A comparison of Dictyostelium discoideum 3'-5' RNA polymerases reveals a conserved tRNAHis guanylyltransferase residue that plays a dual role in catalysis

The 3'-5' RNA polymerase family consists of eukaryotic tRNAHis guanylyltransferase (Thg1) and Thg1 homologs known as Thg1-like proteins (TLPs) that exist in all three domains of life. Thg1 catalyzes an essential reaction adding a G-1 nucleotide to the 5' end of the tRNAHis, forming an identity element for tRNA aminoacylation. All TLPs studied, except Dictyostelium discoideum (Ddi) TLP2, perform in vitro Watson-Crick (WC) dependent addition of multiple nucleotides to repair truncated tRNA. DdiTLP2 has similar activity to Thg1, adding G-1 to mt-tRNAHis, but shares other biochemical properties with other TLPs, including a restriction to making WC base pairs during this reaction. We identified two regions in DdiTLP2 that lacked residues that are conserved in other Thg1/TLP enzymes. DdiTLP2 variants in both regions abolish enzymatic activity of DdiTLP2, indicating these regions are important for DdiTLP2 catalysis. Changing DdiThg1 D150 to the corresponding arginine found in DdiTLP2 causes an unexpected reversal of this enzyme's specificity, with a loss of its ability to incorporate a non-WC base paired G-1 to its physiological substrate, while gaining the ability to add WC base paired G-1 to mt-tRNAHis. Biochemical study of other changes to D150, combined with structural models, suggest a previously unknown role for D150 in controlling substrate specificity at the adenylation step by providing a checkpoint for correct setup of a WC base pair in the active site. Thg1 also appears to have adapted the role of the ancestral D150 residue for a second function, promoting non-WC nucleotide addition to its eukaryotic tRNAHis substrate.