Metal Ion Requirement for Catalysis by 3'-5' RNA Polymerases.

The two-metal ion mechanism for catalysis of RNA and DNA synthesis by 5'-3' polymerases has been extensively characterized. The 3'-5' polymerase family of enzymes, consisting of tRNAHis guanylyltransferase (Thg1) and Thg1-like proteins (TLPs), perform a similar nucleotide addition reaction, but in the reverse direction, adding Watson-Crick base-paired NTPs to the 5'-ends of RNA substrates. However, the effect of divalent cations beyond magnesium has not been described. Here, we examined the effects of five divalent cations (Mg2+, Mn2+, Co2+, Ni2+ and Ca2+) on templated nucleotide addition activity and the kinetics of 5'-activation by ATP catalyzed by recombinantly purified, metal-free TLPs from organisms across diverse domains of life. This work revealed that different TLPs exhibit distinct dependencies on the concentration and identity of divalent metal ions that support effective catalysis. The patterns of metal ion usage demonstrated here for TLPs evince features that are characteristic of both canonical 5'-3' polymerases and DNA/RNA ligases. Similar to 5'-3' polymerases, some metals were also observed to be mutagenic in the context of TLP catalysis. Furthermore, we provide the first direct evidence that both ATP and the NTP poised for nucleotidyl transfer are present in the active site during the 5'-adenylylation. These results provide the first in-depth study of the role of the two-metal ion mechanism in TLP catalysis, which was initially suggested by structures of these enzymes.