Figure 3.

(A) The sequence of sR47, a C/D guide RNA from P.abyssi (6). Note that in the Pyrococcus sRNA database (http://rna.wustl.edu/snoRNAdb/), this RNA was annotated as sR31. The AS and AS′ guide sequences are complementary to putative methylation sites in tRNAGly(UCC) and tRNALeu(CAA), respectively. (B) Binding of L7Ae to sR47 as measured by EMSAs. Uniformly ³²P-labeled RNA was incubated in the presence of L7Ae from P.abyssi and then analyzed by native gel electrophoresis. The L7Ae concentrations were 62.5, 125, 250, 500 and 1000 nM. RNP1 and RNP2 are RNA–protein complexes. Dissociation constants (Materials and Methods) are indicated at the top of each panel. Wild-type (WT) sR47 and the various mutants are indicated at the bottom of each panel. In mut C′/D′, CUGA was changed to CUUC and GUGAUGA was changed to GUUCUGA, thus, disrupting the two AG sheared base pairs. Comparable changes were made in mut C/D. In mut C/D and C′/D′ all four motifs were mutated. The GGA spacer between the D′ and C′ motifs was changed to CCC in the spacer CCC mutation.