Monday, June 21, 2010

Activation of Transcription Initiation III

Activation Domains
Activation domains are another important portion of the regulatory protein that is involved in altering the activity of a promoter.  There are three main types of activation domains:
1.      Acidic, such as Gal4
2.      Glutamine-rich, such as Sp1
3.      Proline-rich, such as CTF

These different types of activation domains have different mechanisms and may also be involved in allowing the regulatory elements to function at a distance.  Importantly, many regulatory proteins may have multiple activation domains. 

As mentioned previously, the mediator complex works to integrate the signals from multiple activation domains and passes this signal along to RNAPII.  There are about 20 different subunits that bind to RNAPII and different activation domains. 

To determine the functional domains of an activator, we can use reporter genes.  Ideally, we would cotransfect a plasmid containing the protein of interest and a plasmid containing a reporter  (such as lacZ) that is transcribed only when the activation domain of the protein of interest is transfected.  In this way, we can examine different regions of proteins to determine the precise domains that are involved in activating transcription. 

An additional way to determine where an activation domain is in a protein is to use a Gal4 hybrid assay.  This method involves using a domain swap, which uses the DNA-binding domain of Gal4 and other domains from the protein of interest.  By measuring the activity of a reporter gene, such as lacZ, we can determine if the domain that is bound to Gal4 is an important activation domain. 

First, we have activators that are recruited to genes, which are involved in regulating transcription and also bind the DNA directly.  Co-activators, in contrast, are recruited to the promoter but do not bind DNA.  They form complexes and can assemble on the DNA-binding proteins.  In this way, co-activators can interact with proteins essential for transcription, such as the machinery, histone modifiers, and chromatin-remodeling complexes.  Important to note is that some co-activators, such as VP16, CBP, and GCN5, have acetyltransferase activity. 

VP16 is a herpesvirus protein that contains an acidic activation domain and interacts with host cell factor (HCF).  When VP16 binds HCT and OCT1, which is a DNA-binding activator but no activation domain, it promotes the assembly of the PIC and helps to initiate transcription by targeting TBP, TFIIB, and TAF40.

GATA4 is another important co-activator that is a zinc finger DNA-binding protein that is involved in heart development.  It works via the recruitment of TBX-5.

Architectural Factors that Affect Transcription
The main way that architectural factors affect transcription is via DNA bending.  These proteins do not have a transactivation domain, as do other regulatory factors, but they do affect the interactions between activators, co-activators, and the PIC.  This is often accomplished by bending the DNA and shortening the distance between cis-acting elements.  Such bending of the DNA allows for transcriptional regulators to act at a distance. 

The HMG proteins are small, abundant proteins that function to change the DNA architecture.  These proteins do not have high sequence specificity and can bind the minor groove to induce a bend in the DNA.  Bending of the DNA facilitates complex assembly and nucleosome remodeling, which may change the rate of transcription.  

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