Fig. 5

Interaction of TS:N20 with zebrafish TS protein in vitro.

A, Gel mobility shift analysis of TS:N20 interaction with TS protein. A 20-nt TS RNA, which included nt 13?32, was synthesized and radio-labeled with ³²P. ³²P-labeled-TS:N20 RNA was incubated in the absence (lane 1) or presence of 100 ng (lane 2) of pure recombinant zebrafish TS protein. A ³²P-labeled 17-nt TS encompassing nt 13?32 with a deletion of the GCU sequence, Δ20-TS RNA, was incubated in the presence of 100 ng of zebrafish TS protein (lane 3). Lanes 4 and 5 represent the other two mutation variants, Δ20?18-TS RNA and Δ20?23-TS RNA, respectively, incubated with 100 ng of pure TS protein. TS:N25 RNA was incubated with 100 ng TS protein in the presence of 100 ng unlabeled full-length TS mRNA (lane 6). Samples were resolved on a non-denaturing 5% acrylamide gel and visualized by autoradiography. The specific RNA?protein complex is indicated (arrow). B, Competition analysis using TS oligomers with TS protein. ³²P-labeled full-length TS RNA (10⁵ cpm) was incubated with 200 ng of pure zebrafish recombinant TS protein in the presence of 0?10-fold molar excess competitor, TS:N20 RNA (panel 1), Δ20-TS RNA (panel 2), Δ20?18-TS RNA (panel 3) and Δ20?23-TS RNA (panel 4). The RNA?protein complex was resolved on a non-denaturing 5% acrylamide gel and visualized by autoradiography.