Isolation of coactivators associated with the TATA-binding protein that mediate transcriptional activation.

Abstract

A key step in the regulation of transcription involves interactions between promoter-selective factors and various components of the transcriptional apparatus. Here we report the requirements for transcriptional activation directed by NTF-1, a developmentally regulated transcription factor in Drosophila. Reconstituted transcription with fractionated Drosophila basal factors reveals that activation by NTF-1 requires factors present in the endogenous TFIID fraction that are distinct from the purified TATA-binding protein (TBP). Glycerol gradient sedimentation and immunoprecipitation analyses indicate that TFIID is a multiprotein complex containing TBP and at least six tightly bound TBP-associated factors (TAFs). Preparations of TBP lacking TAFs after fractionation with denaturants no longer support activation by NTF-1 but retain basal level activity. Addition of immunopurified and renatured TAFs to free TBP restores the ability of NTF-1 to activate transcription without influencing basal transcription. These results suggest that one or more of the TAF polypeptides confer coactivator function.