What really happens during transcription initiation?

From the Hahn lab, Basic Sciences Division, and collaborators

Transcription is the first step of gene expression, where a DNA sequence is copied into messenger RNA. In eukaryotes, transcription initiation starts with the assembly of the transcription pre-initiation complex (PIC) onto promoter DNA. The PIC comprises the general transcription factors and RNA polymerase II (Pol II), which is the main transcription enzyme. The general transcription factor IID (TFIID) is responsible for initially recognizing the core promoter, which begins the assembly of the PIC. Human TFIID is a trilobed complex (lobes A, B, and C) composed of TATA box binding–protein (TBP) and 13 evolutionarily conserved TBP-associated factors (TAF1 to TAF13), with six TAFs present in two copies. Together, TBP and the TAF subunits of TFIID directly interact with promoter DNA with the assistance of TFIIA, forming a platform for assembling the rest of the PIC.  The structure of TFIID and models postulating its functions has largely contributed to our knowledge of how eukaryotic transcription is regulated.  Nevertheless, a key challenge in understanding the molecular underpinnings of the recognition of promoter DNA by TFIID is the lack of a complete structural depiction of the complex.

Basic Sciences Division member Dr. Steve Hahn, whose laboratory has a long-standing interest in transcription, teamed up with Dr. Jeffrey Ranish’s lab at the Institute for Systems Biology and Dr. Eva Nogales’ laboratory at the University of California, Berkeley to tackle this challenge. In their work published in a recent issue of Science, they utilized cryo-electron microscopy (cryo-EM) to describe the various biochemical and/or conformational states of the complex, providing information on both the structure and dynamics of TFIID and its interaction with promoter DNA. Dr. Hahn explained: “The TFIID complex is required for nearly all transcription by RNA polymerase II.  Its functions include recognition of promoter sequences, delivering the TATA binding protein to promoters (even when they lack a TATA box), as a target of transcription activators and, interacting with chromatin.  Until now, the structural basis for many of these functions was unknown.”  


Structure of human TFIID in its canonical and engaged states Figure from publication

The authors report the cryo-EM structure of TFIID with a resolution of 4.3 Å for lobe C, 4.5 Å for lobe B, and 9.8 Å for lobe A. Together with chemical cross-linking mass spectrometry and structure prediction, a complete structural model of the evolutionarily conserved core of TFIID was generated. TFIID is built on a dimeric scaffold of TAFs, containing at its center a TAF6 dimer in lobe C that connects to lobes A and B. By computational sorting of cryo-EM images, the authors characterized the conformational landscape of TFIID in the presence of TFIIA and promoter DNA. Several distinct states were observed, allowing a mechanistic model for TFIID promoter binding to be generated. The authors propose that TFIID first binds the downstream core promoter elements through TAF1 and TAF2. This binding and the flexible attachment of lobe A help position the upstream DNA in proximity to TBP, which scans for a TATA box or its sequence variants. Engagement of upstream core promoter sequences by TBP is facilitated by TFIIA interacting with TAF4 and TAF12 within lobe B. When TBP finally binds the promoter, it releases from lobe A, opening the binding site for TFIIB, which can then recruit Pol II.

Since all the structural work was carried out in Dr. Eva Nogales’ laboratory in California, Dr. Hahn explained how his lab got involved in this collaborative effort: “Former postdoc Sebastian Grünberg, in collaboration with Jeff Ranish’s lab at the Institute for Systems Biology, used crosslinking-mass spectrometry analysis to map protein-protein interactions within the human TFIID complex.  Since the resolution of the cryo-EM data was not sufficient on its own to determine the structure, the crosslinking results were important to identify some of the protein-protein interactions, the subunit positions within the TFIID structure and to develop and validate the final structural model.  Our lab has much experience in the crosslinking-MS analysis and, several years ago, our two labs decided that this approach might be an important component for augmenting the cryo-EM work.  Eva’s lab supplied Sebastian with purified TFIID and Sebastian together with Jie Luo in Ranish’s lab performed the analysis.”

This work has lead to a mechanistic model of how TFIID prevents TBP from nonspecifically engaging with DNA outside of gene promoters, thereby preventing aberrant PIC assembly and erroneous transcription initiation. The model also suggests how TFIID loads TBP onto TATA-less promoters and how activators and chromatin marks may direct TFIID recruitment and PIC assembly. For Dr. Hahn’s lab, this is exciting news; they have a long-standing interest in the mechanism of transcription from promoters lacking a TATA-box. “We work in the yeast system, where TFIID probably functions somewhat differently from human TFIID.  We’ve recently developed a TFIID-dependent yeast transcription system and our long-term goal is to understand how TFIID is incorporated within the larger set of basal factors and polymerase.  This promises to reveal more about how TFIID promotes transcription initiation” revealed Dr. Hahn, when asked about what bodes next for his lab. These new results also lead to innovative ways of thinking about how regulatory factors and chromatin modifications work to modulate transcription.

Patel AB, Louder RK, Greber BJ, Grunberg S, Luo J, Fang J, Liu Y, Ranish J, Hahn S, Nogales E. 2018. Structure of human TFIID and mechanism of TBP loading onto promoter DNA. Science 362, eaau8872

Funding was provided by the National Institutes of Health, the Swiss National Science Foundation, and the Howard Hughes Medical Institute.

Research reported in the publication is a collaboration between Cancer Consortium members Steve Hahn (Fred Hutch) and Jeffrey Ranish (UW).