Extended Data Fig. 7: Kinetic analysis of WT HsDis3L2 against hairpin RNAs.
a, Minimal kinetic model used for global data fitting. b, Gel showing degradation of hairpinA-GCU14 and ss-U34 to completion by Dis3L2 over a longer time course. c, Quantification of substrate disappearance from reaction shown in b. d, Fraction of hairpinA-GCU14 substrate disappearance measured over time at various Dis3L2 concentrations. e, Comparison of functional equilibrium dissociation constants for productive (K1/2P) and non-productive (K1/2NP) binding determined in the global fit of WT Dis3L2 on hairpinA-GCU14 (the data point for x = 15 was omitted due to large uncertainty). f, Correlation of experimental data vs the corresponding data calculated using the kinetic parameters for pre-steady state reactions of Dis3L2 on hairpinA-GCU14. g, Step-plot showing the change in substrate and intermediate species at discrete timepoints during pulse-chase reaction at 50 nM Dis3L2 on hairpinA-GCU14, the initial timepoint was taken before addition of chase at 3 min. h-j, Comparison of Dis3L2 processivity on hairpinA-GCU14, hairpinA-U16, and hairpinB-GCU14 (P HairpinB-GCU14, x = -11 was omitted due to large uncertainty). Error bars are shown as vertical lines. K1/2 errors in (e) were calculated using error propagation from SEM of koff and kon derived from global fit of data from enzyme titrations (9 concentrations with n = 5) and pulse chase experiments (2 concentrations with n = 4). Processivity error for i represents the Standard Deviation of the Mean with n = 3, while errors for h and j were calculated using the error propagation formula from the SEM of kf and koff as described above for panel e.
