A complex apparatus consisting of as many as 50 unique proteins provides accurate and regulatable transcription of eukaryotic genes. The RNA polymerase enzymes (pol I, pol II, and pol III) transcribe information contained in the template strand of DNA into RNA. These polymerases must recognize a specific site in the promoter in order to initiate transcription at the proper nucleotide. In contrast to the situation in prokaryotes though, eukaryotic RNA polymerases alone are not able to discriminate between promoter sequences and other, non-promoter regions of DNA in the test tube. All eukaryotic RNA polymerases require other proteins known as general transcription factors or GTFs. RNA polymerase II requires TFIIA, B, D (or TBP), E, F, and H to both facilitate promoter-specific binding of the enzyme and formation of the PIC. RNA polymerases I and III require their own polymerase-specific GTFs. Moreover, RNA polymerase II and GTFs do not respond to activator proteins and can only catalyze basal or (non)-unregulated transcription in vitro. Another set of proteins—the coactivators, or coregulators—work in conjunction with the DNA-binding transactivator proteins described above to communicate with Pol II/GTFs to regulate the rate of transcription (see below).
Formation of the Pol II Transcription Complex
In bacteria, a σ-factor–polymerase holoenzyme complex, Eσ, selectively and directly binds to promoter DNA to form the PIC. The situation is much more complex in eukaryotic genes. mRNA-encoding genes, which are transcribed by pol II, are described as an example. In the case of pol II-transcribed genes, the function of σ-factors is assumed by a number of proteins. PIC formation requires pol II and the six GTFs (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH). These GTFs serve to promote RNA polymerase II transcription on essentially all genes. Some of these GTFs are composed of multiple subunits. TFIID, which binds to the TATA box promoter element through its TBP subunit, is the only one of these factors that is independently capable of specific, high affinity binding to promoter DNA. TFIID consists of 15 subunits, TBP and 14 TBP Associated Factors, or TAFs.
TBP binds to the TATA box in the minor groove of DNA (most transcription factors bind in the major groove) and causes an approximately 100-degree bend or kink of the DNA helix. This bending is thought to facilitate the interaction of TAFs with other components of the transcription initiation complex, the multicomponent eukaryotic promoter and possibly with factors bound to upstream elements. Although initially defined as a component solely required for transcription of pol II gene promoters, TBP, by virtue of its association with distinct, polymerase-specific sets of TAFs, is also an important component of pol I and pol III transcription initiation complexes even if they do not contain TATA boxes.
The binding of TFIID marks a specific promoter for transcription. Of several subsequent in vitro steps, the first is the binding of TFIIA, then TFIIB to the TFIID-promoter complex. This results in a stable ternary complex, which is then more precisely located and more tightly bound at the transcription initiation site. This complex then attracts and tethers the pol II–TFIIF complex to the promoter. Addition of TFIIE and TFIIH are the final steps in the assembly of the PIC. TFIIE appears to join the complex with pol II–TFIIF, and TFIIH is then recruited. Each of these binding events extends the size of the complex so that finally about 60 bp (from -30 to +30 relative to the +1 TSS) are covered (Figure 36–9). The PIC is now complete and capable of basal transcription initiated from the correct nucleotide. In genes that lack a TATA box, the same factors are required. In such cases, the Inr and/or DPE serve to (see Figure 36–8) position the complex for accurate initiation of transcription.
Promoter Accessibility and Hence PIC Formation Is Often Modulated by Nucleosomes
On certain eukaryotic genes, the transcription machinery (pol II, etc.) cannot access the promoter sequences (ie, TATA–INR–DPE) because these essential promoter elements are wrapped up in nucleosomes (see Figures 35–2, 35–3 and 36–10). Only after transcription factors bind to enhancer DNA upstream of the promoter and recruit chromatin remodeling and modifying coregulatory factors such as the Swi/Snf, SRC-1, p300/CBP (see Chapter 42) P/CAF or other factors, are the repressing nucleosomes removed (Figure 36–10). Once the promoter is “open” following nucleosome eviction, GTFs and RNA polymerase II can bind and initiate mRNA gene transcription. Note that the binding of transactivators and coregulators can be sensitive to, and/or directly control the composition and/or covalent modification status of the DNA and the histones within the nucleosomes in and around the promoter and enhancer, and thereby increase or decrease the ability of all the other components required for PIC formation to interact with a particular gene. This so-called epigenetic code of DNA, histone and protein modifications can contribute importantly to gene transcription control. Indeed, mutations in proteins that catalyze (code writers), remove (code erasers), or differentially bind (code readers) modified DNA and/or histones can lead to human disease.
Nucleosome covalent modifications, remodeling and eviction by chromatin-active coregulators modulate PIC formation and transcription. Shown in A, is an inactive mRNA encoding gene (see X over TSS) with a single dimeric transcription factor (Activator-1; violet ovals) bound to its cognate enhancer binding site (Activator-1). This particular enhancer element was nucleosome-free and hence available for interaction with its cognate activator binding protein. However, this gene is still inactive (X over TSS) due to the fact that a portion of its enhancer (in this illustration the enhancer is bipartite and composed of Activator-1 and Activator-2, DNA-binding sites) and the promoter are covered by nucleosomes. Recall that nucleosomes have ~150 bp of DNA wound around the histone octamer. Hence, the single nucleosome over the promoter will occlude access of the transcription machinery (pol II+GTFs) to the TATA, Inr and/or DPE promoter elements. (B) Enhancer DNA-bound Activator-1 interacts with any of a number of distinct ATP-dependent chromatin remodelers and chromatin-modifying Co-regulator complexes. These coregulators together have the ability to both move, or remodel (ie. change the octameric histone content, and/or remove nucleosomes) through the action of various ATP-dependent remodelers as well as to covalently modify nucleosomal histones using intrinsic acetylases (HAT; resulting in acetylation [Ac]) and methylases (SET; resulting in methylation [Me], among other PTMs; Table 35–1) carried by subunits of these complexes. (C) The resulting changes in nucleosome position and nucleosome occupancy (ie. nucleosome -4 and nucleosome 0), composition (nucleosome -1 and nucleosome +1) thus allow for the binding of the second Activator-2 dimer to Activator-2 DNA sequences, which leads to the binding of the transcription machinery (TFIIA,B,D,E,F,H; Polymerase II and Mediator) to the promoter (TATA-INR-DPE) and the formation of an active PIC, which leads to activated transcription.
Phosphorylation Activates Pol II
Eukaryotic pol II consists of 12 subunits. As noted above the two largest subunits of pol II (MW 220 and 150 kDa) are homologous to the bacterial β′ and β subunits. In addition to the increased number of subunits, eukaryotic pol II differs from its prokaryotic counterpart in that it has a series of heptad repeats with consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser at the carboxyl terminus of the largest pol II subunit. This carboxy terminal domain (CTD) has 26 repeated units in yeast and 52 units in mammals. The CTD is a substrate for several enzymes (kinases, phosphatases, prolyl isomerases, glycosylases); phosphorylation of the CTD was the first CTD PTM discovered. Among other proteins the kinase subunit of TFIIH can modify the CTD. Covalently modified CTD is the binding site for a wide array of proteins, and it has been shown to interact with many mRNA modifying and processing enzymes and nuclear transport proteins. The association of these factors with the CTD of RNA polymerase II (and other components of the basal machinery) thus serves to couple transcription initiation with mRNA capping, splicing, 3′ end formation and transport to the cytoplasm (see below). Pol II polymerization is activated when phosphorylated on the Ser and Thr residues and displays reduced activity when the CTD is dephosphorylated. CTD phosphorylation/dephosphorylation is critical for promoter clearance, elongation, termination, and even appropriate mRNA processing. Pol II lacking the CTD tail is incapable of activating transcription, and cells expressing pol II lacking the CTD are inviable. These results underscore the importance of this domain to mRNA biogenesis.
Pol II can associate with other proteins termed Mediator or Med proteins to form a complex sometimes referred to as the pol II holoenzyme; this complex can form on the promoter or in solution prior to PIC formation (see below). The Med proteins (over 30 proteins; Med1-Med31) are essential for appropriate regulation of pol II transcription by serving myriad roles, both activating and repressing transcription. Thus Mediator, like TFIID is a transcriptional coregulator (Figure 36–11).
Models for the formation of an RNA polymerase II preinitiation complex. Shown at top is a typical mRNA encoding gene transcription unit: enhancer-promoter (TATA)-TSS (bent arrow) and ORF (open reading frame) within the transcribed region. PICs have been shown to form by at least two distinct mechanisms: (A) the stepwise binding of GTFs, pol II, and Mediator (Med), or (B) by the binding of a single multiprotein complex composed of pol II, Med, and the six GTFs. DNA-binding transactivator proteins specifically bind enhancers and in part facilitate PIC formation (or PIC function) by binding directly to the TFIID-TAF subunits or Med subunits of Mediator (not shown, see Figure 36–10); the molecular mechanism(s) by which such protein–protein interactions stimulate transcription remain a subject of intense investigation.
The Role of Transcription Activators & Coregulators
TFIID was originally considered to be a single protein, TBP. However, several pieces of evidence led to the important discovery that TFIID is actually a complex consisting of TBP and the 14 TAFs. The first evidence that TFIID was more complex than just the TBP molecules came from the observation that TBP binds to a 10-bp segment of DNA, immediately over the TATA box of the gene, whereas native holo-TFIID covers a 35 bp or larger region (Figure 36–9). Second, purified recombinant E. coli-expressed TBP has a molecular mass of 20–40 kDa (depending on the species), whereas the native TFIID complex has a mass of about 1000 kDa. Finally, and perhaps most importantly, TBP supports basal transcription but not the augmented transcription provided by certain activators, for example, Sp1 bound to the GC box. TFIID, on the other hand, supports both basal and enhanced transcription by Sp1, Oct1, AP1, CTF, ATF, etc. (Table 36–3). The TAFs are essential for this activator-enhanced transcription. There are likely several forms of TFIID that differ slightly in their complement of TAFs. Thus different combinations of TAFs with TBP—or one of several recently discovered TBP-like factors (TLFs)—bind to different promoters, and recent reports suggest that this may account for the tissue or cell-selective gene activation noted in various promoters and for the different strengths of certain promoters. TAFs, since they are required for the action of activators, are often called coactivators or coregulators. There are thus three classes of transcription factors involved in the regulation of pol II genes: pol II and GTFs, coregulators, and DNA-binding activator-repressors (Table 36–4). How these classes of proteins interact to govern both the site and frequency of transcription is a question of central importance and active investigation. It is currently thought that coregulators both act as a bridge between the DNA-binding transactivators and pol II/GTFs, and act to modify chromatin.
TABLE 36–3Some of the Mammalian RNA Polymerase II Transcription Control Elements, Their Consensus Sequences, and the Factors That Bind to Them |Favorite Table|Download (.pdf) TABLE 36–3 Some of the Mammalian RNA Polymerase II Transcription Control Elements, Their Consensus Sequences, and the Factors That Bind to Them
|Element ||Consensus Sequence ||Factor |
|TATA box ||TATAAA ||TBP/TFIID |
|Inr ||t/ct/cANt/at/ct/c ||TFIID |
|DPE ||a/gGa/tCGTG ||TFIID |
|CAAT box ||CCAATC ||C/EBP∗, NF-Y∗ |
|GC box ||GGGCGG ||Sp1∗ |
| ||CAACTGAC ||Myo D |
| ||t/cGGa/cN5GCCAA ||NF1∗ |
|Ig octamer ||ATGCAAAT ||Oct1, 2, 4, 6∗ |
|AP1 ||TGAg/cTC/AA ||Jun, Fos, ATF∗ |
|Serum response ||GATGCCCATA ||SRF |
|Heat shock ||(NGAAN)3 ||HSF |
TABLE 36–4Three Classes of Transcription Factors Involved in mRNA Gene Transcription |Favorite Table|Download (.pdf) TABLE 36–4 Three Classes of Transcription Factors Involved in mRNA Gene Transcription
|General Mechanisms ||Specific Components |
|Basal components ||RNA Polymerase II, TBP, TFIIA, B, D, E, F, and H |
|Coregulators ||TAFs (TBP + TAFs) = TFIID; certain genes |
| ||Mediator, Meds |
| ||Chromatin modifiers |
| ||Chromatin remodelers |
|Activators ||SP1, ATF, CTF, AP1, etc |
Two Models Can Explain the Assembly of the Preinitiation Complex
The formation of the PIC described above is based on the sequential addition of purified components as observed through in vitro experiments. An essential feature of this model is that PIC assembly takes place on a DNA template where the transcription proteins all have ready access to DNA. Accordingly, transcription activators, which have autonomous DNA binding and activation domains (see Chapter 38), are thought to function by stimulating PIC formation. Here the TAF or mediator complexes are viewed as bridging factors that communicate between the upstream-bound activators, and the GTFs and pol II. This view assumes that there is stepwise assembly of the PIC—promoted by various interactions between activators, coactivators, and PIC components, and is illustrated in panel A of Figure 36–11. This model is supported by observations that many of these proteins can indeed bind to one another in vitro.
Recent evidence suggests that there is another possible mechanism of PIC formation and thus transcription regulation. First, large preassembled complexes of GTFs and pol II are found in cell extracts, and these complexes can associate with the promoter in a single step. Second, the rate of transcription achieved when activators are added to limiting concentrations of pol II holoenzyme can be matched by atificially increasing the concentration of pol II and GTFs in the absence of activators. Thus, at least in vitro, one can establish conditions where activators are not in themselves absolutely essential for PIC formation. These observations led to the “recruitment” hypothesis, which has now been tested experimentally. Simply stated, the role of activators and some coactivators may be solely to recruit a preformed holoenzyme-GTF complex to the promoter. The requirement for an activation domain is circumvented when either a component of TFIID or the pol II holoenzyme is artificially tethered, using recombinant DNA techniques, to the DBD of an activator. This anchoring, through the DBD component of the activator molecule, leads to a transcriptionally competent structure, and there is no further requirement for the activation domain of the activator. In this view, the role of activation domains is to direct preformed holoenzyme–GTF complexes to the promoter; they do not assist in PIC assembly (see panel B, Figure 36–11). In this model, the efficiency of the recruitment process directly determines the rate of transcription at a given promoter.