Fig. 5.

Deconvolution of RNA length and sequence effects on Rrp6p activity. (A) Reaction scheme for competition experiments. (B) Representative competition reactions of A₃ (300 μM; A₃₆, 1 nM; Rrp6p, 220 nM) and A₄ (300 μM; A₃₆, 1 nM; Rrp6p, 220 nM). (C) Observed rate constants (k_obs) for the degradation of A₃₆ at single-nucleotide resolution in the absence (solid circles) and presence of A₃ (open circles) and A₄ (triangles), under conditions noted in A. Datapoints represent an average of 3 independent measurements. Error bars show one SD. (D) Apparent dissociation constants for productive binding (K_1/2^P) of A₃ and A₄ obtained experimentally and calculated values assuming similar scaling of inhibition by A₃ and A₄ (SI Appendix, Fig. S6). (E) Reaction scheme for competition experiments. (F) Representative competition reactions of 5′-GAC-3′ (300 μM; A₃₆, 1 nM; Rrp6p, 220 nM) and 5′-AGAC-3′ (300 μM; A₃₆, 1 nM; Rrp6p, 220 nM). (G) Observed rate constants (k_obs) for the degradation of A₃₆ at single-nucleotide resolution in the absence (solid circles) and presence of 5′-GAC-3′ (open circles) and 5′-AGAC-3′ (triangles), under conditions noted in E. Datapoints represent an average of 3 independent measurements. Error bars show one SD. (H) Apparent dissociation constants for binding of 5′-GAC-3′ and 5′-AGAC-3′ obtained from competitive inhibition experiments. (I) Contributions of upstream RNA (light gray) and 3′-terminal quadruplet (dark gray) to free binding energy for productive binding for each nucleotide for A₃₆. (J) Contributions of upstream RNA (light gray) and 3′-terminal quadruplet (dark gray) to free binding energy for productive binding for each nucleotide for R₃₆ (sequence on the right). (K) Contributions of the upstream RNA component to free binding energy for productive binding for each nucleotide for substrates indicated on the right.