FIG. 6.

TFIIB also interacts with mSin3A. (A) Interaction between GST-TFIIB and radiolabeled mSin3A. Full-length, radiolabeled mSin3A was synthesized in vitro by a coupled transcription-translation protocol. The radiolabeled mSin3A protein was then incubated either with immobilized nonrecombinant GST or with an immobilized GST-TFIIB fusion. Radiolabeled mSin3A protein bound to the GST or GST-TFIIB matrix was eluted with soluble glutathione, resolved by SDS-PAGE, and visualized and quantified by PhosphorImager analysis. The percentage of mSin3A bound is depicted numerically below the lanes and was determined relative to the total radiolabeled mSin3A used in the assay (input). (B) Localization of domains within mSin3A able to bind to TFIIB. Radiolabeled TFIIB was synthesized in vitro by a coupled transcription-translation protocol. The radiolabeled TFIIB protein was then incubated with various domains of mSin3A (as denoted above the lanes), each expressed in bacteria as a GST fusion protein and immobilized on a glutathione-agarose column. Radiolabeled TFIIB bound to the different GST-mSin3A agarose matrices was eluted with soluble glutathione, resolved by SDS-PAGE, and visualized and quantified by PhosphorImager analysis. The binding of TFIIB to nonrecombinant GST (GST) was also tested in parallel as a negative control. The percentage of the input TFIIB bound by each GST fusion is also depicted below each lane.