Figure 5.
The TIF32 CTD directly interacts with helices 16–18 of domain I of 18S rRNA in vitro. (A) Same as Figure 1G, with the HCR1-like domain (HLD) shaded. Arrows beneath the schematic depict the minimal segments required for interactions with the indicated molecules, based on results shown in C and reported previously (Valášek et al. 2002). The lines beneath the arrows depict the segments of TIF32 used in the Northwestern blot analysis of C with amino acid endpoints and clone designations indicated. The binding of each segment to full-length biotinylated 18S rRNA measured in C is summarized on the right. (B) Full-length TIF32 and NIP1 interact specifically with biotinylated 18S rRNA in Streptavidin pull-down assays. Biotinylated 18S rRNA, 25S rRNA, and β-globin mRNA, respectively, were incubated with [35S]-labeled PRT1 (lane 2), TIF32 (lane 5), or NIP1 (lane 8), and the RNA–protein complexes were precipitated with Streptavidin agarose (GIBCO-BRL), washed, and separated by SDS-PAGE. Gels were dried and subjected to autoradiography. The corresponding nonbiotinylated RNAs were used in lanes 3, 6, and 9. Lanes 1, 4, and 7 show 50% of the input amounts of [35S]-labeled proteins added to each reaction (In). Arrowheads indicate the full-length proteins. (C) The TIF32-CTD binds to biotinylated 18S rRNA in Northwestern assays. GST fusions to full-length TIF32 (lane 2), TIF32-Δ1 (lane 3), TIF32-Δ1 + 4 (lane 4), TIF32-Δ3 (lane 5), TIF32-Δ4 (lane 6), and TIF32-Δ6 (lane 7), or GST alone (lane 1), were expressed in Escherichia coli, immobilized on glutathione-Sepharose beads, and separated by SDS-PAGE. The resolved GST proteins were transferred to a membrane, renatured overnight, and incubated with biotinylated full-length 18S rRNA synthesized in vitro. The RNA–protein complexes were visualized by the Brightstar biodetect kit from Ambion. The amounts of fusion proteins used in the assay are shown in the upper panel stained with Gelcode Blue Stain Reagent (Pierce). (D) Same as in C except that various domains of 18S rRNA containing the nucleotides indicated in brackets were biotinylated and tested for binding to GST–TIF34-Δ4 or GST alone in Northwestern assays. Lanes 1 and 2 show the stained fusion proteins used in the assays and lanes 3–14 show the results of Northwestern assays for the indicated fusion proteins and biotinylated probes.