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ttie numbers shewn am tor yeast but are suntei fur oilier eukaryotes including humans.

ttie numbers shewn am tor yeast but are suntei fur oilier eukaryotes including humans.

FIGURE 12-15 TFHB-TBP-promoter complex. This structure shows the TBP protein hound to the TATA sequence, just as we saw in the previous figure. Here, tho genera! transcription factor TR!8 (shown in turquoise) has been added. Tins tripartite complex forms the platform to which other general transcription factors, and Pol t| iLsetf, are recruited during pre-imtiation complex assembly. (Ntlrolov D.B, Chen H, Halay E.D., Usheva AA, Htsatake K.r Lee D.K., Roeder RG, and Burley S.K. 1995. Nature 377: 119 ) image prepared with MolSaipt, BobStrtp!, and Raster 3D Extencfcd DtvA on either side of image modeled by Leemor Joshua-Tor.

asymmetry in the rest of the assembly of the pre-initiation complex and the unidirectional transcription that results. TFIfB also contacts Pol II in the pre-initiation complex. Thus, this protein appears to bridge the TATA-bonnd TBP and polymerase. Recent structural studies suggest thai the N-terminal domain of TF11H inserts into the RNA exit channel of Pul II in a manner analogous to tr3 2 in the bacterial case.

TFTIF. This twc-subunit factor associates with Pol If and is recruited to the promoter together with that enzyme (and other factors). Binding of Pol 11-TFfIF stabilizes the DNA-TBP-TFIÍB complex and is required before TFI1E and TFIJH are recruited to the pre-initiation complex (Figure 12-13).

TFIÍE and TFIIH. TFHE, which, like TFHF, consists of two subunils, binds next, and has roles in the recruitment and regulation of TFIIH. TFIIH controls the ATP-dependent transition of the pre-initiation complex to the open complex. It is also the largest and most complex of the general transcription faclors—it has nine subunits and a molecular mass comparable to that of the polymerase itself! Within TFIIH are two subunits that function as ATHases, and another that is a protein kinase, with roles in promoter me)ling and escape, as described above. Together with other factors, the ATPase subunits are also involved in nucleotide mismatch repair (see Chapter 9),

In Vivo, Transcription Initiation Requires Additional Proteins, Including the Mediator Complex

Thus far we have described what is needed for Pol 11 to initiate transcription from a naked DNA template in vitro. But we have already noted that high, regulated levels of transcription in vivo requite, additionally, the Mediator Complex, transcriptional regulatory proteins, and, m many cases, nucleosome-mod ¡tying enzymes [which are themselves often parts of large protein complexes) (Figure 12-16). The characteristics of various modifying complexes are given in Table 7.7.

One reason for these additional requirements js that the DNA template in vivo is packaged into nucleosomes and chromatin, as we discussed in Chapter 7. This condition complicates binding to the promoter of polymerase and its associated factors, 'transcriptional regulatory

FIGURE 12-16 Assembly of the pre-initiation complex in presence of Mediator, nudeosome modifiers and re modelers, and transcriptional activators.

In addition to the general transcnption factors shewr, in figure 12-13, transcriptional activators bound to sites near the gene recruit nucleasomes modifying and remodeling complexes, and the Mediator Complex, which together hdp form the pre-Fiitiation complex.

proteins called activators help recruit polymerase lo the promoter, stabilizing its binding there. This recruitment is mediated tlirough interactions between DNA-bound activators and parts of the transcription machinery. Often the interaction is with the Mediator Complex thence its name). Mediator is associated with the C,TD "tail" of the large polymerase subunit through one surface, while presenting other surfaces for interaction with DNA-bound activators. This explains the need for Mediator lo achieve significant transcription in vivo.

Despite this central role in transcriptional activation, deletion of individual subunits of Mediator often leads to loss of expression of only a small subset of genes, different for each subunit (it is made up of many subunits). This resuit likely reflects the fact that different activators are believed to interact with different Mediator subunits to bring polymerase to different genes. In addition. Mediator aids initiation by regulating the CTD kinase in TFfffl.

The need for nucleosome modifiers and remodellers also differs at different promoters or even at the same promoter under different circumstances. When and where required, these complexes are also recruited by the DNA-bound activators.

We will discuss the role of Mediator and modifiers in stimulating transcription in Chapter 17. We now consider some of the structural and functional properties of Mediator,

Mediator Consists of Many Subunits, Some Conserved from Yeast to Human

As shown in Figure 12-17, the yeast and human Mediator each include more than 20 subunits, of which 7 show significant sequence homology be! ween the two organisms. [The names of the subunits arc different in each case, reflecting the experimental approaches that led to their identification.) Very few of these subunits have any identified function. Only one, (Srb4), is essential for transcription of essentially all Pol 11 genes in vivo. Low-resohitlon structural comparisons suggest both Mediators have a similar shape, and both are very large—even bigger than RNA polymerase itself.

The Mediator from both yeast and humans is organized in modules. These modules can be dissociated from one another under certain conditions in vitro. This observation, together with the fact that human Mediator varies in its composition (and size) depending on how it is isolated, has led to the idea that there are various forms of Mediator (particularly in metazoans), each containing subsets of Mediator subunits. Furthermore, it has been argued lhat the different forms are involved in regulating different subsets of genes, or responding to

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