Initiation by Pol II Requires General Transcription Factors

As discussed earlier, the purified E. coli core RNA polymerase, which lacks the σ⁷⁰ subunit, cannot initiate transcription. However, when the core polymerase is associated with σ⁷⁰, the resulting holoenzyme can initiate transcription in vitro from strong promoters. Thus, the σ⁷⁰ subunit of E. coli RNA polymerase functions as an initiation factor; that is, it is required for transcription to begin but is released from the template after polymerization of the initial 10 or so ribonucleotide triphosphates. In contrast, in vitro transcription by purified eukaryotic RNA polymerase II requires the addition of several initiation factors that are separated from the polymerase during purification. These initiation factors, which position Pol II at transcription-initiation sites, are called general transcription factors, because they are thought to be required for transcription of most genes that are transcribed by this type of polymerase. In contrast, the transcription factors discussed in the previous section bind to specific sites in a limited number of genes. A transcription-initiation complex comprises an RNA polymerase and various general transcription factors bound to the promoter region.

Many general transcription factors required for Pol II to initiate transcription from most TATA-box promoters in vitro have been isolated and characterized. These proteins are designated TFIIA, TFIIB, etc., and most are multimeric proteins. TFIID is the largest with a mass of ≈750 kDa. It consists of a single 38-kDa TATA box – binding protein (TBP) and eleven TBP-associated factors (TAFs), which have not been extensively characterized. General transcription factors with similar activities have been isolated from cultured human cells, rat liver, Drosophila embryos, and yeast. The genes encoding these proteins in yeast have been sequenced as part of the complete yeast genome sequence, and many of the cDNAs encoding human and Drosophila general transcription factors have been cloned and sequenced. In all cases, equivalent general transcription factors from different eukaryotes are highly conserved. Although general transcription factors allow Pol II to initiate transcription in vitro at the same start sites used in vivo, additional proteins are required for transcription initiation in vivo.

Proteins Comprising the Pol II Transcription-Initiation Complex Assemble in a Specific Order in Vitro

In most biochemical studies on assembly of the Pol II initiation complex, researchers have used isolated TBP rather than the complete, multisubunit TFIID, which is difficult to purify. Binding of TBP and the other general transcription factors on promoters with consensus TATA boxes has been analyzed by DNase I footprinting and electrophoretic mobility shift assays. These studies demonstrate that the Pol II initiation complex is assembled in vitro in the stepwise sequence depicted in Figure 10-50.

Figure 10-50

Stepwise assembly of a transcription-initiation complex from isolated RNA polymerase II (Pol II) and general transcription factors. Once the complete transcription-initiation complex has (more...)

In in vitro experiments with isolated general transcription factors, TBP is the first protein to bind to a TATA-box promoter. All eukaryotic TBPs analyzed to date have very similar C-terminal domains of 180 residues. The sequence of this region is 80 percent identical in the yeast and human proteins, and most differences are conservative substitutions. This conserved C-terminal domain functions as well as the full-length protein in in vitro transcription. (The N-terminal domain of TBP, which varies greatly in sequence and length among different eukaryotes, functions in the transcription of genes encoding snRNAs, which are discussed Chapter 11.) Figure 10-51 shows a model of the conserved C-terminal domain of TBP complexed to TATA-box DNA, based on x-ray crystallographic analysis. TBP is a monomer that folds into a saddle-shape structure; the two halves of the molecule exhibit an overall dyad symmetry but are not identical, unlike dimeric transcription factors. Like the HMGI and other DNA-bending proteins that participate in formation of enhancesomes, TBP interacts with the minor groove in DNA, bending the helix considerably.

Figure 10-51

Structure of the conserved C-terminal domain of TBP bound to TATA-box DNA. Although TBP is a monomer, the polypeptide backbone of this domain is folded into a conformation that has an approximate (more...)

Once TBP has bound to the TATA box, TFIIB can bind (see Figure 10-50). TFIIB is a monomeric protein, slightly smaller than TBP. Its C-terminal domain makes contact with both DNA and the bound TBP (Figure 10-52). The N-terminal domain of TFIIB extends toward the start site, but its three-dimensional structure is not yet known. Following TFIIB binding, a pre-formed complex of TFIIF (an α₂β₂ tetramer) and Pol II binds, positioning the polymerase over the start site. In the resulting preinitiation complex, which is stable, the two largest subunits of Pol II (L and L′) interact with the promoter DNA along an ≈240 Å channel extending upstream and downstream of the start site (Figure 10-53). At most promoters, two more general transcription factors must bind before the DNA duplex can be separated to expose the template strand. First to bind is TFIIE (an α₂β₂ tetramer), creating a docking site for TFIIH, another multimeric factor containing nine subunits. Binding of TFIIH completes assembly of the transcription-initiation complex in vitro (see Figure 10-50).

Figure 10-52

Structure of the complex formed between TBP, promoter DNA, and TFIIB. In in vitro transcription systems, TFIIB binds to the assembled TBP – promoter DNA complex. Shown (more...)

Figure 10-53

Structural model of the complex composed of promoter DNA, TBP, TFIIB, and RNA polymerase II indicating the relative sizes of the components. Two views of the complex, rotated ≈180° (more...)

In the presence of ATP, the helicase activities of two TFIIH subunits unwind the DNA duplex at the start site, allowing Pol II to form an open complex. If the remaining ribonucleoside triphosphates are added, Pol II begins transcribing the template strand. As the polymerase transcribes away from the promoter region, another subunit of TFIIH phosphorylates the Pol II CTD at multiple sites (see Figure 10-50). In the minimal in vitro transcription assay containing only these general transcription factors and purified RNA polymerase II, TBP remains bound to the TATA-box as the polymerase transcribes away from the promoter region, but the other general transcription factors dissociate. As discussed below, transcription initiation in vivo requires additional factors; it is not yet clear which factors remain associated with promoter regions following each round of transcription initiation in the cell.

Remarkably, many of the subunits of the complex TFIIH protein are also required for two other distinct processes in eukaryotic cells: the activation of protein kinases required for entry into the S phase of the cell cycle (Chapter 13) and transcription-linked repair of DNA damage by the excision-repair pathway (Section 12.4). Indeed the first subunits of TFIIH to be cloned from humans were identified because mutations in the genes encoding them cause defects in the repair of damaged DNA. In normal individuals, when a transcribing RNA polymerase becomes stalled at a region of damaged template DNA, a subcomplex of TFIIH is thought to recognize the stalled polymerase and then recruit other proteins that function with TFIIH in the excision-repair process. In the absence of functional TFIIH, the excision-repair of damaged DNA in transcriptionally active genes is impaired. As a result, affected individuals have extreme skin sensitivity to sunlight (a common cause of DNA damage) and exhibit a high incidence of cancer. Depending on the severity of the defect in TFIIH function, these individuals may suffer from diseases such as xeroderma pigmentosum, trichothiodystrophy, or Cockayne syndrome.

A Pol II Holoenzyme Multiprotein Complex Functions in Vivo

The model shown in Figure 10-50 of eukaryotic transcription initiation in vitro seems far more complex than bacterial transcription initiation. Moreover, genetic and biochemical studies in yeast have revealed additional proteins called Srbs and Meds, which form a complex of approximately 20 polypeptides. This complex, termed Mediator, associates with the CTD region of Pol II. Gene knockout experiments subsequently demonstrated that most of the Srb/Med proteins in this complex are required for cell viability. Further experiments with temperature-sensitive mutants showed that some Srb proteins are essential for transcription initiation by Pol II in vivo. For example, shifting yeast cells carrying a temperature-sensitive mutation in an Srb gene from the permissive to the nonpermissive temperature resulted in an immediate cessation of initiation by Pol II.

Researchers also have isolated an ≈2-MDa multiprotein complex from yeast cells that includes Pol II, the Mediator complex, TFIIB, TFIIF, and TFIIH. A total of ≈50 polypeptides are present in this very large protein complex, which is stable in the absence of promoter DNA. The Pol II in this complex has an unphosphorylated CTD tail; this is the form of the enzyme that initiates transcription, as opposed to the chain-elongating form, which has a phosphorylated tail. Similar high-molecular-weight complexes containing a portion of the total cellular Pol II have been observed in extracts from nuclei of cultured human cells. These observations have led to the hypothesis that most of the proteins required for transcription initiation in cells are preassembled in an ≈2-MDa holoenzyme complex that then binds to promoter DNA in a single binding step.

Another general transcription factor, TFIIA, which is not required for initiation in vitro, is required for initiation by Pol II in vivo. Purified TFIIA forms a complex with TBP and TATA-box DNA. X-ray crystallography of the complex shows that TFIIA interacts with TBP and DNA on the opposite side of TBP from where the other general transcription factors and Pol II bind (see Figure 10-52). It is likely that in assembly of transcription-initiation complexes in higher eukaryotic cells TFIIA and TFIID, with its multiple TAF subunits, bind first to the promoter DNA and then all the components of the holoenzyme mentioned above subsequently bind. This amounts to a multiprotein complex of some 60 – 70 polypeptides with a mass of ≈3 MDa, nearly as large as a eukaryotic ribosome.

SUMMARY

•  RNA polymerase II requires several general transcription factors to locate the proper start site in a DNA template and initiate transcription. These include TFIID, which binds to a TATA-box through its TATA-box binding subunit, TBP.
•  Transcription of protein-coding genes by RNA polymerase II can be initiated in vitro by sequential binding of the following in the indicated order: TBP, which binds to TATA-box DNA; TFIIB; a complex of Pol II and TFIIF; TFIIE; and finally TFIIH (see Figure 10-50).
•  The helicase activities of two TFIIH subunits separate the template strands at the start site in most promoters, a process that requires hydrolysis of ATP. As Pol II begins transcribing away from the start site, its CTD is phosphorylated by another TFIIH subunit.
•  Initiation by Pol II in vivo requires the multiprotein Mediator complex, which associates with the unphosphorylated CTD of Pol II, forming a very large holoenzyme complex that also includes most of the general transcription factors. This pre-assembled holoenzyme is thought to bind to promoter DNA in a single step in vivo.
•  The Pol II transcription-initiation complex that assembles on promoters in vivo may comprise as many as 60 – 70 polypeptides with a total mass similar to that of a ribosome.