Characterization of tRNA splicing enzymes RNA ligase and tRNA 2′-phosphotransferase from the pathogenic fungi Mucorales

Effect of pH and magnesium on single-turnover pRNA_2′p ligation. (A) pH dependence. Reaction mixtures (10 µL) containing either 50 mM Tris-acetate (pH 4.5, 5.0, 5.5, 6.0, or 6.5) or 50 mM Tris-HCl (pH 7.0, 7.5, 8.0, 8.5, 9.0, or 9.5), 5 mM MgCl₂, 1 pmol 5′ ³²P-labeled pRNA_2′p, and 10 pmol MciRNL were incubated at 37°C for 1 min. (B) Magnesium dependence. Reaction mixtures (10 µL) containing 50 mM Tris-acetate, pH 6.5, 1 pmol 5′ ³²P-labeled pRNA_2′p, 10 pmol MciRNL, and MgCl₂ as specified or 5 mM EDTA were incubated at 37°C for 1 min. The reaction products were analyzed by urea-PAGE and visualized by scanning the gel with a Fujifilm FLA-7000 imaging device.