Reconstitution of partial TAF/TBP complexes and holo-TFIID reveals a mechanism of transcriptional activation

Transcriptional activation by sequence-specific enhancer binding proteins is mediated by a novel class of transcription factors, coactivators, some of which are subunits of the essential basal transcription factor TFIID. TFIID is a multiprotein complex comprised of the TATA-binding protein (TBP) and at least eight TBP associated factors (TAFs). Importantly, one or more TAF subunits in the TFIID complex is able to confer coactivator activities.; To investigate the mechanism of activation, I developed an in vitro protein-protein interaction assay to search for potential targets of the transcription factor Sp1. My studies show that Sp1 specifically interacts with the TAF{dollar}sb{lcub}rm II{rcub}{dollar}110 subunit of TFIID. Importantly, these results provide the first evidence for a direct physical interaction between an activator and a putative coactivator. To decipher the molecular basis of this interaction, I performed a functional domain analysis. The results indicate the existence of multiple contacts between Sp1 and TAF{dollar}sb{lcub}rm II{rcub}{dollar}110 that contributes to the highly specific nature of this interaction.; In an effort to further dissect the structure and function of TFIID, I optimized the expression and purification of all nine recombinant subunits of TFIID. As a crucial step toward functionally reconstituting TFIID activity, I participated in a collaborative effort to identify various TAF-TAF and TAF-TBP interactions in the TFIID complex. Our current view is that the TFIID complex is assembled by a plethora of TAF-TAF interactions and only a limited number of TAF-TBP interactions.; Using this knowledge, I succeeded in assembling various partial TBP/TAF complexes. First, I showed that TAF{dollar}sb{lcub}rm II{rcub}{dollar}110 is essential for Sp1 dependent activation in a reconstituted in vitro transcription reaction, demonstrating the functional significance of the activator-TAF interaction in the activation process. Interestingly, TAF{dollar}sb{lcub}rm II{rcub}{dollar}150 is also important for Sp1 activation even though this subunit does not contact Sp1. These results suggest that activation may require multiple TAFs, some of which make direct contact with activators, while others have a distinct function in the activation process. Two ternary complexes containing either TAF{dollar}sb{lcub}rm II{rcub}{dollar}150 or TAF{dollar}sb{lcub}rm II{rcub}{dollar}60 mediate activation by the transcription factor NTF-1, but not by Sp1. This suggests that distinct types of activators have different TAF requirements. This result also reveals that a single activator can function via dual or multiple pathways involving distinct TAFs. In addition, an important lesson from my studies on the function of TAFs in Sp1 and NTF-1 mediated activation is that a single TAF can perform multiple distinct functions in activation. For example, in the case of NTF-1, TAF{dollar}sb{lcub}rm II{rcub}{dollar}150 serves as an adaptor that contacts NTF-1. Although not contacted by Sp1, this subunit is also required to transmit the Sp1 activation signal to the transcriptional apparatus.; Finally, reconstituted holo-TFIID containing all nine recombinant subunits mediates activation by a variety of distinct activators that I tested, indicating that purified recombinant TFIID is able to recapitulate the transcriptional properties of endogenous TFIID. In conclusion, my studies have led to a better understanding of the mechanism by which upstream activators communicate with the basal machinery to activate transcription.