The HD Domain of the Escherichia coli tRNA Nucleotidyltransferase Has 2′,3′-Cyclic Phosphodiesterase, 2′-Nucleotidase, and Phosphatase Activities.

In all mature tRNAs, the 3′-terminal CCA sequence is synthesized or repaired by a template-independent nucleotidyltransferase (ATP(CTP):tRNA nucleotidyltransferase; EC 2.7.7.25). The Escherichia coli enzyme comprises two domains: an N-terminal domain containing the nucleotidyltransferase activity and an uncharacterized C-terminal HD domain. The HD motif defines a superfamily of metal-dependent phosphohydrolases that includes a variety of uncharacterized proteins and domains associated with nucleotidyltransferases and helicases from bacteria, archaea, and eukaryotes. The C-terminal HD domain in E. coli tRNA nucleotidyltransferase demonstrated Ni2+-dependent phosphatase activity toward pyrophosphate, canonical 5′-nucleoside tri- and diphosphates, NADP, and 2′-AMP. Assays with phosphodiesterase substrates revealed surprising metal-independent phosphodiesterase activity toward 2′,3′-cAMP, -cGMP, and -cCMP. Without metal or in the presence of Mg2+, the tRNA nucleotidyltransferase hydrolyzed 2′,3´-cyclic substrates with the formation of 2′-nucleotides, whereas in the presence of Ni2+, the protein also produced some 3′-nucleotides. Mutations at the conserved His-255 and Asp-256 residues comprising the C-terminal HD domain of this protein inactivated both phosphodiesterase and phosphatase activities, indicating that these activities are associated with the HD domain. Low concentrations of the E. coli tRNA (10 nM) had a strong inhibiting effect on both phosphatase and phosphodiesterase activities. The competitive character of inhibition by tRNA suggests that it might be a natural substrate for these activities. This inhibition was completely abolished by the addition of Mg2+, Mn2+, or Ca2+, but not Ni2+. The data suggest that the phosphohydrolase activities of the HD domain of the E. coli tRNA nucleotidyltransferase are involved in the repair of the 3′-CCA end of tRNA. [ABSTRACT FROM AUTHOR]