A generic promoter can in principle be expressed in any cell. The accessory proteins that are required for polymerase II to initiate at such a promoter define the general transcription factors involved in the mechanics of binding to DNA and initiating transcription. The general factors are described as TFIIX, where "X" is a letter that identifies the individual factor. A generic promoter functions at only a low efficiency; activators are required for a proper level of function. The activators are not described systematically, but have casual names reflecting their histories of identification.

We may expect any sequence components involved in the binding of RNA polymerase and general transcription factors to be conserved at most or all promoters. As with bacterial promoters, when promoters for RNA polymerase II are compared, homologies in the regions near the startpoint are restricted to rather short sequences. These elements correspond with the sequences implicated in promoter function by mutation. Figure 20.14 shows the minimal sequence for a pol II promoter, which has two sequence elements.

At the startpoint, there is no extensive homology of sequence, but there is a tendency for the first base of mRNA to be A, flanked on either side by pyrimidines. (This description is also valid for the CAT start sequence of bacterial promoters.) This region is called the initiator (Inr), and may be described in the general form Py₂CAPy₅. The Inr is contained between positions –3 and +5. A promoter consisting only of the Inr has the simplest possible form recognizable by RNA polymerase II.

Most promoters have a sequence called the TATA box, usually located ~25 bp upstream of the startpoint. It constitutes the only upstream promoter element that has a relatively fixed location with respect to the startpoint. The core sequence is TATAA, usually followed by three more A·T base pairs. The TATA box tends to be surrounded by G·C-rich sequences, which could be a factor in its function. It is almost identical with the –10 sequence found in bacterial promoters; in fact, it could pass for one except for the difference in its location at –25 instead of –10.

Single base substitutions in the TATA box act as strong down mutations. Some mutations reverse the orientation of an A·T pair, so base composition alone is not sufficient for its function. So the TATA box comprises an element whose behavior is analogous to our concept of the bacterial promoter: a short, well-defined sequence just upstream of the startpoint, which is necessary for transcription. The minority of promoters that do not contain a TATA element are called TATA-less promoters.

Figure 20.14 The minimal pol II promoter has a TATA box ~25 bp upstream of the InR. The TATA box has the consensus sequence of TATAA. The Inr has pyrimidines (Y) surrounding the CA at the startpoint. The sequence shows the coding strand.

The first step in complex formation at a promoter containing a TATA box is binding of the factor TFIID to a region that extends upstream from the TATA sequence. TFIID contains two types of component. Recognition of the TATA box is conferred by the TATA-binding protein (TBP), a small protein of ~30 kD. The other subunits are called TAFs (for TBP-associated factors). Some TAFs are stoichiometric with TBP; others are present in lesser amounts. TFIIDs containing different TAFs could recognize different promoters. Some (substoichiometric) TAFs are tissue-specific. The total mass of TFIID typically is ~800 kD, containing TBP and 11 TAFs, varying in mass from 30-250 kD. The TAFs in TFIID are named in the form TAF_II00, where "00" gives the molecular mass of the subunit. The TAF_IIs are not confined exclusively to TFIID; certain TAF_IIs are found also in protein complexes that act to modify the structure of chromatin prior to transcription (see 21 Regulation of transcription) (651, 653, 657; for review see 225, 1709).

Positioning factors that consist of TBP associated with a set of TAFs are responsible for identifying all classes of promoters. TFIIIB (for pol III promoters) and SL1 (for pol I promoters) may both be viewed as consisting of TBP associated with a particular group of proteins that substitute for the TAFs that are found in TFIID (for review see 1709). TBP is the key component, and is incorporated at each type of promoter by a different mechanism. In the case of promoters for RNA polymerase II, the key feature in positioning is the fixed distance of the TATA box from the startpoint.

Figure 20.15 shows that the positioning factor recognizes the promoter in a different way in each case. At promoters for RNA polymerase III, TFIIIB binds adjacent to TFIIIC. At promoters for RNA polymerase I, SL1 binds in conjunction with UBF. TFIID is solely responsible for recognizing promoters for RNA polymerase II. At a promoter that has a TATA element, TBP binds specifically to DNA, but at other promoters it may be incorporated by association with other proteins that bind to DNA. Whatever its means of entry into the initiation complex, it has the common purpose of interaction with the RNA polymerase.

TFIID is ubiquitous, but not unique. All multicellular eukaryotes also express an alternative complex, which has TLF (TBP like factor) instead of TBP (1708). A TLF is typically ~60% similar to TBP. It probably initiates complex formation by the usual set of TFII factors.However, TLF does not bind to the TATA box, and we do not yet know how it works. Drosophila also has a third factor, TRF1, which behaves in the same way as TBP and binds its own set of TAFs, to form a complex that functions as an alternative to TFIID at a a specific set of promoters (1707).

This section updated 4-30-2001

Figure 20.15 RNA polymerases are positioned at all promoters by a factor that contains TBP.

TBP has the unusual property of binding to DNA in the minor groove. (Virtually all known DNA-binding proteins bind in the wide groove.) The crystal structure of TBP suggests a detailed model for its binding to DNA. Figure 20.16 shows that it surrounds one face of DNA, forming a "saddle" around the double helix. In effect, the inner surface of TBP binds to DNA, and the larger outer surface is available to extend contacts to other proteins. The DNA-binding site consists of sequences that are conserved between species, while the variable N-terminal tail is exposed to interact with other proteins (647, 648, 649).

TBP not only sits in the minor groove, but also bends the DNA by ~80°, as illustrated in Figure 20.17. The TATA box bends towards the major groove, widening the minor groove. The distortion is restricted to the 8 bp of the TATA box; at each end of the sequence, the minor groove has its usual width of ~5 Å, but at the center of the sequence the minor groove is >9 Å. This is a deformation of the structure, but does not actually separate the strands of DNA, because base pairing is maintained.

Within TFIID as a free protein complex, the factor TAF_II230 binds to TBP, where it occupies the concave DNA-binding surface. In fact, the structure of the binding site, which lies in the N-terminal domain of TAF_II230, mimics the surface of the minor groove in DNA. This molecular mimicry allows TAF_II230 to control the ability of TBP to bind to DNA; the N-terminal domain of TAF_II230 must be displaced from the DNA-binding surface of TBP in order for TFIID to bind to DNA (654).

Figure 20.16 A view in cross-section shows that TBP surrounds DNA from the side of the narrow groove. TBP consists of two related (40% identical) conserved domains, which are shown in light and dark blue. The N-terminal region varies extensively and is shown in green. The two strands of the DNA double helix are in light and dark grey. Photograph kindly provided by Stephen Burley.

Figure 20.17 The cocrystal structure of TBP with DNA from -40 to the startpoint shows a bend at the TATA box that widens the narrow groove where TBP binds. Photograph provided by Stephen Burley.

Initiation requires the transcription factors to act in a defined order to build a complex that is joined by RNA polymerase. The series of events can be followed by the increasing size of the protein complex associated with DNA. Footprinting of the DNA regions protected by each complex suggests the model summarized in Figure 20.18. As each TFII factor joins the complex, an increasing length of DNA is covered. RNA polymerase is incorporated at a late stage (644; for review see 223, 226).

Addition of TFIIB gives some partial protection of the region of the template strand in the vicinity of the startpoint, from –10 to +10. This suggests that TFIIB is bound downstream of the TATA box, perhaps loosely associated with DNA and asymmetrically oriented with regard to the two DNA strands. The crystal structure shown in Figure 20.19 confirms this model. TFIIB binds adjacent to TBP, extending contacts along one face of DNA. It may provide the surface that is in turn recognized by RNA polymerase. (In archaea, the homologue of TFIIB actually makes sequence-specific contacts with the promoter. (652))

Although assembly can take place just at the core promoter in vitro, this reaction is not sufficient for transcription in vivo, where interactions with activators that recognize the more upstream elements are required. The activators interact with the basal apparatus at various stages during its assembly (see 20.20 Activators interact with the basal apparatus).

Figure 20.18 An initiation complex assembles at promoters for RNA polymerase II by an ordered sequence of association with transcription factors.

Figure 20.19 Two views of the ternary complex of TFIIB-TBP-DNA show that TFIIB binds along the bent face of DNA. The two strands of DNA are green and yellow, TBP is blue, and TFIIB is red and purple. Photograph kindly provided by Stephen Burley.

Most of the transcription factors are required solely to bind RNA polymerase to the promoter, but some act at a later stage. Binding of TFIIE causes the boundary of the region protected downstream to be extended by another turn of the double helix, to +30. Two further factors, TFIIH and TFIIJ, join the complex after TFIIE. They do not change the pattern of binding to DNA. TFIIH has several activities, including an ATPase, a helicase, and a kinase activity that can phosphorylate the CTD tail of RNA polymerase II; it is also involved in repair of damage to DNA (see 20.11 A connection between transcription and repair) (650).

The initiation reaction, as defined by formation of the first phosphodiester bond, occurs once RNA polymerase has bound. Figure 20.20 proposes a model in which phosphorylation of the tail is needed to release RNA polymerase II from the transcription factors so that it can make the transition to the elongating form. Most of the transcription factors are released from the promoter at this stage.

On a linear template, ATP hydrolysis, TFIIE, and the helicase activity of TFIIH (provided by the XPB subunit) are required for polymerase movement. This requirement is bypassed with a supercoiled template. This suggests that TFIIE and TFIIH are required to melt DNA to allow polymerase movement to begin (946). TFIIH is an exceptional factor that may play a role also in elongation.

RNA polymerase II stutters at some genes when it starts transcription. (The result is not dissimilar to the abortive initiation of bacterial RNA polymerase discussed in 9.10 Sigma factor controls binding to DNA, although the mechanism is different.) At many genes, RNA polymerase II terminates after a short distance. The short RNA product is degraded rapidly. To extend elongation into the gene, a kinase called P-TEFb is required (for review see 948). This kinase is a member of the cdk family that controls the cell cycle (see 27 Cell cycle and growth regulation). P-TEFb acts on the CTD, to phosphorylate it further. We do not yet understand why this effect is required at some promoters but not others or how it is regulated.

Figure 20.20 Phosphorylation of the CTD by the kinase activity of TFIIH may be needed to release RNA polymerase to start transcription.

In bacteria, the repair activity is provided by the uvr excision-repair system (see 14 Recombination and repair). Preferential repair is abolished by mutations in the gene mfd, whose product provides the link from RNA polymerase to the Uvr enzymes (for review see 224).

Figure 20.21 shows a model for the link between transcription and repair. When RNA polymerase encounters DNA damage in the template strand, it stalls because it cannot use the damaged sequences as a template to direct complementary base pairing. This explains the specificity of the effect for the template strand (damage in the nontemplate strand does not impede progress of the RNA polymerase).

The Mfd protein has two roles. First, it displaces the ternary complex of RNA polymerase from DNA. Second, it causes the UvrABC enzyme to bind to the damaged DNA. This leads to repair of DNA by the excision-repair mechanism (see Figure 14.28). After the DNA has been repaired, the next RNA polymerase to traverse the gene is able to produce a normal transcript (661).

Figure 20.22 shows that the basic factor involved in transcription consists of a core (of 5 subunits) associated with other subunits that have a kinase activity; this complex also includes a repair subunit. The kinase catalytic subunit that phosphorylates the CTD of RNA polymerase belongs to a group of kinases that are involved in cell cycle control (see 27 Cell cycle and growth regulation). It is possible that this connection influences transcription in response to the stage of the cell cycle.

The alternative complex consists of the core associated with a large group of proteins that are coded by repair genes. These include a subunit (XPC) that recognizes damaged DNA, which provides the coupling function that enables a template strand to be preferentially repaired when RNA polymerase becomes stalled at damaged DNA. Other proteins associated with the complex include endonucleases (XPG, XPF, ERCC1). Homologous proteins are found in the complexes in yeast (where they are often identified by rad mutations that are defective in repair) and in man (where they are identified by mutations that cause diseases resulting from deficiencies in repairing damaged DNA) (662, 663). (Subunits with the name XP are coded by genes in which mutations cause the disease xeroderma pigmentosum (see 14.22 Eukaryotic repair systems). The basic model for repair is animated in Figure 14.37.

The repair function may require modification or degradation of RNA polymerase. The large subunit of RNA polymerase is degraded when the enzyme stalls at sites of UV damage. We do not yet understand the connection between the transcription/repair apparatus as such and the degradation of RNA polymerase. It is possible that removal of the polymerase is necessary once it has become stalled (664).

Another disease that can be caused by mutations in XPD is trichothiodystrophy, which has little in common with XP or Cockayne’s (it involves mental retardation and is marked by changes in the structure of hair). All of this marks XPD as a pleiotropic protein, in which different mutations can affect different functions. In fact, XPD is required for the stability of the TFIIH complex during transcription, but the helicase activity as such is not needed. Mutations that prevent XPD from stabilizing the complex cause the severe disease of trichothiodystrophy. The helicase activity is required for the repair function. Mutations that affect the helicase activity cause the repair deficiency that results in XP or Cockayne’s syndrome (for review see 1641).

Figure 20.21 Mfd recognizes a stalled RNA polymerase and directs DNA repair to the damaged template strand.

Figure 20.22 The TFIIH core may associate with a kinase at initiation and associate with a repair complex when damaged DNA is encountered.

A promoter for RNA polymerase II consists of two types of region. The startpoint itself is identified by the Inr and/or by the TATA box close by. In conjunction with the general transcription factors, RNA polymerase II forms an initiation complex surrounding the startpoint, as we have just described. The efficiency and specificity with which a promoter is recognized, however, depend upon short sequences, farther upstream, which are recognized by a different group of factors, usually called activators. Usually the target sequences are ~100 bp upstream of the startpoint, but sometimes they are more distant. Binding of activators at these sites may influence the formation of the initiation complex at (probably) any one of several stages.

An analysis of a typical promoter is summarized in Figure 20.23. Individual base substitutions were introduced at almost every position in the 100 bp upstream of the -globin startpoint. The striking result is that most mutations do not affect the ability of the promoter to initiate transcription. Down mutations occur in three locations, corresponding to three short discrete elements. The two upstream elements have a greater effect on the level of transcription than the element closest to the startpoint. Up mutations occur in only one of the elements. We conclude that the three short sequences centered at –30, –75, and –90 constitute the promoter. Each of them corresponds to the consensus sequence for a common type of promoter element.

The sequence at –75 is the CAAT box. Named for its consensus sequence, it was one of the first common elements to be described. It is often located close to –80, but it can function at distances that vary considerably from the startpoint. It functions in either orientation. Susceptibility to mutations suggests that the CAAT box plays a strong role in determining the efficiency of the promoter, but does not influence its specificity.

The GC box at –90 contains the sequence GGGCGG. Often multiple copies are present in the promoter, and they occur in either orientation. It too is a relatively common promoter component.

Figure 20.23 Saturation mutagenesis of the upstream region of the -globin promoter identifies three short regions (centered at -30, -75, and -90) that are needed to initiate transcription. These correspond to the TATA, CAAT, and GC boxes.

Promoters are organized on a principle of "mix and match." A variety of elements can contribute to promoter function, but none is essential for all promoters. Some examples are summarized in Figure 20.24. Four types of element are found altogether in these promoters: TATA, GC boxes, CAAT boxes, and the octamer (an 8 bp element). The elements found in any individual promoter differ in number, location, and orientation. No element is common to all of the promoters. Aalthough the promoter conveys directional information (transcription proceeds only in the downstream direction), the GC and CAAT boxes seem to be able to function in either orientation. This implies that the elements function solely as DNA-binding sites to bring transcription factors into the vicinity of the startpoint; the structure of a factor must be flexible enough to allow it to make protein-protein contacts with the basal apparatus irrespective of the way in which its DNA-binding domain is oriented and its exact distance from the startpoint.

Figure 20.24 Promoters contain different combinations of TATA boxes, CAAT boxes, GC boxes, and other elements.

We have considered the promoter so far as an isolated region responsible for binding RNA polymerase. But eukaryotic promoters do not necessarily function alone. In at least some cases, the activity of a promoter is enormously increased by the presence of an enhancer, which consists of another group of elements, but located at a variable distance from those regarded as comprising part of the promoter itself (665; for review see 219).

The concept that the enhancer is distinct from the promoter reflects two characteristics. The position of the enhancer relative to the promoter need not be fixed, but can vary substantially. Figure 20.25 shows that it can be either upstream or downstream. And it can function in either orientation (that is, it can be inverted) relative to the promoter. Manipulations of DNA show that an enhancer can stimulate any promoter placed in its vicinity. In natural genomes, enhancers can be located within genes (that is, just downstream of the promoter) or tens of kilobases away in either direction.

Elements analogous to enhancers, called upstream activator sequences (UAS), are found in yeast. They can function in either orientation, at variable distances upstream of the promoter, but cannot function when located downstream. They have a regulatory role: in several cases the UAS is bound by the regulatory protein(s) that activates the genes downstream.

Figure 20.25 An enhancer can activate a promoter from upstream or downstream locations, and its sequence can be inverted relative to the promoter,