Interactions with the Transcription Apparatus

The pathways by which specific transcriptional activators and co-activators influence transcription initiation remain little understood and, for the most part, an item of speculation. There are two main pathways in discussion which possibly act cooperatively  

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. 

The other model views the activators and coactivators as responsible for the stable and defined spatial arrangement of proteins in the holo-complex and for the induction of topology in the DNA which promotes initiation. 

It can be assumed that the extent to which either pathway is used depends on the structure of the specific gene, as well as on the structure of the chromatin. 

The activating domain of transcriptional activators requires specific binding partners within the transcription apparatus. Unequivocal identification of these binding partners has proven difficult because of the large number of proteins that participate in the formation of the pre-initiation complex. 

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Fig. 1.21. Structural and functional principles of transcription activators. Typical transcription activators of encaryotes possess a DNA-binding domain, an effector domain and a transactivating domain. An incoming signal is registered by the effector domain and transformed into a change in affinity for DNA. In the active state, the transcription activator is capable of binding to its cognate DNA-binding element. Protein-protein interactions with the transcription apparatus bound to the promoter mediate a stimnlation of transcription initiation.

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 picture sketched above for the function of the receptors of the retinoids and the T3- and Vitamin Ds-hormones is in many points still incomplete. The cooperation of the activated receptors with other transcription factors, their interactions with the transcription apparatus and the influence of chromatin structure are still poorly understood. 

Transcriptional activators bind specifically to cognate DNA elements variably located relative to the promoter (see 1.4.2.2) and can interact directly or indirectly with the transcription apparatus. Transcriptional activators depend on the occmrence of regulatory DNA elements for their action and perform their function on specific genes. They are thus termed specific transcriptional activators, to distinguish them from proteins that activate transcription independent of specific DNA elements (see 1.4.3.2). 

The specific transcriptional activators (see 1.4.3.1), represented by the GAL4 protein of yeast, are sequence-specific DNA-binding proteins. They possess both a DNA-binding domain and a trans-activating domain to allow them to interact directly with the transcription apparatus. 

Among the proteins that form part of the transcription machinery are found some cell factors that are produced in limited quantities. They are called cofactors of transcription (NCoA, for nuclear-receptor coactivator NCoI, for nuclear-receptor coinhibitor), formerly known as transcription intermediary factors (TIF) (McDonnell et al. 2002 McKenna et al. 1999). They constitute one of the classes of proteins that form part of the transcription machinery. These proteins are utilized by diverse types of intensifiers, that is to say, by sequences of DNA that anchor transcription factors, of which HRE are a particular case (Gruber et al. 2002 Mester et al. 1995). They do not interact directly with the DNA, but they do with the receptors and with the other elements of the transcription apparatus (Fig. 1.9). 

Direct repressors interact with the basal components of the transcription apparatus or with transcriptional activators to inhibit their activity. Specific repressors, analogous to transcriptional activators, are constructed modularly, with a DNA-binding domain and a repressor domain. The repressive character of such domains has been proven in domain swapping experiments. The mechanism of specific repression remains speculative. The following mechanisms are, however, conceivable ... 

Due to the complex structure of the initiation complex it remains imclear which interactions are responsible for the first mechanism. The coupling between the transactivat-ing domain and the initiation complex can be direct or indirect. There is evidence which indicates that proteins with co-activator function mediate the interaction between HRE-bound receptors and the transcription initiation apparatus. One such protein is RIP-140, which mediates the transcription activation of the estrogen receptor. The AF2 domain can also directly contact the transcriptional apparatus. One component of the RNA polymerase II holoenzyme, the SUGl protein, has been identified as a binding partner for the AF2 domain. The SUGl protein has the function of a co-acti-vator in transcription initiation and is considered a mediator (see 1.4.3.2). 

The trans-activating domains of transcriptional activators are also common substrates for phosphorylation by protein kinases. The detailed mechanism by which the phosphorylation affects the interactions with the basal transcription apparatus is known in very few cases. The reason for this is the difficulty of identifying the specific interaction partner in the complex transcription apparatus. 

In the course of p53 activation, numerous specific protein-protein interactions are engaged to perform the growth-controlling and pro-apoptotic functions of p53. Of these, the interaction with the MDM2 protein stands out, as discussed above. Other important protein-protein interactions include the binding of p53 to components of the basal transcription apparatus, e. g., the TFIIH p62 subunit, interaction with proteins of nucleotide excision repair (XPB, XPD), and the interaction with transcriptional coactivators. 

Chromatin remodeling, transcription factor modification by various enzyme activities, and the communication between the nuclear receptors and the basal transcription apparatus are accomplished by protein-protein interactions with one or more of a class of coregulator molecules. The number of these coregulator molecules now exceeds 100, not counting species variations and splice variants. The first of these to be described was the CREB-binding protein, CBP. CBP, through an amino terminal domain, binds to phosphorylated serine 137 of CREB and mediates transactivation in response to cAMP. It thus is described as a coactivator. CBP and... 

DNA helicases are a class of enzymes necessary for fundamental DNA transactions such as DNA replication, transcription, repair, and recombination. Moreover, among the components of the DNA replication, repair, recombination or transcription apparatus, the first that may encounter a site of DNA damage are the DNA helicases. Thus, a complete understanding of the effect of cisplatin lesions on DNA metabolism requires a biochemical analysis of their interaction with this class of proteins. At least three reports have investigated the effects of cisplatin intrastrand lesions on the activity of DNA helicases implicated either in repair or in recombination. 

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