Kinetics of healing and sealing a 20-mer HORNA>p substrate by KlaTrl1. A reaction mixture (240 µL) containing 50 mM Tris-HCl, pH 8.0, 2 mM DTT, 10 mM MgCl2, 100 µM ATP, 100 µM GTP, 20 nM 3′ 32P-labeled 20-mer HORNA>p substrate (depicted at the bottom of panel A with the 32P-label denoted by •), and 1 µM KlaTrl1 was incubated at 22°C. Aliquots (20 µL) were withdrawn at the times specified and quenched immediately with an equal volume of 90% formamide, 30 mM EDTA. The time 0 sample was withdrawn and quenched prior to adding KlaTrl1. The products were analyzed by urea-PAGE. An autoradiogram of the gel is shown in panel A. The position and identities of the radiolabeled substrate and products are indicated on the left and right. The extent of ligation ([circle + multimers]/total RNA) is plotted as a function of time in panel B. Each datum is the average of three separate time-course experiments ±SEM. A nonlinear regression curve fit of the data to a single exponential is shown.
