L7Ae is dispensable for modification of strongly associated substrates by C/D RNPs. (A) Substrate interactions of sR1c, SL12, and Ct snR61 RNAs. Gibbs free energy (ΔG) is indicated for each guide-substrate duplex. The D substrate of sR1c was mutated to change the GC pair at position 3 of the guide-substrate duplex to a GU or GG pair. (B) Activity of sR1c and sR1c-ex where the D and D′ guides were exchanged. The D or D′ substrates and their premethylated versions were modified at 70°C by Ss C/D RNPs assembled with (w/) or without (w/o) L7Ae. Lanes 1–3 are copied from Figure 1C. (C) The D substrate mutants (S) and their premethylated versions (M) were modified at 70°C by sR1c RNPs assembled with (w/) or without (w/o) L7Ae. The relative modification levels were normalized to modification of wild-type (GC) substrate by sR1c holo-RNP. Averages and standard deviations were calculated from three replications. (D) Modification at 70°C and 50°C. sR1c, SL12, and Ct snR61 RNPs were assembled with or without L7Ae and assayed on their cognate D and D′ substrates and mixed premethylated substrates. The relative modification levels were normalized to the reactions catalyzed by holo-RNPs at 70°C. Averages and standard deviations were calculated from two replications.
