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

Trl1 titration. Reaction mixtures (20 µL) containing 50 mM Tris–HCl (pH 7.5), 2 mM DTT, 10 mM MgCl₂, 100 µM ATP, 100 µM GTP, 20 nM 20-mer _HORNA>p (depicted at the bottom), and 0.5. 1, 2, 5, 10, 20, 50, or 100 nM Trl1 (from left to right in each titration series) were incubated for 30 min at 22°C. Trl1 was omitted from control reactions in lanes (–). The products were analyzed by urea–PAGE. An autoradiograph of the gel is shown. The positions and identities of the _HORNA>p substrate (*), 5′-phosphorylated kinase product (5′-P), and the ligation products (circle and multimers) are indicated.