Machinery of gene transcription

Only gene transcription and its control will be considered here. What is discussed here and in the following chapters 10 and 11 on the regulation of gene transcription is only a brief survey of those aspects most relevant for cell signalling. In the genome era in which we live, the analysis of gene expression is the central issue. Excellent and comprehensive treatments of this field are available (ref. 1). 

Nuclear genes encoding messenger RNAs for proteins are transcribed in eukaryotes by RNA polymerase II (Pol II). This is an enzyme complex comprising at least 12 subunits. Pol II, like the other DNA-directed RNA polymerases (I and III), cannot recognize 

Transcriptional Splicing Eupoil/lmport Lifetime Translational Lifetime 

How do the distantly bound transcription factors contact the basic transcriptional machinery and instruct Pol II when to start transcription Although there are linkers to establish these contacts, the chromatin complex itself helps to make the contacts. DNA is highly condensed, suprahelical, and often bends on binding a dimeric transcription factor, bringing the transcriptional regulators closer together (Fig. 9.4). 

Interaction of the TATA-box-binding protein (TBP) with promoter DNA is rather inefficient and appears to be the rate-limiting step for the start of transcription. TBP must actually dissociate first from the TFIID complex before it can bind to the TATA-box DNA. Dissociation of TBP is facilitated by the dimeric structure of TFIID, when it is not bound to DNA, and by the interaction of TBP with TFIIA. 

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The interaction of external signals with membrane receptors generates second messengers. These messengers either modify the cell concentration of ions or metabolites or alter the functional state of a chain of several molecules that act as intermediaries. These intermediaries may modify the intensity of determined biochemical reactions or, in other cases, are integrated into the machinery of gene transcription and alter the expression of specific genes. The consequences of these activities can lead to the induction of cell division. 

The participation of the nuclear receptors in the machinery of gene transcription takes place by means of specific domains of the molecule known as transactivators (abbreviation for transcription activators). These are made up of sequences of amino acids that interact by means of protein-protein contacts with other transcription factors. The artificial alteration of these sequences has as a consequence the inability of the hormone to induce gene expression (Beato et al. 1996 Klug et al. 1987 Lones et al. 1995). 

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). 

Taniguchi T, Takaoka A. 2002. The interferon-alpha/beta system in antiviral responses A multimodal machinery of gene regulation by the IRF family of transcription factors. Curr Opin Immunol. 14 111-116. 

The human HS cycle can be considered broadly as a period which leads to the dramatic shift in activities of the transcriptional and translational machinery followed by eventual recovery and resumption of original activities preceding stress. Figure 1 depicts many of the key events in the HS cycle for a typical human cell line such as cervical carcinoma-derived HeLa cells. Most cells respond in an identical fashion, but some cell types that have distinctive HS responses. These differences are manifested by shifts in the relative concentrations of accumulated HS proteins and possibly in the pattern of posttranslational modifications. In all cases, however, the cellular stress response is heralded by induction of a specific transcription factor whose DNA binding activity facilitates increased expression of one or more of the stress-inducible genes. 

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... 

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. 

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. 

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). 

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

The fact that a receptor dimer identifies a HRE does not assure, by itself, the transcription of the gene. This is a necessary, but insufficient, condition. Once the dimer-HRE interaction has been produced, the machinery of transcription needs to be assembled, requiring the binding of other intermediary cofactors. Some of these are tissue specific, and others recognize only a particular receptor dimer, thus obviating others that could recognize the same HRE. 

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