FIG 8.

Misincorporation of natural ribonucleotides by POLRMT. (A) The primer and templates used in the assay. (B) An image of the results of primer extension assays performed with the highest NTP concentrations tested that demonstrated the misincorporation of natural rNTP under conditions of different mispairs. The templates used in the reactions are indicated on the top of the gel. The natural rNTPs and 3′-dNTP used in each reaction are indicated at the bottom of each lane. “First” indicates the first correct nucleotide incorporated; “second” indicates the second nucleotide incorporated (misincorporated). POLRMT (20 nM) and 10 nM P/T were used in the assay. The concentration of the first ribonucleotide to be incorporated was 1 μM, and the concentrations of the "second" 3′-dNTPs or natural rNTPs to be incorporated were 100 μM and 1,000 μM, respectively. The reactions were performed at 22°C for 1 h, and the products were resolved by denaturing PAGE. The locations of the 13-mer primer and of the 14- and 15-mer first and second nucleotide extension products, respectively, are indicated on the left. The efficiency of extension was evaluated on the basis of the extension of the 14-mer products as a percentage of the disappearance of the 14-mer in the reactions and is indicated as percent extension.