Fig. 7: Kinetic profile of structured RNA degradation by wild-type HsDis3L2 and mutants at single-nucleotide resolution.
a, Schematic of hairpinA–GCU14. For simplicity, the kinetic data are numbered from 16 (3′ end) to 0 (single strand–double strand junction) to denote the nucleotide position. b, Two representative gels from presteady-state nuclease titration assays with 1 nM 5′ P32-radiolabeled hairpinA–GCU14 and 25 nM HsDis3L2 and HsDis3, respectively. The overall lengths of the species and the single-stranded overhang lengths are indicated on the left and right of the panels, respectively. Each experiment was repeated independently at multiple concentrations of each enzyme with n = 2. c, Processivity (P) of wild-type Dis3L2 versus Dis3. d, Dissociation rate constants (koff) and forward rate constants (kf) of Dis3L2. e, Association rate constants (kon) of wild-type human Dis3L2. kon data point x = −8 was removed due to a large uncertainty value. The x axis shows the number of nucleotides from the start of the double-stranded stem. f, Domain composition of wild-type (WT) human Dis3L2 and the ΔCSD and Δ123H deletion mutants. g, Representative gels from pulse–chase reactions of wild-type human Dis3L2, ΔCSD and Δ123H at a 50 nM concentration with 1 nM radiolabeled hairpinA–GCU14. Cold chase was added to the reaction at the 3-min timepoint to a final concentration of 5,000 nM. Measurements were taken prechase at 3 min (pink dot) and postchase at 4, 5, 7.5 and 10 min (blue gradient dots; light to dark, respectively). Each experiment was repeated independently at two concentrations of each enzyme with n = 3. h, Processivity (P) of wild-type Dis3L2 versus ΔCSD. i, Processivity (P) of wild-type Dis3L2 versus Δ123H. The error bars in plots d and e represent s.e.m. from the global fit of data from the enzyme titrations (nine Dis3L2 concentrations with n = 5) and pulse–chase experiments (two Dis3L2 concentrations with n = 4). The error bars for the processivity plots in c, h and i show propagated errors calculated from the s.e.m. of kf and koff derived from the same global fit of the data.
