FIGURE 1.

Effect of Ssh10b binding on RNA structure. A, binding of Ssh10b to partial B2 RNA, rGrG(rArCrUrG)₈, and 30-nt oligo(rA). Ssh10b was incubated with a 5′-³²P-labeled RNA fragment for 10 min at 22 °C. Samples were subjected to electrophoresis in a 12 or 15% polyacrylamide gel. The gel was exposed to x-ray film. Protein concentrations in lanes 1–7 were 0, 0.01, 0.04, 0.16, 0.63, 2.5, and 10 μm, respectively. Apparent K_D values represent an average of at least three independent measurements. B, a diagram showing the predicted secondary structure of the partial B2 RNA. Sites cleaved by RNase T1 or modified by DMS in the absence of Ssh10b are marked by ▿ or ○, respectively, whereas sites that became sensitive to RNase T1 cleavage or DMS modification in the presence of Ssh10b are indicated by ▾ or ●, respectively. The sequence of the RNA used for crystallization is indicated by the gray rectangle. C, footprinting analysis of RNase T1 cleavage of partial B2 RNA in the presence of Ssh10b. 5′-³²P-Labeled B2 RNA was incubated with Ssh10b at 22 °C for 10 min. RNase T1 (1 unit) was added, and the mixture was incubated at 22 °C for 15 min. RNase T1 ladders were prepared by carrying out the cleavage reaction under denaturing conditions. RNA ladders were generated by incubating the radiolabeled RNA for 5 min at 90 °C under alkaline conditions. Reaction products were subjected to electrophoresis in 15% polyacrylamide containing 8 m urea. The gel was exposed to x-ray film. D, footprinting analysis of DMS modification of partial B2 RNA in the presence of Ssh10b. B2 RNA was incubated with Ssh10b at 22 °C for 10 min. DMS (0.5%) was added, and the samples were incubated at 22 °C for 5 min. Modifications were mapped by primer extension with reverse transcriptase. Reaction products were subjected to electrophoresis in 15% polyacrylamide containing 8 m urea. The gel was exposed to x-ray film.