Mechanistic studies of mammalian transcriptional repressor RBP (CBF1) and transcriptional coactivator TFIIA

Five general transcription factors (GTFs), TFIID, TFIIB, TFIIF, TFIIE and TFIIH are required by RNA pol II to specifically recognize and initiate transcription from protein coding genes. A sixth factor, TFIIA, has been shown to have stimulatory activities.; Transcription initiation is viewed as a master regulatory step since both activators and repressors of transcription converge here to regulate the levels of gene expression. RBP is a cellular protein recently characterized as a transcriptional repressor in mammalian cells. We have focused our studies on determining the mechanism by which RBP represses transcription, taking the simple promoter from the adenovirus plX gene (plX) as a model. Our results show that even though RBP binds DNA very close to other transcription factors that are crucial for activated plX transcription, RBP does not repress by occluding their binding to the promoter. Instead, RBP interacts with two transcriptional coactivators: TFIIA, and the dTAF{dollar}sb{lcub}rm II{rcub}{dollar}110 component of TFIID. In addition, RBP represses by affecting the early stages of transcription preinitiation complex formation, since addition of TFIIA to the TFIID-DNA subcomplex is necessary and sufficient to preclude repression by RBP. Taken together, these studies suggest that upon interaction with TFIIA and dTAF{dollar}sb{lcub}rm II{rcub}{dollar}110, RBP perturbs optimal interactions between these two proteins and other factors within the transcription preinitiation complex required for transcriptional activation.; We have also studied TFIIA. This protein consists of three subunits of 37 kDa {dollar}(alpha){dollar}, 19 kDa {dollar}(beta){dollar}, and 14 kDa {dollar}(gamma){dollar} in size. Functional assays have shown that TFIIA has three associated activities: (a) stabilizes the binding of TFIID (or TBP) to DNA, (b) antirepression, and (c) coactivator of transcription. During purification of native TFIIA from HeLa cell nuclear extract, a form of TFIIA containing only the {dollar}beta{dollar} and {dollar}gamma{dollar} subunits was isolated (mini-TFIIA). Functional studies showed that mini-TFIIA is able to interact with TBP and to overcome transcriptional repression; however, it is deficient in supporting transcriptional activation. These studies indicate that TFIIA has two distinct separable functions: antirepression and transcriptional coactivation. Moreover, we found that the {dollar}alpha{dollar} subunit is required for the coactivator function whereas for antirepression, this subunit is dispensable.