Tuesday, June 22, 2010

Initiation of Eukaryotic Transcription IV

Changing Up the Chromatin
All of the factors that affect transcription that have been described are involved in changing the ability of RNA polymerase and transcription factors to bind to and initiate DNA transcription.   An additional method of modulating transcription is via changes to the chromatin (see writings on that here).  Histones and other proteins are able to change the packaging of DNA and, thereby, affect transcription.  The compaction of the DNA directly affects the accessibility of it to RNA polymerase and transcription factors.  Euchromatin is the active form of chromatin that is not fully compacted; in contrast heterochromatin is generally not active or accessible.  The presence of histone H1 (the linker histone, and, let’s be honest, everyone’s favorite histone) also affects the compaction of the chromatin.

How exactly do histones affect transcription?  Put simply, they prevent other proteins from binding the DNA, especially the DNA that is facing the histone core.  Further, DNA is wrapped around histones, which changes the structure of the DNA.  Such distortions can affect binding sites and preclude transcription factor binding.  If the transcription factors and RNA polymerase are to bind to the DNA, the chromatin must be unraveled and the DNA must become accessible.  One way to do this is to move the histones out of the way, opening up binding sites.  Histones are incredibly dynamic and move around on the DNA frequently, wrapping and unwrapping different sequences.  With the fluctuations of the chromatin, transcription factors can bind while the histones “breathe.”

An additional mechanism to allow access to the DNA is via histone modifications on the N-terminal domains.  The N-termini of the histones contain lysine groups that affect DNA-histone interactions.  Therefore, affecting the charged residues via acetylation or methylation, for example, will change the interactions between the histones and DNA.  Acetylation of a histone tail effectively neutralizes its charge such that its structure is altered and no longer binds DNA as tightly.  Modifications on histones can also form binding sites for transcription sites.  Chromodomains bind to methylated lysine residues, while bromodomains bind acetylated lysines.  There are a number of different modifications that affect transcription, and new effects are still being elucidated.

-          Acetylation of lysine / arginine: transcription induction
-          Phosphorylation of serine, threonine, or tyrosine: transcription induction; chromatin compaction
-          Methylation of arginine: transcription induction
-          Methylation of lysine: gene silencing
-          Ubiquitination of H2A and H2B: degradation; transcription; growth regulation
-          ADP-ribosylation: histone repelled from DNA at sites of repair

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. 

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.  

Tuesday, June 15, 2010

Activation of Transcription Initiation II

Does the Protein Bind DNA?
To determine what DNA sequence a specific protein binds, an electromobility shift assay, or EMSA, is implemented.  This method involves labeling DNA fragments with the sequence of interest and then mixing this DNA sequence with the DNA-binding protein.  We can then run an acrylamide gel with and without the DNA-binding protein, and if the protein does bind the DNA, we should note a shift in the mobility of the DNA, as detected by autoradiography.  Namely, the DNA will become heavier, with the protein bound to it, meaning that it will not travel through the gel as quickly.  Therefore, we should see a shift up.  If the protein does not bind the specific DNA of interest, we should note that the two bands migrate through the gel at the same rate.   We can further test the specificity of the interaction by adding unlabelled competitor DNA:  if we still note a shift in the band, the DNA-binding protein is very specific to the DNA sequence of interest. 

Another method to determine if a protein binds to a DNA sequence is to perform a chromatin immunoprecipitation, which is an extension of an immunoprecipitation.  This technique involves several steps, starting with crosslinking of proteins and DNA.   If the DNA-binding protein is located on the DNA, it will be crosslinked with the DNA (via formaldehyde).  Post-crosslinking, the DNA is sheared using sonication (enzymes can also be used), and the protein of interest is immunoprecipitated.  Crosslinking is then reversed, and we can purify the DNA that was bound to this protein.  Performing PCR on the purified DNA will indicate if a specific DNA region is associated with the DNA-binding protein.

Chromatin immunoprecipitation simplified
1.      Formaldehyde crosslink the DNA and bound proteins
2.      Sonicate to break up DNA
3.      Perform immunoprecipitation with antibodies against the protein of interest
4.      Reverse crosslinking (via high salt, for example)
5.      Purify DNA that co-immunoprecipitated
6.      Perform PCR with gene-specific primers

Friday, June 11, 2010

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.  

Tuesday, June 8, 2010

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.  

Monday, June 7, 2010

More about Eukaryotic Transcription Initiation

Is it just me or does eucaryotic just look funny without the k?
Compared to prokaryotes (discussed previously), transcription in eukaryotes is complicated due to chromatin, multiple complexes, regulatory proteins, and a lack of transcription-translation coupling (one is in the nucleus; one is in the cytoplasm).  The complex that general transcribes genes into RNA is RNA polymerase II, which binds to specific sequences on the eukaryotic genome.  Genes in eukaryotes have several components, including enhancers, promoters and proximal elements, the TATA box, and the exons and introns of the gene.  The regulatory sequences surrounding a gene determine its transcription and utilization, accounting for temporal and spatial regulation of gene transcription. 

Specific factors are involved in the initiation of eukaryotic transcription.  Basal transcription factors (GTFs)  are required for transcription from all promoters, regardless of tissue-specificity.  RNA polymerase II (discussed above) is also required for transcription.  TATA-binding protein (TBP), which binds the TATA box, is also important in initiation of transcription, as are TBP-associated factors (TAFs) and coactivators of transcription. 

How do we analyze the activity of regulatory regions of genes?  Reporters, such as luciferase, GFP, or β-galactosidase are reporters, which can be used to measure the amount of transcription from a promoter or regulator element.  By placing a promoter or enhancer upstream of one of these reporters on a plasmid, transforming this plasmid into a cell, and measuring the amount of reporter gene transcribed, one can analyze the promoter activity.  The total amount of reporter protein that is synthesized is directly related to the activity of the promoter. 

Before being able to perform these reporter assays, however, the DNA sequences that regulate transcription must be putatively identified.  This can be accomplished via 5’ deletion analysis, in which DNA fragments upstream of the 5’ untranslated region (UTR) of a gene are introduced to a reporter vector.  As described above, the activity of the promoter is measured as a function of the reporter protein, such as luciferase. 

Additionally, one can perform linker scanning analysis, in which regions of the DNA are mutated with synthetic linker DNA.  These mutations should abolish the activity of the particular region of DNA that they “cover,” and the changes due to these mutations can be analyzed with reporter genes.  Now, with so many regions mapped and analyzed, bioinformatics can be used more frequently to identify shared enhancer sequences.

The core promoter of a gene consists of the site at which RNA polymerase II (RNAPII) binds and initiates transcription.  This site is approximately 35 bp upstream or downstream of the transcription initiation site, which allows RNAPII to interact with the basal transcription machinery. 

(Note: What about RNA polymerases I and III?  RNAPI is involved in the production of rRNA, and RNAPIII with tRNA.  RNAPII is highly abundant and is inhibited by α-amanatin, which interferes with the translocation of RNA and DNA and is found in poisonous mushrooms.  RNAPII synthesizes approximately 50% of the RNA in an active cell.)  


Sunday, June 6, 2010

Initiation of Eukaryotic transcription

We're eukaryotes, right? So let's delve into eukaryotic processes, starting with transcription.  Bacterial transcription is also pretty important, so if you would like a refresher on that topic, check out that post. Since eukaryotes are more complex, transcription will be covered in several posts in order to hammer out all the important details.
Transcription is essential for differentiating cells: all cells contain the same genome but have different expression patterns of the genome.  The differential expression of the genome creates unique protein compositions in each cell type.  While a number of functions are the same in cells and they, therefore, express many of the same proteins, cell specialization is dependent on different protein expression patterns.  For example, some proteins are abundant in specialized cells but not other types of cells.  One method for detecting which proteins are expressed in a cell is via 2D electrophoresis, which separates proteins based on their pI and molecular weight. 

The genes of the human genome are regulated temporally and quantitatively: only approximately 10,000 genes are expressed in any singular cell type.  In fact, the expression levels of nearly every active gene is different in different cell types.  Temporally, expression is regulated by many factors, including cell cycle, external stimuli, tissue types, and embryogenesis.  Genes are also regulated quantitatively, which is determined by the rate of transcription initiation and elongation, as well as the actions of activators and repressors.  Constitutive genes are those that are expressed throughout the cell cycle; inducible genes are transcribed at different levels depending on the position in the cell cycle or developmental stage.

Modulation of gene expression can occur at several stages:
  • When and how frequently a gene is transcribed
  • Splicing
  • Exporting and localization of mRNA
  • Translation initiation
  • Stability of mRNA in the cytoplasm
  •  Activation, degradation, and compartmentalization of proteins


The main method of modulating gene expression is via selective transcription.  Studying transcription can occur via analysis of cDNAs, which can be probed to determine expression levels.  Microarrays have become important for comparing the expression levels of nearly every gene in a genome.  Analysis of microarrays provides a characteristic expression pattern that can even be diagnostic.

Regulation of transcription at the level of initiation
Transcription control can be the result of different expression patterns in different cell types, developmental stages, or in response to stimuli.  As mentioned previously, changing the rate of mRNA transcription initiation is the main mechanism by which the cell regulates gene expression.  Additionally, control at the stage of transcription initiation ensures that the cell does not waste energy producing mRNAs that are not useful to it.

How do we determine transcription rate?  Via run-on transcription analysis.  To perform this type of assay, we must isolate nuclei of the cell of interest, incubate it with radio-labeled ribonucleoside (32P) and then allow the transcriptional machinery to work.  The processes are allowed to continue for a short while in order to ensure that only RNA elongation and not transcription initiation are measured.  Hybridization of the labeled RNA to specific DNA allows for quantification of its relative transcription rate, compared to standards or other genes.  

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