TFIID transcription initiation

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. 

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. 

TATA box. A sequence found in the promoter (part of the 5 flanking region) of many genes. Deletion of this site (the binding site of transcription factor TFIID) causes a marked reduction in transcription, and gives rise to heterogeneous transcription initiation sites. 

Transcriptional activators or repressors 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 the RNA polymerase II holoenzyme to stimulate transcription. Furthermore, contacts to chromatin proteins are formed to change the structure of chromatin during transcription initiation and ongoing elongation. Regulated transcription and chromatin modification generally requires the aid of further protein components, which are commonly termed coactivators (see Section 1.4.4.2). 

Promoters. The promoter sequences in eukaryotic DNA are larger, more complicated, and more variable than those of prokaryotes. Many promoters for RNA polymerase II contain consensus sequences, referred to as the TATA box, which occur about 25-30 bp upstream from the transcription initiation site. As illustrated in Figure 18.23, the binding of the transcription factor TFIID to the... 

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. 

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,... 

The general transcription factor TFllD is believed to be the key link between specific transcription factors and the general preinitiation complex. However, the purification and molecular characterization of TFllD from higher eucaryotes have been hampered by its instability and heterogeneity. All preparations of TFllD contain the TATA box-binding protein in combination with a variety of different proteins called TBP-associated factors, TAFs. When the preinitiation complex has been assembled, strand separation of the DNA duplex occurs at the transcription start site, and RNA polymerase II is released from the promoter to initiate transcription. However, TFIID can remain bound to the core promoter and support rapid reinitiation of transcription by recruiting another molecule of RNA polymerase. 

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.

Transcription factors (such as TFIID for RNA polymerase II) help to initiate transcription. The requirements for termination of transcription in eukaryotes are not well understood. All transcription can be inhibited by actinomycin D. In addition, RNA polymerase II is inhibited by a-amanitin (a toxin from certain mushrooms). These points are summarized in Table 1-3-1,... 

Requires sigma (a) to initiate at a promoter No sigma, but transcription factors (TFIID) bind before RNA polymerase... 

Initiation of transcription Promoter (-10) TATAAT and (-35) sequence Sigma initiation sub-unit required to recognize promoter ib omoter (-25) TATA and (-70) CAAT Transcription fectors (TFIID) bind promoter... 

In eukaryotes, general transcription factors must bind to the promoter to allow RNA polymerase II to bind and form the initiation complex at the start site for transcription. General manscription factors are common to most genes. The general transcription factor TFIID (the TATA fector) must bind to the TATA box before RNA polymerase II can bind. Other examples delude SP-1 and NF-.l that modulate basal transcription of many genes. 

Fig. 1.32. Phosphorylation of the C-terminal domain of RNA polymerase II and the beginning of transcription. The transition from the initiation complex to actual begin of transcription is regulated via phosphorylation of the C-terminal domain (CTD) of RNA polymerase II. In the above model it is assumed that initially a complex is formed between TFIID and a holoenzyme of RNA polymerase consisting of RNA polymerase II and associated factors (mediators, SRB proteins) and the basal transcription factors. Phosphorylation of the C-terminal domain effects the dissociation of the RNA polymerase from the initation complex and the transition to the elongation phase. A protein kinase, which is part of TFIIH, is responsible for the phosphorylation. The nature of the signal that induces phosphorylation of RNA polymerase II remains unknown. SRB suppressor of RNA polymerase B. After Koleske and Young (1995).

In one model it is assumed that transcriptional activators and coactivators increase the efficiency of formation of the pre-initiation complex. This function includes a restructuring of chromatin at the transcription start site. In this context the formation of the TFIID complex at the promoter plays an important role. 

Direct inhibition of the formation of a pre-initiation complex complexation of basal transcription factors, such as TFIID or TFIIB, or competition with TFIIB for binding to the promoter. An example for this type of repression is the negative cofactor NC2 (see 1.4.3.2). Transcription repression can also result from phosphorylation of the basal transcription factors. By this token, the repression of transcription observed during mitosis is attributed to the hyperphosphorylation of TBP and TAFs. 

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