The C-terminal carboxy group of T7 RNA polymerase ensures efficient magnesium ion-dependent catalysis

DNA and RNA polymerases use divalent metal ions for catalysis. Crystal structures of several polymerases reveal that two acidic residues are involved in coordinating two metal ions at the catalytic centre. Bacteriophage RNA polymerases contain a highly conserved C-terminus with the carboxylate positioned near the active site. We examined whether theC-terminal carboxy group of T7 RNA polymerase is important for magnesium ion-dependent catalysis. Introduction of a methyl ester or decarboxylation of the C-terminal carboxy group was achieved with an intein-based protein expression system and an elongation rate assay was developed to test the effects of the modifications. The results show that enzymes with a modified C-terminal carboxy group exhibit a magnesium ion-dependent decrease in catalytic activity.