AnCutA displays a nucleotidyltransferase activity, dependent on the intact aspartates in the active site. (A) Domain organization of different fungal noncanonical nucleotidyltransferases: S. pombe Cid1 poly(U) polymerase, S. cerevisiae Trf4 poly(A) polymerase, and CutA proteins from A. nidulans and T. terrestris (AnCutA and TtCutA, respectively). All proteins contain NTase domain (dark gray rectangle), which is inserted into PAP/OAS1 SBD (light gray rectangle), formed by protein fragments separated in the primary sequence, but located in proximity in the three-dimensional structure. Italicized numbering refers to amino acids located at the borders of NTase (above) and PAP/OAS1 SBD (below) domains. (B) SDS-PAGE analysis of the recombinant full-length AnCutA WT and DADA mutant (with aspartates at positions 203 and 205 substituted with alanines) variants purified by nickel-column affinity chromatography. Protein preparations were analyzed together with PageRuler Prestained Protein Ladder (Thermo Fisher Scientific) as a molecular weight marker (lane MW). Masses of individual bands are indicated on the right. Position of band corresponding to the full-length protein is marked with an open arrow. (C) Analysis of nucleotidyltransferase activity, performed for AnCutA WT and DADA variants with three different RNA oligonucleotides, labeled with fluorescein amidite (FAM) at the 5′-end. Reactions were programmed with individual NTPs or a mixture, as indicated at the top, and terminated after the time points indicated below the lanes.
