Extended Data Fig. 10: Contribution of the CSDs and trihelix-and-linker to degradation of structured substrates by human Dis3L2.
a–c, Kinetic profile for hairpinA-GCU14 degradation by the ΔCSD mutant at single-nt resolution: a, forward rate constants (orange squares), b, dissociation rate constants (pink triangles), and, c, association rate constants (purple circles) compared to WT Dis3L2 (gray), (the datapoints for kon at x = −3 and −4 were omitted due to large uncertainty). Error bars mark the SEM derived from a global fit of data from enzyme titrations (8 ∆CSD concentrations with n = 2) and pulse-chase experiments (2 ∆CSD concentrations with n = 3). d–f, Kinetic profile for hairpinA-GCU14 degradation by Δ123H (trihelix deletion) mutant at single-nt resolution: d, forward rate constants (orange squares), e, dissociation rate constants (pink triangles), and, f, association rate constants (purple circles) compared to WT Dis3L2. Error bars mark the SEM derived from a global fit of data from enzyme titrations (9 ∆123H concentrations with n = 3) and pulse-chase experiments (2 ∆123H concentrations with n = 4). g, Representative gels showing the degradation profile by 250 nM Δ123H mutant against 1 nM hairpinA-GCU14 and U34 substrates. Each experiment was repeated independently with similar results for n = 2 h–j, Fraction of substrate disappearance, fraction of intermediates, and fraction of end-product accumulation over time from reactions shown in g, k, Representative pulse-chase experiments for 1 nM hairpinA-GCU14 and 100 nM Dis3L2 helix mutants: Δ1H (Δ612-634), Δ2H (Δ632-647), Δ3H (Δ654-665) and ∆123H. Measurements were taken pre-chase at 5 min (pink dot), and post-chase at 6, 7, 10, and 15 min (blue gradient dots). Each experiment was repeated independently with similar results for n = 2.
