Abstract
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 tRNA(His) 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, yet the effect of divalent cations beyond magnesium has not been described. Here, we examined the effects of five divalent cations (Mg(2+), Mn(2+), Co(2+), Ni(2+) and Ca(2+)) on templated nucleotide addition activity and kinetics of 5'-activation by ATP catalyzed by recombinantly purified, metal-free TLPs from organisms from 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 seen 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 that was first suggested by structures of these enzymes.