Role of Human General Transcription Factors and Cofactors in Eukaryotic Transcription
Several general transcription factors, including TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, are essential for transcription on most eukaryotic promoters by RNA polymerase II (Pol II).
The core promoter-binding factor TFIID, which is comprised of the TATA-binding protein (TBP) and a dozen or so evolutionarily conserved Pol II-specific TBP-associated factors (TAFs), has intrinsic activity to recognize the TATA box, initiator, and downstream promoter elements and initiates preinitiation complex assembly on both TATA-containing and TATA-less promoters. TFIID also functions as a general cofactor in transducing regulatory signals to the general transcription machinery and plays a crucial role for transcription from chromatin templates due to multiple enzymatic activities inherent to its TAF components.
These diverse features have implicated TFIID as a central player in eukaryotic transcription and as a core promoter-defining factor.
To dissect the mechanisms of gene activation and repression in mammalian cells, we have employed human cell-free transcription systems reconstituted either with:
a) individually purified general transcription components (TFIIA, TFIIB, TBP, TFIIE, TFIIF, and PC4 coactivator) and highly purified epitope-tagged protein complexes (TFIID, TFIIH, and pol II)
b) with TFIID and a preassembled Pol II holoenzyme complex that contains components of the general transcription machinery as well as SWI/SNF chromatin remodeling factor, GCN5 histone acetyltransferase, and Mediator complex.
Our objectives are to define the biochemical activities of general transcription components in modulating gene activity and to uncover the combinatorial nature of eukaryotic gene regulation using well-defined cell-free transcription systems reconstituted with purified human proteins.