FIGURE 3.

Phosphate backbone probing of A.n. COB pre-RNA. (A) Portion of a gel showing iodine cleavage of phosphorothioate substituted RNAs. A.n. COB pre-RNA was transcribed in the presence of one phosphorothioate analog and its 5′ end labeled. A.n. COB RNAs were incubated in various concentrations of Mg²⁺ as well as in the presence of I-AniI and then subjected to cleavage with iodine. The products were separated by denaturing gel electrophoresis and quantified. Nucleotide numbers are indicated at the side of the gel and the identity of the phosophorothioate substitution at the top. Lanes 1, 6, 11, and 16, mock-treated RNA; lanes 2, 7, 12, and 17, 0 mM Mg²⁺; lanes 3, 8, 13, and 18, 5 mM Mg²⁺; lanes 4, 9, 14, and 19, 75 mM Mg²⁺; lanes 5, 10, 15, and 20, 5 mM Mg²⁺ plus I-AniI. (B) Example of the quantification of the iodine mapping data. The histogram shows PhosophorImager counts in each lane that were normalized and expressed as a ratio relative to 0 mM Mg²⁺. (C) Summary of iodine cleavage data on the secondary structure of A.n. COB pre-RNA. Colored shading shows condition-specific protections, and positions that are cleaved under all conditions are demarcated by a gray oval. Three independent experiments were performed, and positions were scored as protected if a twofold or greater reduction in iodine cleavage relative to 0 mM Mg²⁺ was observed in each experiment. The relative amount of cleavage for unmarked positions could not be assigned because of inconsistencies between experiments, degradation, or poor resolution in the gel. Regions expected to be within a solvent inaccessible core include J3/4, J4/5, J5/4, J6/7, J8/7, the A-rich bulge in P5a, and the P7 helix. A portion of the P9.1 helix is shown schematically. (D) Position of putative I-AniI binding sites on the three-dimensional structure of the Tetrahymena intron. Red shading includes protected phosphate residues that lie on one side of the intron core. Other I-AniI-specific protections (blue shading) are on the opposite side of this surface and are likely due to interactions with the P1 helix.