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. 

Co-activators

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.  

Activation of Transcription Initiation

We’ve briefly covered the initiation of transcription but the process is more complex than transcription factors floating to a promoter and starting up RNAPII.  In thermodynamic terms (ouch, I know), the transcriptional activator proteins shift the equilibrium of free transcription factors to the formation of the preinitiation complex (PIC):  these activators increase or decrease the association rate of proteins and affect the formation of the PIC.  Proteins can affect transcription initiation by altering accessibility to the promoter or changing the stability of the PIC.  Activators (also called transcription factors and gene regulatory proteins) bind to specific DNA sequences and promote transcription.  Co-activators interact with these activators to promote transcription, without interacting directly with the DNA.  Essentially, activators and co-activators function to recruit, position, and modify GTFs and RNAPII by altering the transcriptional machinery, bending the DNA, or changing the chromatin structure.

Regulatory elements are important in eukaryotes and come in several forms.  The core promoter contains the start site of transcription and the TATA box.  Here is where GTFs and RNAPII bind to form the PIC.  The proximal promoter (or the upstream activator sequence in yeast) is located within 200 bp upstream of the start site and contains sites for regulatory factors to bind.  Finally, enhancer sequences exist from 200 to 50 000 bp from the start site and can also bind regulatory factors.  Enhances act independent of function, and they can act at a distance due to DNA looping.

What are the components of a transcriptional activator protein?

TAD: trans-activation domain

DBD: DNA-binding domain

NLS: nuclear localization signal

Regulatory domains: catalytic function of the activator protein

Dimerization domain: for dimerization of activators (especially important for DNA-binding)

DNA-Binding Domains

The DNA-binding domain can read DNA sequences and has several structural motifs: the helix-turn-helix (HTH), homeodomain, zinc finger, basic leucine zipper, and helix-loop-helix.  In general, these domains contain an alpha helix that fits snuggly in the major groove of the DNA and makes specific contacts with the DNA.  These DBDs can thereby recognize response elements in the DNA to carry out their functions. 

The helix-turn-helix motif binds DNA as a monomer and recognizes DNA via a C-terminal helix, and the N-terminal helix positions the C-terminal helix in the major groove of the DNA.  In contrast, the homeodomain binds DNA as a monomer and contains three helices, one of which binds the DNA and the other two bind other proteins or the DNA backbone. 

The zinc finger motif uses a zinc ion to coordinate the structure of the protein.  The Cys2/His2 zinc finger motifs act as a monomer or a dimer and use cysteine and histidine to coordinate the zinc and bind the DNA major groove.  Additionally, Cys4 zinc finger motifs also act as monomers or dimers and coordinate the zinc with four cysteine residues to allow for DNA interactions.  The basic helix-loop-helix domain and leucine zipper motifs are additional DNA-binding motifs that act as dimers and are commonly found in DNA-binding proteins.  

RNA Polymerase and Basal Transcription

Part 3 of 3.  Of part 1 of 4.  So I guess it's like part 3 of 12, but that sounds too intimidating.  Let's stick with 3 of 3.

RNA Polymerase II

RNA polymerases in general consist of about 10 subunits and making a protein of greater than 500 kDa.  Five subunits are common to all of the three polymerases.  However, RNAPII contains the all-important C-terminal domain (CTD): YSPTSPS, which is repeated 52 times in mammals (26 times in yeast).  RNAPII that can initiate transcription has a CTD that is unphosphorylated, but upon initiation and movement of the polymerase from the promoter, the CTD becomes phosphorylated.  RNAPII alone, however, is not enough to initiate transcription, as it requires a number of other factors for transcription actually begin.  These include six GTPS: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH.  Once these and RNAPII have assembled at the promoter, the pre-initiation complex (PIC) has formed, which allows for basal transcription.  How often this PIC is formed is regulated by upstream activator and repressor proteins. 

Motifs Required for Basal Transcription

A number of DNA sequences are necessary for the core promoter to actually lead to transcription of a gene:

The TATA box: located at about -25, it binds the TBP and is found mainly in tissue-specific genes.  Consensus sequence of TATA(A/T)AA(G/A).  This element is involved in positioning RNAPII to start transcription, so any mutations in this region can be devastating to transcriptional activity. 

The BRE (TFIIB response element): located at about -32 to -35, binds TFIIB

The INR (initiator): located at -2, binds TFIID, and can stimulate TATA box activities, though weakly.  Used by about 65% of genes in place of a TATA box. 

The DPE (downstream promoter elements): located roughly from +28 to +32 and stimulate gene transcription.

The Steps in Transcription Initiation

Formation of the preinitiation complex (PIC) is the initial step in transcriptional initiation and involves the assembly of GTFs on the gene:

1. TBP binds the minor groove of the TATA box, causing a bend in the DNA and promoting the binding of more factors
2. About 10 TAFs bind TBP to form TFIID
3. TFIIA binds TFIID complex
4. TFIIB binds the TFIID-TFIIA complex
5. TFIIF recruites RNAPII to the promoter
6. TFIIE and TFIIH join to form the functional PIC

TFIIH acts as a helicase to promote initiation and also has kinase activity to phosphorylate the CTD of RNAPII for promoter clearance.

TAFs are a diverse set of proteins that affect the ability of TBP to interact with the promoter, and these TAFs are particularly important when there is no TATA box on the gene.  These proteins can act as co-activators, functioning to recruit TFIID or interact with other transcription factors, for example.  Additionally, other TAFs have acetyltransferase, kinase, and ubiquitin-conjugating activities. 

Mediator is a large protein complex that stimulates or inhibits the activity of RNAPII.  Other activators and inhibitors of transcription interact with mediator, sometimes at a long distance, and these signals are integrated to promote or inhibit RNAPII activity.  While not all subunits of mediator are necessary for transcription, some are required. 

After the formation of the PIC, transcription begins and the promoter is cleared, at which point the CTD on RNAPII is phosphorylated and the GTFs are released, except for TBP.