Figure 3.

Direct interaction between recombinant K-Rev and the K-RRE in vitro. (A) The RNA gel-shift procedure used has been described (25). All reactions contained 1 μg of yeast tRNA and 4 μg of rRNA as nonspecific competitors. A ³²P-labeled 434-nt K-RRE probe or a 293-nt V-RRE probe was prepared by in vitro transcription, and ≈1 ng (4 × 10⁴ cpm) of probe was then added to a premade in vitro reaction containing no (lanes 1 and 6, Neg.), ≈3 ng (lanes 2 and 7), ≈9 ng (lanes 3 and 8), ≈27 ng (lanes 4 and 9), or ≈81 ng (lanes 5 and 10) of GST-K-Rev. After incubation for 10 min at 4°C, reaction products were resolved on a 5% native polyacrylamide gel and visualized by autoradiography. C1 and C2 indicate distinct protein complexes. (B) The procedure was performed as described for A, except that the reaction mix, containing 9 ng of GST-K-Rev, was preincubated for 10 min at 4°C with an unlabeled K-RRE or H-RRE competitor RNA. (lane 1, Neg.) No added GST-K-Rev; (lane 2) no additional RNA competitor; (lanes 3 and 7) ≈3 ng of RNA competitor; (lanes 4 and 8) ≈12 ng of RNA competitor; (lanes 5 and 9) ≈50 ng of RNA competitor; (lanes 6 and 10) ≈200 ng of RNA competitor.