TFIIH

Given that these proteins have properly assembled, the initiation complex is ready to start transcription. How does the enzyme get started A component of TFIID, again a multi-subunit complex TFIIH, unwinds the DNA and phosphorylates serine-5 of the C-terminal tail (CTD) of the largest polymerase subunit (Rpbl). Serine-5 phosphorylation and phosphorylation of serine-2 (by pTEFb) are required to release the enzyme from the other components of the initiation complex and to start RNA synthesis. 

Garcia-Martinez LF, Mavankal G, Neveu JM, Lane WS, Ivanov D, Gaynor RB (1997) Purification of a Tat-associated kinase reveals a TFIIH complex that modulates HIV-1 transcription. 

Gervais, V., et al.. Solution structure of the N-terminal domain of the human TFIIH MATl subunit new insights into the RING finger family. J Biol Chem, 2001, 276(10), 7457-64. 

Wada, T., Orphanides, G., Hasegawa, I., Kim, D.K., Shima, D., Yamaguchi, Y., Fukuda, A., Hisatake, K., Oh, S., Reinberg, D., and Handa, H. (2000) FACT relieves DSIF/NELF-mediated inhibition of transcriptional elongation and reveals functional differences between P-TEFb and TFIIH. Mol. Cell 5, 1067-1072. 

This consists of DNA-dependent RNA polymerase II and basal transcription factors (TFIIX, X = A - H). First, the basal factor TFIID binds to the promoter. TFIID, a large complex of numerous proteins, contains TATA boxbinding protein (TBP) and so-called TAFs (TBP-associated factors). The polymerase is attached to this core with the help of TFIIB. Before transcription starts, additional TFs have to bind, including TFIIH, which has heli-case activity and separates the two strands of DNA during elongation. In all, some 35 differ-... 

TFim 2 34,57 binding of TFIIH, modulation of activities of TFIIH... 

TFIIH 9 35-89 hehcase, protein kinase and ATPase-activity promoter unwinding, promoter clearance ( )... 

TFIIE binds TFIIH to assist the latter with the melting of the promoter. 

The binding of TFIIH completes the formation of the pre-initiation complex. TFIIH is a multi-protein complex with a variable composition (see 1.4.2.5) and which possesses protein kinase, ATPase and heUcase activities. The heUcase activity of TFIIH is required for the melting of the promoter. 

General transcription initiation factors TFIIB, TFIIE, TFIIF and TFIIH have been identified as components of the RNA polymerase 11 holoenzyme of yeast. Various forms of the yeast holoenzyme contain further proteins, known as mediators or SRB proteins (SRB, suppressor of RNA polymerase B). The mediators fimction as coactivators (see 1.4.3.2). The holoenzyme is difficult to define structurally because the proteins accessory to the core enzyme (see table 1) may not be permanently associated with RNA polymerase II. 

Fig. 1.32. Phosphorylation of the C-terminal domain of RNA polymerase II and the beginning of transcription. The transition from the initiation complex to actual begin of transcription is regulated via phosphorylation of the C-terminal domain (CTD) of RNA polymerase II. In the above model it is assumed that initially a complex is formed between TFIID and a holoenzyme of RNA polymerase consisting of RNA polymerase II and associated factors (mediators, SRB proteins) and the basal transcription factors. Phosphorylation of the C-terminal domain effects the dissociation of the RNA polymerase from the initation complex and the transition to the elongation phase. A protein kinase, which is part of TFIIH, is responsible for the phosphorylation. The nature of the signal that induces phosphorylation of RNA polymerase II remains unknown. SRB suppressor of RNA polymerase B. After Koleske and Young (1995).

The protein kinase responsible for phosphorylation at the C-terminus is localized in TFIIH. It has been shown (Shiekhattar et al., 1995) that TFIIH contains a Ser/Thr-spe-cific protein kinase termed CDK7 (or M015). CDK7 belongs to the family of cychn-dependent protein kinases (see 14.2.1). The cognate cyclin, cyclin H, is also foimd in TFIIH. 

TFIIH is a multi-protein complex consisting of at least eight different subimits (review Drapkin and Reinberg, 1994 Svjestrup et al, 1996). 

A more in-depth characterization of the subimits brought surprising results. Some of the polypeptides of TFIIH could be identified as proteins shown in other experiments to participate in repair of DNA damage, as well as in the regulation of the cell cycle (CDK7/Cyclin H, see above). TFIIH itself, or other individual components of TFIIH, thus participate in the following fundamental processes in the cell ... 

The picture of the structure and function of TFIIH is a varied and complex one. The mechanistic details are long not imderstood. 

Drapkin, R. and Reinberg, D. Tire multifunctional TFIIH complex and transcriptional control (1994) Trends Biochem. Sd. 19, 504-508... 

Svjestrup, Q., Vichi,P. and Egli,J.-M. The multiple roles of transcription/repair factor TFIIH (1996) Trends Biochem.Sd. 71, 346-350... 

TFIIE 2 34,000, 57,000 Recruits TFIIH hasATPase and helicase activities... 

TFIIH 12 35,000-89,000 binding of Pol II to nonspecific DNA sequences Unwinds DNA at promoter (helicase activity) ... 

RNA Strand Initiation and Promoter Clearance TFIIH has an additional function during the initiation phase. A kinase activity in one of its subunits phosphorylates Pol II at many places in the CTD (Fig. 26-9). Several other protein kinases, including CDK9 (cyclin-dependent kinase 9), which is part of the complex pTEFb (positive transcription elongation/actor b), also phosphorylate the... 

During synthesis of the initial 60 to 70 nucleotides of RNA, first TFIIE and then TFIIH is released, and Pol II enters the elongation phase of transcription. 

Diverse Functions of TFIIH In eukaryotes, the repair of damaged DNA (see Table 25-5) is more efficient within genes that are actively being transcribed than for other damaged DNA, and the template strand is repaired somewhat more efficiently than the nontemplate strand. These remarkable observations are explained by the alternative roles of the TFIIH subunits. Not only does TFIIH participate in the formation of the closed complex during assembly of a transcription complex (as described above), but some of its subunits are also essential components of the separate nucleotide-excision repair complex (see Fig. 25-24). 

When Pol II transcription halts at the site of a DNA lesion, TFIIH can interact with the lesion and recruit the entire nucleotide-excision repair complex. Genetic loss of certain TFIIH subunits can produce human diseases. Some examples are xeroderma pigmentosum (see Box 25-1) and Cockayne s syndrome, which is characterized by arrested growth, photosensitivity, and neurological disorders. ... 

TATA-Binding Protein The first component to bind in the assembly of a preinitiation complex at the TATA box of a typical Pol II promoter is the TATA-binding protein (TBP). The complete complex includes the basal (or general) transcription factors TFIIB, TFIIE, TFIIF, TFIIH Pol II and perhaps TFIIA (not all of the factors are shown in Fig. 28-27). This minimal preinitiation complex, however, is often insufficient for the initiation of transcription and generally does not form at all if the promoter is obscured within chromatin. Positive regulation leading to transcription is imposed by the transactivators and coactivators. 

Another important coactivator consists of 20 or more polypeptides in a protein complex called mediator (Fig. 28-27) the 20 core polypeptides are highly conserved from fungi to humans. Mediator binds tightly to the carboxyl-terminal domain (CTD) of the largest subunit of Pol II. The mediator complex is required for both basal and regulated transcription at promoters used by Pol II, and it also stimulates the phosphorylation of the CTD by TFIIH. Both mediator and TFIID are required at some promoters. As with TFIID, some DNA-binding transactivators interact with one or more components of the mediator complex. Coactivator complexes function at or near the promoter s TATA box. 

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