Nucleotide specificity of the human terminal nucleotidyltransferase Gld2 (TUT2)

Insertion of an active site histidine into Gld2 alters nucleotide specificity. (A) Gld2 and Gld2-His purification. Recombinant His-tagged Gld2 and the Gld2-His mutant were purified via Ni-NTA chromatography to apparent homogeneity as judged by SDS-PAGE. (B,C) Nucleotide preference of Gld2 and Gld2-His. Recombinant enzymes were incubated with and without RNA substrates and radiolabeled ATP or UTP. Formation of α-³²P labeled RNA products was monitored by electrophoretic separation and subsequent phosphorimaging. RNA substrates were let-7a (B) and 15A (C).