TFIID subunits

Detailed biochemical studies revealed how the Pol II preinitiation complex, comprising a Pol II molecule and general transcription factors bound to a promoter region of DNA, is assembled. In these studies DNase I footprinting and electrophoretic mobility shift assays were used to determine the order in which Pol II and general transcription factors bound to TATA-box promoters. Because the complete, multisubunit TFIID is difficult to purify, researchers used only the isolated TBP component of this general transcription factor in these experiments. Pol II can initiate transcription in vitro in the absence of the other TFIID subunits. 

At present, approximately 13 polypeptides have been isolated as TAFs in all species studied, and they are listed in Table I (Tora, 2002). Earlier studies were performed almost exclusively in Drosophila, human cells, and budding yeast, whereas more recent studies in Caenorhabditis elegans indicate that it is also a useful model system, especially for studying the role of TAFs during development (Walker et al., 2001). In some species, additional TAFs have been isolated, but these have not been found universally, and it remains to be seen whether they are bona fide TFIID subunits. 

The side of the p sheet that faces away from DNA is covered by two long a helices. One of these helices contains a number of basic residues from the middle segment of the polypeptide chain while the second helix is formed by the C-terminal residues. Residues from these two helices and from the short loop that joins the two motifs (red in Figure 9.4) are likely candidates for interactions with other subunits of the TFIID complex, and with specific transcription factors. 

Given that these proteins have properly assembled, the initiation complex is ready to start transcription. How does the enzyme get started A component of TFIID, again a multi-subunit complex TFIIH, unwinds the DNA and phosphorylates serine-5 of the C-terminal tail (CTD) of the largest polymerase subunit (Rpbl). Serine-5 phosphorylation and phosphorylation of serine-2 (by pTEFb) are required to release the enzyme from the other components of the initiation complex and to start RNA synthesis. 

Figure 37-10. Two models for assembly of the active transcription complex and for how activators and coactivators might enhance transcription. Shown here as a small oval is TBP, which contains TFIID, a large oval that contains all the components of the basal transcription complex illustrated in Figure 37-9 (ie, RNAPII andTFIIA,TFIIB, TFIIE,TFIIF, and TFIIFI). Panel A The basal transcription complex is assembled on the promoter after the TBP subunit of TFIID is bound to the TATA box. Several TAFs (coactivators) are associated with TBP. In this example, a transcription activator, CTF, is shown bound to the CAAT box, forming a loop complex by interacting with a TAF bound to TBP. Panel B The recruitment model. The transcription activator CTF binds to the CAAT box and interacts with a coactivator (TAF in this case). This allows for an interaction with the preformed TBP-basal transcription complex. TBP can now bind to the TATA box, and the assembled complex is fully active.

TAF 250 TBP-Associated Factor II 250. Another direct connection between acetylation and transcriptional activation was demonstrated with the discovery that one of the TAFII (TATA-binding protein [TBP]-associated factor) subunits of the general transcription factor TFIID is itself a HAT. TFIID is one of the general factors required for the assembly of the RNA polymerase II transcription preinitiation... 

The best-characterized coactivator is the transcription factor TFIID (Fig. 28-27). In eukaryotes, TFIID is a large complex that includes TBP and ten or more TBP-associated factors (TAFs). Some TAFs resemble histones and may play a role in displacing nucleosomes during the activation of transcription. Many DNA-binding transactivators aid in transcription initiation by interacting with one or more TAFs. The requirement for TAFs to initiate transcription can vary greatly from one gene to another. Some promoters require TFIID, some do not, and some require only subsets of the TFIID TAF subunits. 

Another important coactivator consists of 20 or more polypeptides in a protein complex called mediator (Fig. 28-27) the 20 core polypeptides are highly conserved from fungi to humans. Mediator binds tightly to the carboxyl-terminal domain (CTD) of the largest subunit of Pol II. The mediator complex is required for both basal and regulated transcription at promoters used by Pol II, and it also stimulates the phosphorylation of the CTD by TFIIH. Both mediator and TFIID are required at some promoters. As with TFIID, some DNA-binding transactivators interact with one or more components of the mediator complex. Coactivator complexes function at or near the promoter s TATA box. 

TBP TATA binding protein, the protein subunit of TFIID that binds TATAA sequence at -30 bp from the start of transcription. 

The TAF subunits of TFIID appear to play a role in initiating transcription from promoters that lack a TATA box. For Instance, some TAF subunits contact the initiator element In promoters where It occurs, probably explaining how such sequences can replace a TATA box. Additional TFIID TAF subunits can bind to a consensus sequence A/G-G-A/T-C-G-T-G centered 30 base pairs downstream from the tran-... 

In vivo transcription Initiation by Pol II also requires TFIIA and, in metazoans, a complete TFIID protein, including its multiple TAF subunits as well as the TBP subunit. 

TFIID and its individual subunits have been subjected to intense study. As will be discussed below, complementary information from biochemical, genetic, and structural studies has shown, and will continue to give, a clearer picture of the forms and functions of this crucial component of the transcription machinery. 

The purification of TFIID was soon followed by the identification of its subunits (reviewed in Albright and Tjian, 2000 Burley and Roeder, 1996). The amino acid sequences of many TAFs revealed a high level of conservation. TFIID structure has changed little through evolution, with all... 

Fig. 1. Possible subunit structure of TFIID. On the basis of immuno-EM studies (Leurent et al., 2002), yeast two-hybrid data (Andel et al., 1999 Brand et al., 1999 Yatherajam et al., 2003), and in vitro protein interaction data (see text), a model of TFIID interactions was constructed. TAFs that appear in both TFIID and the SAGA HAT complex are shaded. BD1 and BD2 signify the bromodomains found in higher eukaryotic TAF1 or in yeast Bdfl. The exact stoichiometry of histone-like TAFs within each lobe is unclear some data indicate an octamer-like structure, whereas others support two tetramers.

---