Genes VII

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 20.11. 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).

Commitment to a promoter is initiated when TFIID binds the TATA box. When TFIIA joins the complex, TFIID becomes able to protect a region extending farther upstream. TFIIA may activate TBP by relieving the repression that is caused by the TAF_II230.

Addition of TFIIB gives some partial protection of the region of the template strand in the vicinity of the startpoint, from V10 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 20.12 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))

The factor TFIIF consists of two subunits. The larger subunit (RAP74) has an ATP-dependent DNA helicase activity that could be involved in melting the DNA at initiation. The smaller subunit (RAP38) has some homology to the regions of bacterial sigma factor that contact the core polymerase; it binds tightly to RNA polymerase II. TFIIF may bring RNA polymerase II to the assembling transcription complex and provide the means by which it binds. The complex of TBP and TAFs may interact with the CTD tail of RNA polymerase, and interaction with TFIIB may also be important when TFIIF/polymerase joins the complex.

Polymerase binding extends the sites that are protected downstream to +15 on the template strand and +20 on the nontemplate strand. The enzyme extends the full length of the complex, since additional protection is seen at the upstream boundary.

The initiation reaction, as defined by formation of the first phosphodiester bond, can occur at this stage. Some further general factors, TFIIE and TFIIH, are required for promoter clearance Xto allow RNA polymerase to commence movement away from the promoter.

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 next section) (650).

Most of the TFII factors are released before RNA polymerase II leaves the promoter. Figure 20.13 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. 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 initiatiaton of bacterial RNA polymerase discussed in 9.4 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.

The CTD may coordinate processing of RNA with transcription. The capping enzyme (guanylyl transferase), which adds the G residue to the 5′ end of newly synthesized mRNA, binds to the phosphorylated CTD: this may be important in enabling it to modify the 5′ end as soon as it is synthesized. Some splicing factors bind to the CTD and so do some components of the cleavage/polyadenylation apparatus, suggesting that it may be a general focus for connecting other processes with transcription.

The general process of initiation is similar to that catalyzed by bacterial RNA polymerase. Binding of RNA polymerase generates a closed complex, which is converted at a later stage to an open complex in which the DNA strands have been separated. In the bacterial reaction, formation of the open complex completes the necessary structural change to DNA; a difference in the eukaryotic reaction is that further unwinding of the template is needed after 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 for the initial melting of DNA that allows polymerase movement to begin (946).

What happens at TATA-less promoters? The same general transcription factors, including TFIID, are needed. The Inr provides the positioning element; TFIID binds to it via an ability of one or more of the TAFs to recognize the Inr directly. The function of TBP at these promoters is more like that at promoters for RNA polymerase I and at internal promoters for RNA polymerase III.

Many of the general factors consist of multiple subunits, so the total number of polypeptides involved in the basal apparatus is rather large. There are probably ~20 polypeptides with a total mass of ~500 kD. Remember that RNA polymerase II itself has ~10 subunits with a mass of ~500 kD, so we see that initiation involves the assembly of an extremely large complex.

Assembly of the RNA polymerase II initiation complex provides an interesting contrast with prokaryotic transcription. Bacterial RNA polymerase is essentially a coherent aggregate with intrinsic ability to bind DNA; the sigma factor, needed for initiation but not for elongation, becomes part of the enzyme before DNA is bound, although it is later released. But RNA polymerase II can bind to the promoter only after separate transcription factors have bound. The factors play a role analogous to that of bacterial sigma factor Xto allow the basic polymerase to recognize DNA specifically at promoter sequences Xbut have evolved more independence. Indeed, the factors are primarily responsible for the specificity of promoter recognition. The process of assembling the transcription complex reminds us of ribosome subunit assembly, in which ribosomal proteins must bind to rRNA (or to other proteins in the complex) in a certain order. Only some of the factors participate in protein-DNA contacts (and only TBP makes sequence-specific contacts); thus protein-protein interactions are important in the assembly of the complex.

The sequences in the vicinity of the startpoint comprise a "core" promoter at which the basal transcription apparatus is assembled. When a TATA box is present, it determines the location of the startpoint. Its deletion causes the site of initiation to become erratic, although any overall reduction in transcription is relatively small. Indeed, some TATA-less promoters lack unique startpoints; initiation occurs instead at any one of a cluster of startpoints. The TATA box aligns the RNA polymerase (via the interaction with TFIID and other factors) so that it initiates at the proper site. This explains why its location is fixed with respect to the startpoint. Binding of TBP to TATA is the predominant feature in recognition of the promoter, but two large TAFS (TAF_II250 and TAF_II150) also contact DNA in the vicinity of the startpoint and influence the efficiency of the reaction.

Although assembly can take place just at the core promoter in vitro, this reaction is not sufficient for transcription in vivo, where interactions with other factors that recognize the more upstream elements are required. These factors interact with the basal apparatus at various stages during its assembly.

This section updated 4-3-2000