Transcriptional machinery

This early biological result spurred a variety of biochemical studies of the interactions of various polyamides with the basal transcription machinery and TE-DNA complexes. Two studies have used promoter scanning to identify sites where polyamide binding inhibits transcription [64, 65]. The method uses a series of DNA constructs with designed polyamide binding sites at varying distances from... 

Upon binding, the artificial transcription factor recruits the necessary transcriptional machinery for gene activation. (Bottom, left) Ball-and-stick model for a polyamide conjugated to the VP2 activation domain. Symbols are as in Fig. 3.4. (Bottom, right) Structure of the polyamide-VP2 conjugate with the polyproline linker domain in brackets... 

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.

Finally, the binding of specific transcription factors to cognate DNA elements may result in disruption of nucleosomal structure. Many eukaryotic genes have multiple protein-binding DNA elements. The serial binding of transcription factors to these elements—in a combinatorial fashion—may either directly disrupt the structure of the nucleosome or prevent its re-formation or recruit, via protein-protein interactions, multiprotein coactivator complexes that have the ability to covalently modify or remodel nucleosomes. These reactions result in chromatin-level structural changes that in the end increase DNA accessibifity to other factors and the transcription machinery. 

In the case of plasmid DNA the intracellular target (i.e., transcription machinery) is located in the nucleus. Plasmid DNA is a much larger molecule than oligonucleotides, and... 

There are now numerous examples of proteins that are unstructured or only partially structured under physiological conditions yet are nevertheless functional (Dunker and Obradovic, 2001 Wright and Dyson, 1999). In many cases, such intrinsically disordered proteins adopt folded structures upon binding to their biological targets. As the proteins that constitute the transcriptional machinery have become... 

Intracellular pathways after escape from the endolysosomal system into the cytosol are less clear. Obvious bottlenecks include, in the case of gene transfer (pDNA delivery), cytosolic transport to the perinuclear area, nuclear uptake, and nuclear presentation of the pDNA to the transcriptional machinery in bioactive form. In the case of siRNA (or mRNA and some other nucleic acids such as oligonucleotides), cytosolic accessibility for the required function is essential. Besides cytosolic transport [176, 177] and the nuclear import of large nucleic acid molecules [178-180], incorporation of functional nuclear import peptide domains has been evaluated [181-186]. Another bottleneck, nucleic acid unpackaging [187], i.e., partial or complete dissociation from the polymeric carrier, which is required for biological accessibility of the delivered nucleic acid, will be discussed in Sect. 3.3. 

Fig. 1 Bioresponsive polyplexes. (a) Systemic circulation of shielded polyplexes in blood stream and attachment to cell surface receptor (b) endocytosis into endosomes, deshielding by cleavage of PEG linkers and activation of membrane-destabilizing component by acidic pH or other means (c) endosomal escape into cytosol (d) siRNA transfer to form a cytosolic RNA-induced silencing complex complex (e) cytosolic migration and intranuclear import of pDNA (/) presentation of pDNA in accessible form to the transcription machinery...

As was previously established, the spatial structure of the receptor domains is altered by interaction with the hormone, with DNA, with other proteins, and by the state of the receptor phosphorylation. Different states of folding suppose that the receptor exhibits different surfaces that permit it to gain or to lose affinity for DNA sequences or for proteins, as they are components of the native receptor or of the transcriptional machinery. The different properties that... 

At least seven proteins, besides the RNA-polymerase II, participate in the transcription machinery. The initiation of the transcription occurs when the transcriptional complex in the promoter region of the gene has been stabilized. The receptor dimer forms a complex of high affinity with the sequence of the HRE. This binding provides a firm base for the anchorage and stabilization of the transcriptional complex. The dimeric structure of the receptor acquires affinity to attract different coactivators that bring together the proteins of the transcriptional complex (Fig. 1.9). 

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). 

The participation of the different cofactors that form part of the transcription machinery is not homogeneous. Some, like pl60, can interact with both transcription activator domains of the receptor, TAF1 and TAF2, even though they utilize different pi60 domains. Others, like CBP/p300, do not enter into... 

An additional variable to consider is how the subsequent interaction of the coactivators with other proteins of the transcription machinery is affected. This interaction occurs in the context of the promoter of each particular gene. 

The bond of the receptor dimer with the nucleotide sequence of the HRE in the promoter region of the gene is what directs the assembly of the proteins (up to 19) that yields the transcription machinery. The operation of the machinery depends on the continual, sequential reestablishment of protein-protein contacts. Each new interaction depends on whether the previous proteins had assembled themselves correctly in such a way that the protein under consideration does not bind unless the prior interactions have created the appropriate surface of contact. 

The context of the gene promoter being considered that has some specific conditions for accepting activation by particular conformations of the transcription machinery. 

Many aspects relating to the specificity and intensity of gene transcription in response to hormones remain open. Nevertheless, a prudent conclusion permits establishing that two definite elements are intervening the interaction of the receptor dimer with the palindrome and several protein-protein interactions that are produced between the dimer and the remaining components of the transcription machinery (Beato 1989). 

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