Figure 3

Recombinant purified Spt8, but not Spt3, protein is sufficient to interact with TBP. (A) Spt8 pull-down experiment using Strep-tagged TBP immobilized on Strep-Tactin resin shows that wild-type TBP (lanes 4 and 5) and TBP 153I mutant (lanes 8 and 9) bind to TBP but that TBP R171E is defective in this interaction (lanes 6 and 7). Input, supernatant (unbound) and bound fractions are labeled I, S and B, respectively. Spt8 proteins were detected via anti-Spt8 antibodies. (B) Same comments as for (A) except that Spt3 protein was used and detected using anti-HIS antibodies which recognized the C-terminal hexahistidine tag engineered on the recombinant Spt3. (C) Schematic of Spt8 and Spt8 (210–602) showing location of the seven WD40 repeats predicted by CD-Search (Marchler-Bauer and Bryant, 2004). The N-terminal acidic region is shown in light gray, and the predicted WD40 repeats in gray. (D) GST pull-down experiment shows that Spt8 (210–602) is sufficient for specific, direct interactions with TBP. GST only or GST-Spt8 (210–602) immobilized on glutathione–Sepharose was incubated with Strep-tagged TBP, TBP R171E or TBP T153I proteins, and visualized with anti-Strep antibodies (IBA) by Western blotting.