Base-pairing interactions between substrate RNA and H/ACA guide RNA modulate the kinetics of pseudouridylation, but not the affinity of substrate binding by H/ACA small nucleolar ribonucleoproteins

Affinity of the snR34 H/ACA snoRNP for short substrate RNA variants. Affinity of the snoRNP was determined for each substrate RNA variant using nitrocellulose filtration. Each panel shows a representative data set of triplicate experiments. Full data sets are shown in Supplemental Figure S1. The dissociation constant (K_D) was determined by fitting the data to a hyperbolic equation (smooth lines—see Materials and Methods). Dissociation constants are listed in Table 1. (A) 3′ hairpin substrate wild-type. (B) 3′ hairpin substrate Δ1–2. (C) 3′ hairpin substrate Δ1–4. (D) 3′ hairpin substrate Δ14–17. (E) 3′ hairpin substrate Δ16–17. (F) 3′ hairpin substrate Δ12–17. (G) 3′ hairpin substrate Δ1,12–17. (H) 3′ hairpin substrate Δ1–2,12–17. (I) 3′ hairpin substrate 10CC-GG. (J) 3′ hairpin substrate 7CU-GA. (K) 3′ hairpin substrate G7 insert. (L) 5′ hairpin substrate wild-type. (M) 5′ hairpin substrate YRA1.