FIG 2.

NHC-TP is incorporated into RNA by POLRMT. The primer extension reaction mixtures contained 10 nM P/T (shown at the top) and 20 nM POLRMT, and the reactions were performed in the presence of 1 μM ATP as the first ribonucleotide and increasing concentrations (in micromolar) of 3′-dCTP or NHC-TP (EIDD-2061), as indicated under each lane. The products were resolved by denaturing PAGE. The migrations of the 21-mer primer and the 22- and 23-mer first and second ribonucleotide extension products are indicated on the right. (Bottom) Quantitative analysis of nucleotide analog incorporation. The incorporation efficiency was evaluated based on the extension of 22-mer to 23-mer products. The measured K_1/2 values are shown on the right of the graph. The discrimination between the analog and 3′-dCTP, D*_analog, was calculated as K_{1/2, analog}/K_{1/2, 3′-dCTP} (where K_1/2 is defined as the analog triphosphate concentration resulting in 50% product extension). The D*_EIDD-2061 in this experiment is 13.2. In second analogous experiment repeat, the D*_EIDD-2061 was measured to be 11.5.