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. 2013 Jan 17;152(1-2):120-31.
doi: 10.1016/j.cell.2012.12.005.

Human TFIID binds to core promoter DNA in a reorganized structural state

Michael A Cianfrocco  1 , George A Kassavetis, Patricia Grob, Jie Fang, Tamar Juven-Gershon, James T Kadonaga, Eva Nogales
Affiliations

Human TFIID binds to core promoter DNA in a reorganized structural state

Michael A Cianfrocco et al. Cell. .

Abstract

A mechanistic description of metazoan transcription is essential for understanding the molecular processes that govern cellular decisions. To provide structural insights into the DNA recognition step of transcription initiation, we used single-particle electron microscopy (EM) to visualize human TFIID with promoter DNA. This analysis revealed that TFIID coexists in two predominant and distinct structural states that differ by a 100 Å translocation of TFIID's lobe A. The transition between these structural states is modulated by TFIIA, as the presence of TFIIA and promoter DNA facilitates the formation of a rearranged state of TFIID that enables promoter recognition and binding. DNA labeling and footprinting, together with cryo-EM studies, were used to map the locations of TATA, Initiator (Inr), motif ten element (MTE), and downstream core promoter element (DPE) promoter motifs within the TFIID-TFIIA-DNA structure. The existence of two structurally and functionally distinct forms of TFIID suggests that the different conformers may serve as specific targets for the action of regulatory factors.

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Figures

Figure 1
Figure 1. Lobe A exists in a range of positions relative to a stable BC core within the purified TFIID complex
2D reference-free cryo-EM class averages of TFIID shown alongside contour models to indicate lobe positions within each average. Lobe A is colored yellow whereas the stable BC core is colored blue. Scale bar in (A) is 200 Å. See also Figure S1 & Supplementary Movie 1.
Figure 2
Figure 2. TFIID’s conformational landscape changes in response to TFIIA and SCP DNA
Distribution of lobe A positions relative to the stable BC core for TFIID (A), TFIID-SCP (B), TFIID-TFIIA (C), and TFIID-TFIIA-SCP samples (D). Inset within (A): Class averages corresponding to specific lobe A measurements from the TFIID sample. See also Figure S1.
Figure 3
Figure 3. TFIIA-mediated binding of SCP DNA to the rearranged state of TFIID
Cryo-EM 2D reference-free class averages of the rearranged conformation (A) and the canonical conformation (B) shown alongside contour models. 3D reconstructions of the rearranged conformation for TFIID-TFIIA-SCP at 32 Å (C) and TFIID at 35 Å (D), where the density attributed to DNA in (C) is shown in green. 3D reconstructions of the canonical conformation for TFIID-TFIIA-SCP at 32 Å (E) and TFIID at 35 Å (F). The stable BC core is colored blue for each model. Lobe A is colored yellow for (C) and (D) and orange for (E) and (F). Scale bar is 200Å. See also Figures S2 & S3.
Figure 4
Figure 4. Organization of promoter DNA and TFIIA within the TFIID-IIA-SCP complex
(A) Cryo-EM reconstruction of TFIID-IIA-SCP(−66) at 35 Å (left column) aligned with the cryo-EM reconstruction of TFIID-IIA-SCP (center) and their difference map (right). DNA density is colored in green, whereas lobe A and the BC core are colored in yellow and blue, respectively. Scale bar is 100 Å. 2D reference-free class averages for the rearranged TFIID-TFIIA-SCP complex with Nanogold labels on SCP DNA at +45 (B), SCP DNA at TATA (C), and TFIIA (D). (E) 2D reference-free class average for the canonical state of TFIID-TFIIA-SCP containing Nanogold labeled on TFIIA. 3D models are shown alongside high defocus averages (density threshold at σ = 3.5) with a gold sphere marking the localization of Nanogold for each experiment. See Extended Experimental Procedures for a detailed discussion of generating low defocus class averages. Summary of gold labeling results for (B – D) are shown in (A), 2nd row.
Figure 5
Figure 5. Core promoter architecture dictates TFIIA-dependent and TFIIA- independent interactions of TFIID with core promoter DNA
DNase I (A) and MPE-Fe (B) footprinting of TFIID-SCP and TFIID-TFIIA-SCP. (C) DNase I footprinting on ‘wild type’ and ‘mutant’ SCP DNA sequences. 5′ labeled downstream probes were analyzed for DNase I protection in the presence or absence of TFIIA for wild type, mutant TATA (mTATA), Inr (mInr), and MTE/DPE (mMTE/DPE). See also Figures S4 & S6.
Figure 6
Figure 6. Structural description of the rearranged TFIID-IIA-SCP complex relative to the TSS
Promoter DNA for SCP(−66) docked into the TFIID-TFIIA-SCP(−66) map (shown in mesh). (A) & (B): DNA models were taken from Figure 5C for sequences from −66 to +45. Asterisk (*) in (B) indicates DNase I hypersensitive site at +3. Black lines in (B) indicate regions of continuous protection along SCP helix. (C) The proposed location of the crystal structures of TBP-TFIIA-TFIIB on TATA box DNA is in close proximity to Inr (PDB accession codes 1VOL and 1NVP). The unresolved DNA path between Inr and TATA is indicated by a dotted line. See also Supplementary Movie 2.
Figure 7
Figure 7. Model of TFIID’s interaction with core promoter DNA in a conformation- and TFIIA-dependent fashion
Top row (A – C) describes conformations adopted by TFIID-TFIIA, while bottom row (D – F) describes TFIID alone. (A & B): TFIIA stabilizes TFIID in the canonical conformation. The addition of SCP DNA stabilizes the rearranged conformation for the ternary complex TFIID-TFIIA-SCP (C). In contrast to TFIID-TFIIA (A & B), TFIID adopts a conformational landscape that populates canonical and rearranged states equally (D & E). Upon addition, SCP DNA is bound by TFIID in the rearranged conformation (F). Brackets ([ ]) denote that (F) was observed only through biochemical footprinting.
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