Asp36 is essential for MciKIN activity. (A) Aliquots (10 µg) of purified recombinant wild-type, D36A, and D36N MciKIN proteins were analyzed by SDS-PAGE. The Coomassie blue-stained gel is shown. The positions and sizes (kDa) of marker polypeptides are indicated on the left. (B) Reaction mixtures (10 µL) containing 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 2 mM DTT, 1 mM MgCl2, 0.1 mM GTP, 100 nM (1 pmol) 10-mer 5′-OH RNA substrate 3′-labeled with Cy5, and wild-type, D36A, or D36N MciKIN proteins as specified were incubated at 37˚C for 5 min. The reactions were quenched with formamide/EDTA, and the products were analyzed by urea-PAGE. The positions of the 10-mer HORNA substrate and the 10-mer pRNA product are indicated.
