TLPs catalyze efficient 3′–5′ polymerase activity with RNA duplexes. (A) A representative time course of 3′–5 polymerase activity measured with AcaTLP2 and DdiTLP4 (15 μM) with the 7 bp RNA duplex assayed under single turnover conditions (at least 100-fold excess [Enzyme] over [RNA]) with individual time points measured from 0 to 120 min (time points shown in this assay correspond to 0.5, 1, 2, 5, 10, 20, 30, 45, 60, 120 min). The band corresponding to unreacted substrate (S) or substrate in reactions that contain template (ST) is indicated by the black bar on the side of the time course. Extension products are as indicated by added nucleotides shown in green. Reaction A2-G shows the migration of the single-nucleotide addition product observed in the absence of GTP, as shown previously. (B) Determination of kobs values for 15 μM enzyme (AcaTLP2, circles; DdiTLP4, squares) from the average percent total nucleotide addition products observed in triplicate reactions measured with the 8 and 7 bp substrates, as indicated. The data were fit to Equation 1 to yield kobs for each enzyme with the indicated RNA.
