TFIID components

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 37-9. The eukaryotic basal transcription complex. Formation of the basal transcription complex begins when TFIID binds to the TATA box. It directs the assembly of several other components by protein-DNA and protein-protein interactions. The entire complex spans DNA from position -30 to +30 relative to the initiation site (+1, marked by bent arrow). The atomic level, x-ray-derived structures of RNA polymerase II alone and ofTBP bound to TATA promoter DNA in the presence of either TFIIB or TFIIA have all been solved at 3 A resolution. The structure of TFIID complexes have been determined by electron microscopy at 30 A resolution. Thus, the molecular structures of the transcription machinery are beginning to be elucidated. Much of this structural information is consistent with the models presented here.

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.

The formation of the PIC described above is based on the sequential addition of purified components in in vitro experiments. An essential feature of this model is that the assembly takes place on the DNA template. Accordingly, transcription activators, which have autonomous DNA binding and activation domains (see Chapter 39), are thought to function by stimulating either PIC formation or PIC function. The TAF coactivators are viewed as bridging factors that communicate between the upstream activators, the proteins associated with pol II, or the many other components of TFIID. This view, which assumes that there is stepwise assembly of the PIC—promoted by various interactions between activators, coactivators, and PIC components— is illustrated in panel A of Figure 37-10. This model was supported by observations that many of these proteins could indeed bind to one another in vitro. 

MLL (also named ALL-1, HRX, and HTRX), the human homolog of Drosophila trithorax, is a SET domain protein that methylates H3 at Lys-4. The enzymatic activity was enhanced with H3 acetylated at Lys-9 or Lys-14. MLL is a component of a large multiprotein complex composed of greater than 29 proteins, including TFIID, SWI/SNF remodeling complex and NuRD, a histone deacetylase complex. MLL binds to the promoter of Hox genes and regulates their expression [202,203]. 

Transcriptional activators can intervene as regulators at various steps in the initiation of transcription. They can interact with components of TFIID, as well as with components of RNA polymerase II, to stimulate transcription. Regulated transcription generally requires the aid of further protein components, which are commonly termed coactivators (see 1.4.3.2). An understanding of the details of coactivator function is only just emerging. 

Fig. 1.34 Activators and coactivators of transcription intiation. The figure shows the function of three groups of proteins that function as coactivators. The general cofactors mediate the interactions between the specific transcription activators and the TFIID complex as well as with various forms of the RNA polymerase II holoenzyme. The TAFs are components of the TFIID complex and serve as contact points for specific transcription activators. The mediators are components of various forms of holoenzymes of RNA polymerase II. SRB proteins belong to the class of mediators, which, among other things, interacts with the CTD of RNA polymerase. The simphfied diagram does not show the interactions with chromatin.

The TAFs are components of TFIID (see table 1.1) and are required for a regulated transcription (review Verriijzer and Tijan 1996, Bmley and Roeder, 1996 ). Thus, the stimulation of transcription by the transcriptional activators Spl and NTF-1 depends upon the presence of specific TAFs in the TFllD complex. The TAFs mediate interactions between the transcriptional activators and the TFllD complex in many cases direct protein-protein interactions could be demonstrated between the activators and TAFs. Some of the TAFs possess additional enzymatic activities which allow them to participate in the regulation of transcription. By this token, the histone acetylase and protein kinase activity of TAFn250 is ascribed a regulatory function in the remodeling of chromatin and in the control of the activity of the basal transcription factors. 

In the N-terminal region of p53, there is a transactivation domain which p53 uses to make contact with the transcription apparatus. Different protein binding sites have been identified in this region. These include binding sites for components of the TFIID complex and for coactivators such as the CBP/p300 coactivator (see 1.4.6). 

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. 

Fortunately, the complicated process of the assembly of the many components of PIC at transcription initiation sites can be simplified by substituting the multicomponent TFIID complex by TBP. The TBP—TATA-box complex represents a reduced PIC which can actually carry out transcription in vitro. 

The TATA-box-bInding protein (TBP). a component of TFIID, recognizes the TATA box.] After assembly, TFIIH opens the DNA double helix and phosphorylates the carboxyl-termina domain (CTD). allowing the polymerase to leave the promoter and begin transcription. 

Retention of transcription factors in mitotic chromosomes could provide one component of this molecular memory. Immunocytochemical and subcellular fraction of mitotic Hela cells indicated that some TFIID remained associated with mitotic chromatin (Segil et al., 1996). Thus, upon entry into interphase, TFIID could nucleate the formation of a productive transcription complex for a gene expressed in G2 of the prior cell cycle. The retention of other transcription factors Or enhancer proteins could promote the same effect. 

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. 

Transcriptional initiation of RNA pol II-dependent genes in eukaryotes involves the assembly of a large protein complex at the promoter that requires as many as eight multisubunit components RNA pol II, TFIID, TFTTB,... 

Fig. 14.7. Transcription apparatus. The TATA-binding protein (TBP), a component of TFIID, binds to the TATA box. Transcription factors TFII A and B bind to TBP. RNA polymerase binds, then TFII E, F, and H bind. This complex can transcribe at a basal level. Some coactivator proteins are present as a component of TFIID, and these can bind to other regulatory DNA binding proteins (called specific transcription factors or transcriptional activators).

Histone acetylase activity has been associated with a number of transcription factors and co-activators. The proteins ACTR (activator of the thyroid and retinoic acid receptor) and SRC-1 (steroid receptor co-activator) are involved in activation of transcription by several ligand-bound nuclear receptors, and both contain histone acetylase activity. TAF250, a component of TFIID, also contains histone acetylase activity, as does the co-activator p300/CBP (CREB binding protein), which interacts with the transcription factor CREB. 

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. 

New evidence indicates that the major mediators in remodeler recruitment may be bromo and chromo domain-containing proteins. Bromo domains mediate protein binding to acetyl-lysines in histones and other proteins (Jacobson et al., 2000), and chromo domains have been shown to bind methyl-lysines (Jacobs et al., 2002). For instance, TAF1, a component of TFIID, contains two tandem bromo domains that bind selectively to multiply acetylated H4 peptides (Jacobson et al., 2000). Furthermore, the Gcn5 bromo domain preferentially binds acetylated H4 K16 (Owen et al., 2000). 

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