A difference between the enhancer and a typical promoter is presented by the density of regulatory elements. Figure 20.26 summarizes the susceptibility of the SV40 enhancer to damage by mutation; and we see that a much greater proportion of its sites directly influences its function than is the case with the promoter analyzed in the same way in Figure 20.23. There is a corresponding increase in the density of protein-binding sites. Many of these sites are common elements in promoters; for example, AP1 and the octamer.

A difference between enhancers and promoters may be that an enhancer shows greater cooperativity between the binding of factors. A complex that assembles at the enhancer that responds to IFN (interferon) assembles cooperatively to form a functional structure called the enhanceosome. Binding of the nonhistone protein HMGI(Y) bends the DNA into a structure that then binds several activators (NF-B, IRF, ATF-Jun). In contrast with the "mix and match" construction of promoters, all of these components are required to create an active structure at the enhancer. These components do not themselves directly bind to RNA polymerase, but they create a surface that binds a coactivating complex. The coactivator binds RNA polymerase II and recruits it to the pre-initiation complex of basal transcription factors that is assembling at the promoter. We discuss the function of coactivators in more detail in 20.20 Activators interact with the basal apparatus.

Figure 20.26 An enhancer contains several structural motifs. The histogram plots the effect of all mutations that reduce enhancer function to <75% of wild type. Binding sites for proteins are indicated below the histogram.

How can an enhancer stimulate initiation at a promoter that can be located any distance away on either side of it? When enhancers were first discovered, several possibilities were considered for their action as elements distinctly different from promoters (for review see 1715):

Now we take the view that enhancer function involves the same sort of interaction with the basal apparatus as the interactions sponsored by upstream promoter elements. Enhancers are modular, like promoters. Some elements are found in both enhancers and promoters. Some individual elements found in promoters share with enhancers the ability to function at variable distance and in either orientation. So the distinction between enhancers and promoters is blurred: enhancers might be viewed as containing promoter elements that are grouped closely together, with the ability to function at increased distances from the startpoint (666).

The essential role of the enhancer may be to increase the concentration of activator in the vicinity of the promoter (vicinity in this sense being a relative term). Two types of experiment illustrated in Figure 20.27 suggest that this is the case.

A fragment of DNA that contains an enhancer at one end and a promoter at the other is not effectively transcribed, but the enhancer can stimulate transcription from the promoter when they are connected by a protein bridge. Since structural effects, such as changes in supercoiling, could not be transmitted across such a bridge, this suggests that the critical feature is bringing the enhancer and promoter into close proximity (667).

What limits the activity of an enhancer? Typically it works upon the nearest promoter. There are situations in which an enhancer is located between two promoters, but activates only one of them on the basis of specific protein-protein contacts between the complexes bound at the two elements. The action of an enhancer may be limited by an insulator—an element in DNA that prevents it from acting on promoters beyond (see 21.18 Insulators block enhancer actions).

Figure 20.27 An enhancer may function by bringing proteins into the vicinity of the promoter. An enhancer does not act on a promoter at the opposite end of a long linear DNA, but becomes effective when the DNA is joined into a circle by a protein bridge. An enhancer and promoter on separate circular DNAs do not interact, but can interact when the two molecules are catenated.

We can now extend the view that initiation of transcription requires interactions among proteins bound at the promoter or enhancer by dividing the factors required for transcription into several classes (for review see 1710, 1712). Figure 20.28 summarizes their properties:

• Basal factors, together with RNA polymerase, bind at the startpoint and TATA box (see 20.9 The basal apparatus assembles at the promoter).
• Activators bind to sites in the promoter or in enhancers (see 20.12 Short sequence elements bind activators).
• Coactivators provide a connection between activators and the basal apparatus (see 20.20 Activators interact with the basal apparatus).
• Some regulators act to make changes in chromatin (see 21.15 Histone acetylation and deacetylation control chromatin activity).

Figure 20.28 Factors involved in gene expression include RNA polymerase and the basal apparatus, activators that bind directly to DNA at the promoter ot at enhancers, co-activators that bind to both activators and the basal apparatus, and regulators that act on chromatin structure.

Upstream promoter elements may be an appreciable distance from the startpoint, and in many cases may be oriented in either direction. Enhancers may be even farther away and always show orientation independence. This organization has implications for both the DNA and proteins. The DNA may be looped or condensed in some way to allow the formation of the transcription complex. And the domains of the activator may be connected in a flexible way, as illustrated diagrammatically in Figure 20.29. The main point here is that the DNA-binding and activating domains are independent, and connected in a way that allows the activating domain to interact with the basal apparatus irrespective of the orientation and exact location of the DNA-binding domain.

Figure 20.30 illustrates an experiment to answer this question. The activator GAL4 has a DNA-binding domain that recognizes a UAS; an activating domain stimulates initiation at the target promoter. The bacterial repressor LexA has an N-terminal DNA-binding domain that recognizes a specific operator; binding of LexA at this operator represses the adjacent promoter. In a "swap" experiment, the DNA-binding domain of LaxA can be substituted for the DNA-binding domain of GAL4. The hybrid gene can then be introduced into yeast together with a target gene that contains either the UAS or a LexA operator.

This result fits the modular view of transcription activators. The DNA-binding domain serves to bring the protein into the right location. Precisely how or where it is bound to DNA is irrelevant, but, once it is there, the transcription-activating domain can play its role. According to this view, it does not matter whether the transcription-activating domain is brought to the vicinity of the promoter by recognition of a UAS via the DNA-binding domain of GAL4 or by recognition of a LexA operator via the LexA specificity module. The ability of the two types of module to function in hybrid proteins suggests that each domain of the protein folds independently into an active structure that is not influenced by the rest of the protein (for review see 230, 232).

The idea that activator have independent domains that bind DNA and that activate transcription is reinforced by the ability of the tat protein of HIV to stimulate initiation without binding DNA at all. The tat protein binds to a region of secondary structure in the RNA product; the part of the RNA required for tat action is called the tar sequence. A model for the role of the tat-tar interaction in stimulating transcription is shown in Figure 20.31.

Figure 20.29 DNA-binding and activating functions in a transcription factor may comprise independent domains of the protein.

Figure 20.30 The ability of GAL4 to activate transcription is independent of its specificity for binding DNA. When the GAL4 DNA-binding domain is replaced by the LexA DNA-binding domain, the hybrid protein can activate transcription when a LexA operator is placed near a promoter.

Figure 20.31 The activating domain of the tat protein of HIV can stimulate initiation if it is tethered in the vicinity by binding to the RNA product of a previous round of transcription. Activation is independent of the means

The model of domain independence is the basis for an extremely useful assay for detecting protein interactions. In effect, we replace the connecting domain in Figure 20.29 with a protein-protein interaction. The principle is illustrated in Figure 20.32. We fuse one of the proteins to be tested to a DNA-binding domain. We fuse the other protein to a transcription-activating domain. (This is done by linking the appropriate coding sequences in each case and making synthetic proteins by expressing each hybrid gene.) If the two proteins that are being tested can interact with one another, the two hybrid proteins will interact. This is reflected in the name of the technique: the two hybrid assay (952). The protein with the DNA-domain binds to a reporter gene that has a simple promoter containing its target site. But it cannot activate the gene by itself. Activation occurs only if the second hybrid binds to the first hybrid to bring the activation domain to the promoter. Any reporter gene can be used where the product is readily assayed, and this technique has given rise to several automated procedures for rapidly testing protein-protein interactions.

Run Animation
Figure 20.32 The two hybrid technique tests the ability of two proteins to interact by incorporating them into hybrid proteins where one has a DNA-binding domain and the other has a transcription-activating domain.

Run Animation
Figure 20.32 The two hybrid technique tests the ability of two proteins to interact by incorporating them into hybrid proteins where one has a DNA-binding domain and the other has a transcription-activating domain.

An activator may work directly when it consists of a DNA-binding domain linked to a transcription-activating domain, as illustrated in Figure 20.29. In other cases, the activator does not itself have a transcription-activating domain, but binds another protein—a coactivator—that has the transcription-activating domain. Figure 20.33 shows the action of such an activator. We may regard the coactivator as transcription factors whose specificity is conferred by the ability to bind to DNA-binding transcription factors instead of directly to DNA. A particular activator may require a specific coactivator.

But although the protein components are organized differently, the mechanism is the same. An activator that contacts the basal apparatus directly has an activation domain covalently connected to the DNA-binding domain. When an activator works through a coactivator, the connections involve noncovalent binding between protein subunits (compare Figure 20.29 and Figure 20.33). The same interactions are responsible for activation, irrespective of whether the various domains are present in the same protein subunit or divided into multiple protein subunits (645).

A transcription-activating domain works by making protein-protein contacts with general transcription factors that promote assembly of the basal apparatus (for review see 218, 221). Contact with the basal apparatus may be made with any one of several basal factors, typically TFIID, TFIIB, or TFIIA. All of these factors participate in a relatively early stage of assembly of the basal apparatus (see Figure 20.18). Figure 20.34 illustrates the situation when such a contact is made. It suggests that the major effect of activators is to influence the assembly of the basal apparatus, presumably by increasing a rate-limiting step (as opposed to acting after assembly of the basal apparatus to influence its activity) (646, 659).

The activating domains of the yeast activators GAL4 and GCN4 have multiple negative charges, giving rise to their description as "acidic activators." Another particularly effective activator of this type is carried by the VP16 protein of the Herpes Simplex Virus. (VP16 does not itself have a DNA-binding domain, but interacts with the transcription apparatus via an intermediary protein.) Experiments to characterize acidic activator functions have often made use of the VP16 activating region linked to a DNA-binding motif (658).

Adding up all the components required for efficient transcription—basal factors, RNA polymerase, activators, coactivators—we get a very large apparatus, consisting of >40 proteins. Is it feasible for this apparatus to assemble step by step at the promoter? Some activators, coactivators, and basal factors may assemble stepwise at the promoter, but then may be joined by a very large complex consisting of RNA polymerase preassembled with further activators and coactivators, as illustrated in in Figure 20.35 (for review see 1710).

Several forms of RNA polymerase have been found in which the enzyme is associated with various transcription factors. The most prominent "holoenzyme complex" in yeast consists of RNA polymerase associated with a 20-subunit complex called mediator (1713; for review see 1711). The mediator includes products of several genes in which mutations block transcription, including some SRB loci (so named because many of their genes were originally identified as suppressors of mutations in RNA polymerase B.) The name was suggested by its ability to mediate the effects of activators. Mediator is necessary for transcription of most yeast genes. Homologous complexes are required for the transcription of most higher eukaryotic genes. Mediator interacts with the CTD domain of RNA polymerase. It is probably released when a polymerase starts elongation. And in addition to these complexes, which consist of factors acting directly on transcription, there are complexes in which RNA polymerase and activators are associated with factors that act to manipulate the structure of chromatin (see 21 Regulation of transcription).

This section updated 4-30-2001

Figure 20.33 An activator may bind a coactivator that contacts the basal apparatus.

Figure 20.34 Activators may work at different stages of initiation, by contacting the TAFs of TFIID or contacting TFIIB.

Figure 20.35 RNA polymerase exists as a holoenzyme containing many activators.

Repression of transcription in eukaryotes is generally accomplished at the level of influencing chromatin structure; regulator proteins that function like trans-acting bacterial repressors to block transcription are relatively rare, but some examples are known. One case is the global repressor NC2/Dr1/DRAP1, a heterodimer that binds to TBP to prevent it from interacting with other components of the basal apparatus (1741, 1742, 1743). The importance of this interaction is suggested by the lethality of null mutations in the genes that code for the repressor in yeast.

In a more specific case, the CAAT sequence is a target for regulation. Two copies of this element are found in the promoter of a gene for histone H2B (see Figure 20.24) that is expressed only during spermatogenesis in a sea urchin. CAAT-binding factors can be extracted from testis tissue and also from embryonic tissues, but only the former can bind to the CAAT box. In the embryonic tissues, another protein, called the CAAT-displacement protein (CDP), binds to the CAAT boxes, preventing the activator from recognizing them.

Figure 20.36 illustrates the consequences for gene expression. In testis, the promoter is bound by transcription factors at the TATA box, CAAT boxes, and octamer sequences. In embryonic tissue, the exclusion of the CAAT-binding factor from the promoter prevents a transcription complex from being assembled. The analogy with the effect of a bacterial repressor in preventing RNA polymerase from initiating at the promoter is obvious. These results also make the point that the function of a protein in binding to a known promoter element cannot be assumed: it may be an activator, a repressor, or even irrelevant to gene transcription.

Figure 20.36 A transcription complex involves recognition of several elements in the sea urchin H2B promoter in testis. Binding of the CAAT displacement factor in embryo prevents the CAAT-binding factor from binding, so an active complex cannot form.