Characterization of the tRNA ligases of pathogenic fungi Aspergillus fumigatus and Coccidioides immitis

NTP donor specificity of the AfuTrl1 and CimTrl1 5′ kinase reaction. Reaction mixtures (20 µL) containing 50 mM Tris–HCl (pH 7.5), 2 mM DTT, 10 mM MgCl₂, 20 nM 10-mer _HORNA>p, 200 nM CimTrl1 (panel A) or AfuTrl1 (panel B) KIN-CPD enzyme, and 0, 0.1, 1, 10, or 100 µM of the indicated NTP (ATP, CTP, GTP, or UTP) were incubated at 22°C for 2 min. The products were analyzed by urea–PAGE. The extents of 5′ phosphorylation were quantified by scanning the gel and are plotted in bar graph format as a function of NTP concentration. Each datum is the average of three separate experiments ±S.E.M.