Structural basis of promoter recognition by human TFIID

The eukaryotic transcription initiation machinery is a cornerstone of the central dogma of biology and a primary control point for gene regulation in cells. In eukaryotic cells, initiation of transcription by RNA polymerase II (Pol II) requires a vast assemblage of proteins called the transcription preinitiation complex (PIC), which functions to recognize the promoter region of a gene, and to place the template DNA strand into the catalytic center of Pol II so that it can begin transcribing./nThe general transcription factor TFIID is a pivotal component of the PIC, and is primarily responsible for recognizing the promoter DNA and nucleating PIC assembly. TFIID is a large multi-subunit complex comprising the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs), which specifically interact with a variety of core promoter DNA sequences, general transcription factors, transcriptional activators, and epigenetic marks. Despite its importance in coordinating transcription initiation, a detailed mechanistic understanding of TFIID’s activities has been elusive. The high degree of conformational plasticity within the TFIID architecture has largely limited structural analysis to low-resolution electron microscopy studies. However, by improving the biochemical preparation of promoter-bound TFIID complexes and by exploiting the latest technological advances within the field of cryo-EM, we have been able to generate high-resolution structures of promoter-bound TFIID, showing with unprecedented detail the molecular means by which TFIID recognizes and binds to promoter DNA and forms a platform onto which the PIC can assemble./nThe subnanometer-resolution structure of the downstream promoter-binding region of TFIID has revealed the structural basis of TFIID’s recognition of conserved promoter sequence motifs, showing how the TAF1 and TAF2 subunits mediate all contacts between TFIID and the downstream core promoter using highly conserved regions rich in positively-charged residues. These structures have also allowed for an accurate model the TFIIA-TBP complex bound to the TATA-box DNA, showing that TFIIA interacts with other subunits within TFIID in order to localize TBP near the upstream region of the promoter, such that it can be properly positioned to enable accurate loading of RNA Pol II. By analyzing these structures in the context of previous structural and biochemical data, we have been able to generate a compelling model that explains the functional importance of TFIID’s large conformational rearrangement for accurate and regulated loading of TBP onto promoter DNA./nBy combining the TFIID structure with structural models of the remaining pieces of the transcription machinery, we have generated the first ever structural model of the full TFIID-based PIC. On top of explaining a large amount of previous biochemical data from various labs, this model yields novel insights into human PIC assembly, including the role of TFIID subunits in recruiting other components of the PIC to the promoter, and how the long observed conformational flexibility of TFIID is likely to be an important property that provides regulatory checkpoints throughout the process of PIC assembly and thus transcription initiation.

Advisor: Nogales, Eva