Yeast total trna

As described previously [42 (link)], the aminoacylation assays were performed at room temperature with 50 nM enzyme in 50 μL reaction mixture contains 50 mM Hepes (pH 7.5), 20 mM KCl, 5 mM MgCl₂, 4 mM ATP, 2 mM DTT, 5 μg/mL pyrophosphatase, 20 μM cold L-serine, 1.34 μM [3H]-serine (1 mCi/mL), and 200 μM yeast total tRNA (Roche Diagnostics, Indianapolis, Indiana, USA). The reaction was initiated by adding 50 nM enzyme into the reaction mixture. Aliquots of 5 μL were applied into 100 μL quench solution (300 mM NaOAc (pH 3.0), 1 mg/mL DNA, and 100 mM EDTA) in the Multi Screen 96-well filter plate (0.45 μm pore size hydrophobic, low-protein-binding membrane; Merck Millipore) at different time points of the reaction. After that, 100 μL of 20% (w/v) trichloroacetic acid (TCA) was added to precipitate the nucleic acids in each well. Plate was then washed 4 times with 200 μL/well of 5% TCA containing 100 mM cold L-serine, followed by 1-time wash with 95% ethanol. Air-dried plate was then treated with 70 μL/well of 100 mM NaOH to elute tRNAs. The eluted tRNAs were centrifuged into a 96-well flexible PET microplate (PerkinElmer, Santa Clara, California, USA) with 150 μL/well of Supermix scintillation mixture. After mixing, the radioactivity in each well of the plate was measured in a PerkinElmer 1450 Liquid Scintillation Counter and Luminescence Counter.

Compound was prepared using Echo 550 in 384-well plates in a series of 10 concentrations. DMSO wells were of negative control, and no enzyme wells were of positive control. For assay with Mtb PheRS, the same method was used as aforementioned. For hFARS1, 50 nM enzyme, 2 μM ATP, 20 μM l-Phe, and 1 mg/ml yeast total tRNA (Roche) were incubated with compound at 37 °C for 1 h. For hFARS2, the same method as hFARS1 except that E. coli total tRNA (Roche) was used at 0.5 mg/ml. Plates were processed for luminescence as aforementioned. In data analysis with the GraphPad Prism, the enzyme remaining activity was calculated as: $\text{Activity}\left(\text{\%}\right)=\left(1-\frac{{\text{Lum}}^{max}-{\text{Lum}}^I}{{\text{Lum}}^{max}-{\text{Lum}}^{min}}\right)\times 100$
Lum^max was luminescence value without PheRS in reaction, Lum^min was the value from DMSO control well, and Lum^I was from inhibitor well. The activity (percentage) was plotted against inhibitor concentration. To calculate IC50, the resulted dose–response curve was fit to Equation 1, where H was the Hillslope factor. $y=y_{min}+\frac{{\text{y}}_{max}\pm {\text{y}}_{min}}{1+{\left(\frac{I{C}_{50}}{x}\right)}^H}$