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Transcriptional control

Gene expression in prokaryotes and eukaryotes is tightly controlled, mostly via transcriptional control whereby the initiation of transcription is highly regulated. In prokaryotes, the promoter is where repressors and activators bind and it is usually near the gene it regulates. In eukaryotes, the promoter is relatively far away and is bound by transcription factors (the eukaryotes equivalent of repressors and activators). In higher eukaryotes, some transcription factors even bind at regulatory sites thousands of base pairs upstream or downstream from the promoter; and the same transcription factor can even bind multiple regulatory sites by bringing two sites close to each other (looping out DNA in the middle). In bacteria, control of gene expression is most often accomplished by regulating transcription initiation from promoters that are ~100 bp of the start site.

How do we know that transcription of a gene most often is what determines whether the encoded protein will be synthesized in a specific type of cell, say albumin in a liver cell or a neurotransmitter receptor in a neuron? If a protein is not expressed, its corresponding gene rarely is transcriptionally active. Thus, control of protein synthesis is at the level of transcriptional control; if a protein is not expressed, then its gene is likely not even expressed. This was figured out by an early experiment using a cDNA microarray. cDNA is single-stranded DNA that has been reverse-transcribed in vitro from cellular mRNA. By soaking a cDNA microarray with labeled RNAs, it is possible to determine which genes are being transcribed in a particular cell; only active genes were being transcribed. Thus, regulation is at the transcriptional level,

Promoters

Promoters can differ in strength, i.e. how actively they promote transcription of their adjacent DNA sequence. Promoter strength is usually a matter of how tightly RNAP and GTFs bind to respective DNA sequences. The more similar the sequences are to a consensus sequence, the stronger the binding is. Gene expression in eukaryotes is controlled by complex interactions between cis-acting elements within the regulatory regions of the DNA, and trans-acting factors that include transcription factors and the basal transcription complex. Upstream control elements that regulate and amplify the formation of the basal transcription complex: The core promoter of protein-encoding genes contains binding sites for the basal transcription complex and RNAPII, and is normally within about 50 bases upstream of the transcription initiation site.

Further transcriptional regulation is provided by upstream control elements (UCEs), usually present within about 200 bases upstream of the initiation site. The core promoter for RNAP II normally (though not always) contains a TATA box, the highly conserved DNA sequence: T A T A T/A A. A similar sequence, though not as highly conserved, is found in the INR (initiator) element, part of the some RNAP II promoters. Some genes also have enhancer elements that can be thousands of bases upstream or downstream of the transcription initiation site.

Enhancer elements

How do enhancer elements function to stimulate transcription? Enhancers do not act on the promoter region itself, but are bound by activator proteins. These activator proteins interact with the mediator complex, which recruits polymerase II and the general transcription factors which then begin transcribing the genes. Enhancers can also be found within introns. An enhancer's orientation may even be reversed without affecting its function. They can be so far or near because the genome can bend around, bringing the enhancer-bound activators close to the mediator complex.

Identifying promoters

The adenovirus promoter experiment is the most famous technique. Another laboratory had been working out techniques to extract protein from nuclei so as to maximize in vitro transcription from isolated DNA (0.3 M KCl + buffers and reducing agents). They used this nuclear extract to perform in vitro transcription on restriction fragments of viral DNA from the region where the promoter was approximately mapped in the previous experiment.

Repressors and activators

Repressors are modular and have separate DNA-binding and repression domains. In repressor-directed histone deactylation, a histone directs the deacetylation of histone tails of chromatin. Activators are also modular and have separate DNA-binding and activation domains. They can direct hyper-acetylation in activator-directed hyperacetylation.

Eukaryotic transcription activator proteins

The two most important elements are the: DNA-binding domain; and activator domain. The flexible protein domain allows eukaryotic DNA sequence control elements to function together even when the number of base pairs between the control elements is altered. Activation domains interact with coregulatory proteins, which can merely stabilize binding of the transcription factor (and even RNAP II) to the DNA or can even be bound to enhancer elements (DNA sequences) near or far, upstream or downstream that then interact with the mediator complex that recruits RNAP II in a productive manner.

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Transcriptional controlComments
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