Identification, characterization, and structure of a tRNA splicing enzyme RNA 5′-OH kinase from the pathogenic fungi Mucorales

Divalent cation requirement. (A) Metal specificity. Reaction mixtures (10 µL) containing 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.1 mM GTP, 100 nM (1 pmol) 10-mer 5′-OH RNA substrate 3′-labeled with Cy5, 5 nM (50 fmol) MciKIN, and either no added divalent cation (lane –) or 1 mM CaCl₂, CoCl₂, CuCl₂, MgCl₂, MnCl₂, NiCl₂, or ZnCl₂ as specified were incubated at 37˚C for 5 min. (B) Inactive metals are inhibitory. Reaction mixtures (10 µL) containing 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.1 mM GTP, 100 nM (1 pmol) 10-mer 5′-OH RNA substrate 3′-labeled with Cy5, 5 nM (50 fmol) MciKIN, and either no added divalent cation (lane –), 2 mM MgCl₂, or 1 mM MgCl₂ plus 1 mM CoCl₂, CuCl₂, NiCl₂, or ZnCl₂ as specified were incubated at 37˚C for 5 min. The reaction products were analyzed by urea-PAGE.