Biochemical and structural bioinformatics studies of fungal CutA nucleotidyltransferases explain their unusual specificity toward CTP and increased tendency for cytidine incorporation at the 3′-terminal positions of synthesized tails

TtCutA_tr3 variants act processively with ATP and distributively in the presence of CTP or UTP. (A) Products of reactions shown in Figure 12B for TtCutA_tr3 WT protein were resolved in 10% high-resolution sequencing gel, showing that the substrate 3′-end can be extended by not more than two cytidines, as indicated on the right (B–D). Nucleotidyltransferase activity assays were performed for WT (B), PAP1 (C), and PUP3 (D) TtCutA_tr3 protein variants, with 40-fold molar excess of the 5′-FAM-labeled ss22-A₄ RNA substrate (position marked with arrows). Reactions were programmed with 100 µM ATP, CTP, or UTP, as indicated above the lanes and terminated after 2–60 min following start of incubation. Lengths of the synthesized tails are indicated on both sides of the gel images.