FIGURE 5.

TRAM3066 assisted RNases to hydrolyse structured RNA. The highly structured PP6 RNA was used as the substrate of endonucleases and labeled with [γ -³²P] ATP. Using the same procedure as in Figure 4A, rEMSA was performed to detect the concentration of TRAM3066 shown on the top of the gel binding radiolabelled PP6 RNA (A). Ribonucleolytic assays were performed as described in the Experimental Procedures. PP6 RNA was first mixed with (+) or without (–) 1,000 pmol of TRAM3066 protein, and 0.2, 0.05, 0.0125, and 0.00625 ng (lanes 1–4 and 5–8) of RNase A (B) or 2, 1, 0.5, 0.25 U (lanes 1–8) of RNase T1 (C) was added. After 10 min of reaction at room temperature, the reaction mixture was electrophoresed on a 6% (RNase A assay) or 10% (RNase T1 assay) acrylamide gel at 150 V at room temperature. Arrows point to full-length PP6 RNA. 0, mock without RNases.