Eukaryotic activators

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In a basal promoter region, basal trancription is very low (almost inactive) without Upstream Activating Sequences and Enhancers, therefore the function of these is to stimulate transcription.  
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In a basal [[Promoter|promoter]] region, basal transcription is very low (almost inactive) without [[Upstream Activating Sequences|Upstream Activating Sequences]] and [[Enhancers|Enhancers]], therefore the function of these is to stimulate [[Transcription|transcription]].  
  
 
There are two classes of Upstream Activating Sequences and Enhancer elements. These are elements which direct the binding of specific activators to enhance transcription, and they include:  
 
There are two classes of Upstream Activating Sequences and Enhancer elements. These are elements which direct the binding of specific activators to enhance transcription, and they include:  
  
1) 'Common' Sequence Elements, which are often located close to the core promoter. These bind activators that are relatively abundant and don't respond to any particular stimuli therefore they are consitutivelly active.  
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#'Common' Sequence Elements, which are often located close to the core promoter. These bind activators that are relatively abundant and don't respond to any particular stimuli, therefore, they are constitutively active.
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#Response Elements which bind factors whose activity is induced in response to specific stimuli, therefore they only activate transcription under certain circumstances.
  
2) Response Elements which bind factors whose activity is induced in response to specific stimuli, therefore they ony activate transcription under certain circumstances.  
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The combination of these elements is what dictates when and at what level a gene is described, and this is known as the combinatorial control of transcription.  
  
The combination of these elements is what dictates when and at what level a gene is described, and this is known as the combinatorial control of transcription. 
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Enhancers function irrespective of location and orientation. Enhancers may be close or many kilobases away from gene being activated. NB: enhancers may also be downstream of the open reading frame.  
  
Enhancers function irrespective of location and orientation. Enhancers may be close or many kilobases away from gene being activated. NB: enhancers may also be downstream of open reading frame.
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Eukaryotic activators are modular with two main domains (joined by a flexible protein domain):  
  
Eukaryotic activators are modular with two main domains (joined by a flexible protein domain):<br>1) Activation domain (some activators have multiple activator domains). This is what allows activators to contact the basal transcription machinery. <br>2) DNA binding domain (most only have 1 DNA binding domain). They have a well defined structure (eg Leucine Zipper, Homeodomain, Helix Loop Helix)<ref>Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.</ref><br>However activation domains lack sequence conservation and structural information, and we can only characterise them according to their amino acid composition. They are thought to be unstructured and flexible, only thought to form their structure once they interact with their target. Furthermore they contain multiple short segments that work together in an additive fashion, meaning that parts of them may be removed and they can still remain functional. (An image showing the different domains and how they interact with DNA is shown [http://images.slideplayer.com/16/5011349/slides/slide_4.jpg here])<br>
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#Activation domain (some activators have multiple activator domains). This is what allows activators to contact the basal transcription machinery.  
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#DNA binding domain (most only have 1 DNA binding domain). They have a well-defined structure (e.g. [[Leucine Zipper|Leucine Zipper]], [[Homeodomain|Homeodomain]], [[Helix Loop Helix|Helix Loop Helix]])<ref>Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.</ref>.&nbsp;However, activation domains lack sequence conservation and structural information, and we can only characterise them according to their [[amino acid|amino acid]] composition. They are thought to be unstructured and flexible, only thought to form their structure once they interact with their target. Furthermore, they contain multiple short segments that work together in an additive fashion, meaning that parts of them may be removed and they can still remain functional. (An image showing the different domains and how they interact with DNA is shown [http://images.slideplayer.com/16/5011349/slides/slide_4.jpg here].)
  
There are multiple in vivo and in vitro methods for the analysis of activators and their function.  
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There are multiple''in vivo'' and ''in vitro'' methods for the analysis of activators and their function.  
  
 
One example is a reporter assay which may be performed as follows: Prepare two plasmids, one with gene encoding the protein thought to be the activator, and one with the reporter gene and the activator's binding site. After this, cotransfect plasmids into chosen cells and if activator binds, there will be reporter gene expression. (An image of this process is shown [https://www.activemotif.com/images/products/lightswitch_promoter_assay_v4_big.jpg here])  
 
One example is a reporter assay which may be performed as follows: Prepare two plasmids, one with gene encoding the protein thought to be the activator, and one with the reporter gene and the activator's binding site. After this, cotransfect plasmids into chosen cells and if activator binds, there will be reporter gene expression. (An image of this process is shown [https://www.activemotif.com/images/products/lightswitch_promoter_assay_v4_big.jpg here])  
  
This measures both the ability of the protein to bind DNA and activate transcription. <br>
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This measures both the ability of the protein to bind DNA and activate transcription.  
  
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Revision as of 17:43, 25 October 2017

In a basal promoter region, basal transcription is very low (almost inactive) without Upstream Activating Sequences and Enhancers, therefore the function of these is to stimulate transcription.

There are two classes of Upstream Activating Sequences and Enhancer elements. These are elements which direct the binding of specific activators to enhance transcription, and they include:

  1. 'Common' Sequence Elements, which are often located close to the core promoter. These bind activators that are relatively abundant and don't respond to any particular stimuli, therefore, they are constitutively active.
  2. Response Elements which bind factors whose activity is induced in response to specific stimuli, therefore they only activate transcription under certain circumstances.

The combination of these elements is what dictates when and at what level a gene is described, and this is known as the combinatorial control of transcription.

Enhancers function irrespective of location and orientation. Enhancers may be close or many kilobases away from gene being activated. NB: enhancers may also be downstream of the open reading frame.

Eukaryotic activators are modular with two main domains (joined by a flexible protein domain):

  1. Activation domain (some activators have multiple activator domains). This is what allows activators to contact the basal transcription machinery.
  2. DNA binding domain (most only have 1 DNA binding domain). They have a well-defined structure (e.g. Leucine Zipper, Homeodomain, Helix Loop Helix)[1]. However, activation domains lack sequence conservation and structural information, and we can only characterise them according to their amino acid composition. They are thought to be unstructured and flexible, only thought to form their structure once they interact with their target. Furthermore, they contain multiple short segments that work together in an additive fashion, meaning that parts of them may be removed and they can still remain functional. (An image showing the different domains and how they interact with DNA is shown here.)

There are multiplein vivo and in vitro methods for the analysis of activators and their function.

One example is a reporter assay which may be performed as follows: Prepare two plasmids, one with gene encoding the protein thought to be the activator, and one with the reporter gene and the activator's binding site. After this, cotransfect plasmids into chosen cells and if activator binds, there will be reporter gene expression. (An image of this process is shown here)

This measures both the ability of the protein to bind DNA and activate transcription.

References

  1. Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.
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