM HEPES (pH 7.5), 20 mM KCl, 4 mM MgCl₂, 2 mM DTT, 3 mM ATP, 20 μM cold glycine, 1 μM L-[³H]-glycine (PerkinElmer, 48.7 Ci/mmol) and 5 μM annealed tRNA^Gly(CCC). 200 nM hSerRS and 50 nM hGlyRS were added last, and all the reactions were performed at room temperature.