Regulation of Gene Expression in Eukaryotes:- DNA-Binding Transactivators and Coactivators Facilitate Assembly of the General Transcription Factors
To continue our exploration of the regulation of gene expression in eukaryotes, we return to the interactions between promoters and RNA polymerase II (Pol II), the enzyme responsible for the synthesis of eukaryotic mRNAs. Although most (but not all) Pol II promoters include the TATA box and Inr (initiator) sequences, with their standard spacing , they vary greatly in both the number and the location of additional sequences required for the regulation of transcription. These additional regulatory sequences are usually called enhancers in higher eukaryotes and upstream activator sequences (UASs) in yeast. A typical enhancer may be found hundreds or even thousands of base pairs upstream from the transcription start site, or may even be downstream, within the gene itself. When bound by the appropriate regulatory proteins, an enhancer increases transcription at nearby promoters regardless of its orientation in the DNA. The UASs of yeast function in a similar way, although generally they must be positioned upstream and within a few hundred base pairs of the transcription start site. An average Pol II promoter may be affected by a half-dozen regulatory sequences of this type, and even more complex promoters are quite common.
Successful binding of active RNA polymerase II holoenzyme at one of its promoters usually requires the action of other proteins (Fig. 1), of three types: (1) basal transcription factors , required at every Pol II promoter; (2) DNA binding transactivators, which bind to enhancers or UASs and facilitate transcription; and (3) coactivators. The latter group act indirectly—not by binding to the DNA—and are required for essential communication between the DNA-binding transactivators and the complex composed of Pol II and the general transcription factors. Furthermore, a variety of repressor proteins can inter fere with communication between the RNA polymerase and the DNA-binding transactivators, resulting in re pression of transcription (Fig. 28–27b). Here we focus on the protein complexes shown in Figure 28–27 and on how they interact to activate transcription.
TATA-Binding Protein The first component to bind in the assembly of a preinitiation complex at the TATA box of a typical Pol II promoter is the TATA-binding protein (TBP). The complete complex includes the basal (or general) transcription factors TFIIB, TFIIE, TFIIF, TFIIH; Pol II; and perhaps TFIIA (not all of the factors are shown in Fig. 1). This minimal preinitiation complex, however, is often insufficient for the initiation of transcription and generally does not form at all if the promoter is obscured within chromatin. Positive regulation leading to transcription is imposed by the trans activators and coactivators.
DNA-Binding Transactivators The requirements for trans activators vary greatly from one promoter to another. A few transactivators are known to facilitate transcription at hundreds of promoters, whereas others are specific for a few promoters. Many transactivators are sensitive to the binding of signal molecules, providing the capacity to activate or deactivate transcription in response to a changing cellular environment. Some enhancers bound by DNA-binding transactivators are quite distant from the promoter’s TATA box. How do the transactivators function at a distance? The answer in most cases seems to be that, as indicated earlier, the intervening DNA is looped so that the various protein complexes can inter act directly. The looping is promoted by certain nonhistone proteins that are abundant in chromatin and bind nonspecifically to DNA. These high mobility group (HMG) proteins (Fig. 1; “high mobility” refers to their electrophoretic mobility in polyacrylamide gels) play an important structural role in chromatin remodeling and transcriptional activation.
FIGURE 1 Eukaryotic promoters and regulatory proteins. RNA polymerase II and its associated general transcription factors form a preinitiation complex at the TATA box and Inr site of the cognate promoters, a process facilitated by DNA-binding transactivators, acting through TFIID and/or mediator. (a) A composite promoter with typical sequence elements and protein complexes found in both yeast and higher eukaryotes. The carboxyl-terminal domain (CTD) of Pol II is an important point of interaction with mediator and other protein complexes. Not shown are the protein complexes required for histone acetylation and chromatin remodeling. For the DNA-binding transactivators, DNA-binding domains are shown in green, activation domains in pink. The interactions symbolized by blue arrows are dis cussed in the text. (b) A wide variety of eukaryotic transcriptional re pressors function by a range of mechanisms. Some bind directly to DNA, displacing a protein complex required for activation; others interact with various parts of the transcription or activation complexes to prevent activation. Possible points of interaction are indicated with red arrows.
Coactivator Protein Complexes Most transcription re quires the presence of additional protein complexes. Some major regulatory protein complexes that interact with Pol II have been defined both genetically and bio chemically. These coactivator complexes act as inter mediaries between the DNA-binding transactivators and the Pol II complex. The best-characterized coactivator is the transcription factor TFIID (Fig. 1). In eukaryotes, TFIID is a large complex that includes TBP and ten or more TBP associated factors (TAFs). Some TAFs resemble his tones and may play a role in displacing nucleosomes during the activation of transcription. Many DNA-binding transactivators aid in transcription initiation by inter acting 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 media tor (Fig. 1); 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 com ponents of the mediator complex. Coactivator complexes function at or near the promoter’s TATA box.
Choreography of Transcriptional Activation We can now begin to piece together the sequence of transcriptional activation events at a typical Pol II promoter. First, crucial remodeling of the chromatin takes place in stages. Some DNA-binding transactivators have significant affinity for their binding sites even when the sites are within condensed chromatin. Binding of one transact veator may facilitate the binding of others, gradually dis placing some nucleosomes.
The bound transactivators can then interact directly with HATs or enzyme complexes such as SWI/SNF (or both), accelerating the remodeling of the surrounding chromatin. In this way a bound transactivator can draw in other components necessary for further chromatin remodeling to permit transcription of specific genes. The bound transactivators, generally acting through complexes such as TFIID or mediator (or both), stabilize the binding of Pol II and its associated transcription factors and greatly facilitate formation of the preinitiation transcription complex. Complexity in these regulatory circuits is the rule rather than the exception, with multiple DNA-bound transactivators promoting transcription.
The script can change from one promoter to another, but most promoters seem to require a precisely ordered assembly of components to initiate transcription. The assembly process is not always fast. At some genes it may take minutes; at certain genes in higher eukaryotes the process can take days.
Reversible Transcriptional Activation Although rarer, some eukaryotic regulatory proteins that bind to Pol II promoters can act as repressors, inhibiting the formation of active preinitiation complexes (Fig. 1b). Some transactivators can adopt different conformations, enabling them to serve as transcriptional activators or re pressors. For example, some steroid hormone receptors (described later) function in the nucleus as DNA binding transactivators, stimulating the transcription of certain genes when a particular steroid hormone signal is present. When the hormone is absent, the receptor proteins revert to a repressor conformation, preventing the formation of preinitiation complexes. In somecases this repression involves interaction with histone deacetylases and other proteins that help restore the surrounding chromatin to its transcriptionally inactive state.
