Complex Pre-initiation

RRL (Promega) is quickly thawed and placed on ice. Twenty microliter reaction mixtures are prepared containing 70% (v/v) RRL, 0.5 /il amino acid mix (minus methionine), 80 mM KOAc, 1.6 [iM methionine, 1 mM GMP-PNP (for 48S pre-initiation complex) or 0.6 mM cyclo-heximide (for 80S complex formation), and 20 fiM of the small molecule hit under study. 

Sutrias-Grau, M., Bianchi, M.E., and Bernues, J. (1999) High mobility group protein 1 interacts specifically with the core domain of human TATA box-binding protein and interferes with transcription factor IIB within the pre-initiation complex. J. Biol. Chem. 274, 1628-1634. 

C. Eukaryotic transcription is more complex than in prokaryotes, mainly in terms of the nature of the RNA polymerases, the assembly of the pre-initiation complex, and the need for processing eukaryotic RNAs. 

Not every eucaryotic promoters possesses a TATA box. For promoters devoid of a TATA box, the initiation sequence is determining for promoter selection and formation of the pre-initiation complex. 

A transcription-competent pre-initiation complex consisting of general transcription initiation factors and RNA polymerase II, can be reconstituted in the test tube from the individual components. As outlined in Fig. 1.31, efficient reconstitution requires a defined order for the addition of the individual components. 

TFIIA and TFIIB support TFIID in the formation of a stable complex with the promotor. TFllB is necessary for the downstream selection of the start site for RNA polymerase 11. Interactions with TFllB ensure correct positioning of the RNA polymerase 11 on the promoter. Crystal structures have been solved for several of the intermediates of the pre-initiation complex (review Sokolev and Burley, 1997), showing, for example, that TBP affects a predominant kink in the DNA (see Fig. 1.16). TFIIB binds to the TBP-DNA complex, contacting both TBP and the DNA. 

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. 

The addition of ATP to the pre-initiation complex leads to a rapid melting of the promoter, initiation of RNA synthesis and dissociation of the RNA polymerase from the promoter. 

The use of a pre-formed holoenzyme complex of the RNA polymerase II appears to be an economical mechanism for the formation of an initiation-competent transcription complex. There are two basic processes involved in the formation of a pre-initiation complex in the cell ... 

Some of the coactivators are ascribed a mediator function between the DNA-boimd transcriptional activators and the pre-initiation complex, while others are attributed an active role in the restructuring of the chromatin. 

An example of a negative cofactor is the NC2 complex, which can repress the basal transcription level. The NC2 complex consists of two subimits, both displaying homology to the histone proteins. The repressive fimction of NC2 is due to its competition with TFIIB and TFIIA for the promoter binding site, thus blocking formation of the pre-initiation complex. 

In one model it is assumed that transcriptional activators and coactivators increase the efficiency of formation of the pre-initiation complex. This function includes a restructuring of chromatin at the transcription start site. In this context the formation of the TFIID complex at the promoter plays an important role. 

Direct inhibition of the formation of a pre-initiation complex complexation of basal transcription factors, such as TFIID or TFIIB, or competition with TFIIB for binding to the promoter. An example for this type of repression is the negative cofactor NC2 (see 1.4.3.2). Transcription repression can also result from phosphorylation of the basal transcription factors. By this token, the repression of transcription observed during mitosis is attributed to the hyperphosphorylation of TBP and TAFs. 

Induction of a chromatin structure that does not allow the efficient formation of the pre-initiation complex e.g. by deacetylation of histones ( see.1.4.6). 

Having introduced some of the most common DNA-binding motifs, let us try to envision what the pre-initiation complex bound to DNA acmaUy looks like. 

Activation of the pre-initiation complex Melting of the DNA in the vicinty of the start site... 

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