Flexibility of the pseudouridylation pocket. (A) Schematic representations of the 5′ pocket of snR81 base-paired with its substrate. Shown are base-pairing between C41U-U2 and wild-type snR81 (a), between C41U-U2 and G65A-snR81 (compensatory mutant) (b), between 41(U)42-U2 and wild-type snR81 (c), between 41(U)42-U2 and 64(A)65-snR81 (compensatory mutant) (d), between 41(UU)42-U2 and wild-type snR81 (e), between 41(UU)42-U2 and 64(AA)65-snR81 (compensatory mutant) (f), between 41(UUU)42-U2 and wild-type snR81 (g), and between mutant 41(UUUU)42-U2 and wild-type snR81 (h). (B) Pseudouridylation assay in the context of snR81 and U2 RNAs shown in (A). Lanes 3 and 4 are mutant U2 (C41U) and wild-type snR81; lanes 5 and 6 are mutant U2 (C41U) and compensatory mutant snR81 (G65A); lanes 7 and 8 and lanes 17 and 18 are mutant U2 [41(U)42] and wild-type snR81; lanes 9 and 10 are mutant U2 [41(U)42] and compensatory mutant snR81 [64(A)65]; lanes 11 and 12 and lanes 19 and 20 are mutant U2 [41(UU)42] and wild-type snR81; lanes 13 and 14 are mutant U2 [41(UU)42] and compensatory mutant snR81 [64(AA)65]; lanes 21 and 22 are mutant U2 [41(UUU)42] and wild-type snR81; lanes 23 and 24 are mutant U2 [41(UUUU)42] and wild-type snR81; lanes 1 and 2 and lanes 15 and 16 are wild-type U2 and wild-type snR81 (controls). Ψ35, Ψ42, and Ψ44 are indicated. The asterisks mark the shifted Ψ42 bands.