FIG 5.
Kinetic profile of HORNAp ligation by wild-type RtcB. Reaction mixtures (10 μl) containing 50 mM Tris-HCl (pH 8.0), 2 mM MnCl2, 100 μM GTP, 1 μM RtcB, and 0.1 μM HORNAp were incubated at 37°C. The reactions were quenched with formamide-EDTA at the times specified. The products were resolved by urea-PAGE and quantified by scanning of the gel. The levels of RNAppG and ligated RNAs are plotted as a function of time. Each datum is the average of results from three experiments ± the standard error of the mean. The data were fit by nonlinear regression in Prism to a two-step kinetic mechanism (HORNAp → HORNAppG → ligated RNA) with rate constants, kguan and klig, as indicated.