FIG. 5.

Nuclease probing of the sgRNA1 promoter secondary structure. (A) Imidazole and T₁ RNase partial digests of 5′-end-labeled transcript of pT7SGP1 containing the sgRNA1 promoter region (negative sense). Gel-purified, end-labeled RNA was incubated in 0 M (0) and 0.8 M (I) imidazole under nondenaturing (native) conditions for 17 h (lanes 3 and 4). The nondenaturing T₁ digest was performed with 0.01 U of the enzyme for 5 min at 37°C (lane 5) (see Materials and Methods). Denaturing digests with the T₁ (cuts after G) and U2 (cuts A > U) RNases generated markers in lanes 1 and 2. The products were separated on a 6% polyacrylamide gel containing 8 M urea. The straight line beside lane 4 indicates predicted base-paired regions; dashed lines represent predicted single-stranded junctions and ambiguous regions; curved lines show predicted loops and bulges. Double and single arrows represent G’s that were cleaved strongly and weakly, respectively, by T₁ nuclease. Filled circles indicate uncut or very weakly cut G’s. (B) Solution structure of the sgRNA1 promoter. Arrowheads represent the T₁ analysis data; triangles represent the imidazole digestion data. Larger and smaller symbols indicate strong and weak cuts, respectively.