FIGURE 4.

Characterization of the natural and unnatural YBL059W lariat isomer ratio in lariat RNA synthesized by the variations of the two-step strategy shown in Figure 3 ▶. Each pair of lanes shows lariat RNA without or with debranching enzyme treatment (±Dbr1p). The ³²P-radiolabel was in the L portion of each lariat product (12% PAGE). The ratio of the two cleavage bands is a direct measure of the ratio between natural and unnatural lariat isomers, because each isomer gives a distinct Dbr1p cleavage band as indicated. In these experiments, the stem secondary structure element (when present) favors formation of the natural lariat isomer. (A) Blocking and capping approaches to favor the natural lariat isomer. (B) Blocking and capping approaches to favor the unnatural lariat isomer. In B, these approaches must work against the natural lariat preference provided by the stem.