Basal transcription initiation complex

Fig. 1.40. Model of repression and activation of transcription. The figure illustrates various mechanisms of repression of transcription, a) genes are in a generally repressed states in inactive chromatin. In a first phase of activation the chromatin is restrnctured. b) The promoter is now accessible for the binding of the basal transcription factors and for RNA polymerase II. c) An initiation complex is formed that contains the central components of the transcription apparatns, bnt which enables transcription only at a low rate, d) the binding of repressors to the transcription initiation complex can prevent fnrther activation of transcription at this step, e) the binding of transcription activators to their DNA elements leads to activation of transcription, f) an active repression is affected by proteins that bind seqnence specifically to DNA elements and in their DNA-bound form inhibit the transcritption preventing interactions with the transcription apparatus.

The entire top side of TBP bound to the TATA-box DNA is out of the way and is fi e to interact with other factors. This leaves a generous surface on TBP available for interactions with the multitude of factors which are parts of the transcription initiation complex. The same is true for the TFIIA/TFIIB-TBP-complexes. The extensive surfaces displayed by the TFIIA/TFIIB-TBP-DNA complexes represent potential sites for binding basal initiation factors, signal-responsive transcriptional activators, co-activators and mediators, and, most importantly, leave room for Pol II. 

Sequences farther upstream from the start site determine how frequently the transcription event occurs. Mutations in these regions reduce the frequency of transcriptional starts tenfold to twentyfold. Typical of these DNA elements are the GC and CAAT boxes, so named because of the DNA sequences involved. As illustrated in Figure 37—7, each of these boxes binds a protein, Spl in the case of the GC box and CTF (or C/EPB,NF1,NFY) by the CAAT box both bind through their distinct DNA binding domains (DBDs). The frequency of transcription initiation is a consequence of these protein-DNA interactions and complex interactions between particular domains of the transcription factors (distinct from the DBD domains—so-called activation domains ADs) of these proteins and the rest of the transcription machinery (RNA polymerase II and the basal factors TFIIA, B, D, E, F). (See... 

Figure 37-9. The eukaryotic basal transcription complex. Formation of the basal transcription complex begins when TFIID binds to the TATA box. It directs the assembly of several other components by protein-DNA and protein-protein interactions. The entire complex spans DNA from position -30 to +30 relative to the initiation site (+1, marked by bent arrow). The atomic level, x-ray-derived structures of RNA polymerase II alone and ofTBP bound to TATA promoter DNA in the presence of either TFIIB or TFIIA have all been solved at 3 A resolution. The structure of TFIID complexes have been determined by electron microscopy at 30 A resolution. Thus, the molecular structures of the transcription machinery are beginning to be elucidated. Much of this structural information is consistent with the models presented here.

In eukaryotes, general transcription factors must bind to the promoter to allow RNA polymerase II to bind and form the initiation complex at the start site for transcription. General manscription factors are common to most genes. The general transcription factor TFIID (the TATA fector) must bind to the TATA box before RNA polymerase II can bind. Other examples delude SP-1 and NF-.l that modulate basal transcription of many genes. 

In contrast to the procaryotes, where the o -holoenzyme of the RNA polymerase can initiate transcription without the aid of accessory factors, the eucaryotic RNA polymerase requires the help of numerous proteins to begin transcription. These proteins are termed basal or general initiation factors of transcription. Together with RNA polymerase II, they participate in the basal transcription apparatus. The various components must associate in a defined order for the formation of a transcription-competent complex, from which a low level of transcription is possible. An increase in the basal transcriptional level requires the effect of specific transcriptional activators, which bind cognate DNA sequences at a variable distance from the promoter. The transcriptional activators themselves require the aid of further protein factors, known as coactivators (see 1.4.3.2), in order to attain full stimulatory activity. 

The purification and structural and functional characterization of the general initiation factors has proven extremely difficult. The specific function of the various factors, as well as their structural role in the entire complex, remains poorly resolved. According to the current model, the general transcription initiation factors, with which an exact start of transcription is possible in vitro, are required for the formation of a basal... 

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 formation of an active, regulation-competent initiation complex for transcription in eucaryotes demands the concerted action of a large number of proteins. It is estimated that more than 50 different proteins participate in the initiation of transcription in eucaryotes. The basal transcription complex, consisting of the general initiation factors, as well as RNA polymerase 11, allows only for a slow transcription rate. For a regulated acceleration of this low transcription rate it is necessary to have - apart from the regulatory DNA-binding proteins - mediation by further co-activator proteins. 

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. 

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. 

Despite this elaborate binding complex, repression is not absolute. Binding of the Lac repressor reduces the rate of transcription initiation by a factor of 10s. If the 02 and 03 sites are eliminated by deletion or mutation, the binding of repressor to CL alone reduces transcription by a factor of about 102. Even in the repressed state, each cell has a few molecules of /3-galactosidase and galactoside permease, presumably synthesized on the rare occasions when the repressor transiently dissociates from the operators. This basal level of transcription is essential to operon regulation. 

TBP bound to the TATA box is the heart of the initiation complex (see Figure 28.19). The surface of the TBP saddle provides docking sites for the binding of other components (Figure 28.21). Additional transcription factors assemble on this nucleus in a defined sequence. TFIIA is recruited, followed by TFIIB and then TFIIF—an ATP-dependent helicase that initially separates the DNA duplex for the polymerase. Finally, RNA polymerase II and then TFIIE join the other factors to form a complex called the basal transcription apparatus. Sometime in the formation of this complex, the carboxyl-terminal domain of the polymerase is phosphorylated on the serine and threonine residues, a process required for successful initiation. The importance of the carboxyl-terminal domain is highlighted by the finding that yeast containing mutant polymerase II with fewer than 10 repeats is not viable. Most of the factors are released before the polymerase leaves the promoter and can then participate in another round of initiation. 

How do the special transcription factors regulate the activities of the basal transcription factors Information on this issue is only beginning to become available. One might expect, for example, to find a scenario where RXR/VDR directly contacts TFIIA, to stimulate the activity of the initiation complex. 

The basal transcription complex described in Chapter 29 initiates transcription at a low frequency. Recall that several general transcription factors (the preinitiation complex) join with KNA polymerase II to form the transcription complex. Additional transcription factors must bind to other sites for a gene to achieve a high rate of mRNA synthesis. In contrast with the regulators of prokaryotic transcription, few eukaryotic transcription factors have any effect on transcription on their own. Instead, each factor recruits other proteins to build up large complexes that interact with the... 

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