Transcription factors (Gene expression)
How can the function of transcription factors be controlled?
*Tissue distribution* Ubiquitous or cell specific distribution *Post translational modification* Phosphorylation or de-phosphorylation Some TF such as STAT proteins must be phosphorylated before they can bind to DNA Acetylation or deacetylation Methylation or demethylation *Ligand binding* Hormonal control Steroids, oestrogens, glucocorticoids *Complex formation* Homo/hetero dimerisation This is where some TF couple up with themselves or others before binding
What are the mechanisms by which transcription factors act?
1. Stabilise or block the RNA polymerase to DNA 2. Catalyse the acetylation or deacetylation of histone proteins This can be done directly or indirectly by HAT and HDAC activity 3. Recruit coactivator or co-repressor proteins to the transcription factor DNA complex
What are mediator complexes?
A Mediator is a large, multisubunit RNA polymerase II transcriptional regulator A mediator has been shown to be an integral and highly evolutionarily conserved component of the Pol II transcriptional machinery with critical roles in multiple stages of transcription, from regulation of assembly of the Pol II initiation complex to regulation of Pol II elongation. These have multiple roles and interact with TFIID, RNA polymerase, activators and other factors.
Define a promotor
A promoter is a sequence of DNA that is located near the site of the transcription initiation of the gene. It serves as the binding site for the RNA polymerase enzyme. RNA polymerase is the enzyme that catalyzes the transcription of the gene. A promoter is always located near and upstream (5') to the transcriptional unit of the gene. Promoter contains special DNA sequences that ensure the specific binding of RNA polymerase at the correct binding site for the correct transcription of the transcriptional unit. Main elements of the promoter region are core promoter element and regulatory elements.
What is a binding site? What is a specific name for a TF binding site?
Contain at least one DNA binding domain which attaches to a specific sequence of DNA adjacent to the genes they regulate. They bind to either enhancer or promotor regions of DNA, adjacent to the genes they regulate. Depending on the TF, this site will be up or downregulate OR they bind to RNA polymerase OR they bind to another TF Response element: transcription factor binding site.
Give the broad structure of transcription factors
DNA binding domain - can bind to DNA individually or as homo/heterodimers - Attaches to specific sequences of DNA Activation or repression domain - This stimulates or stops the transcription of the target gene Regulatory domain - For example, they can have a hormone or cAMP binding site - Or a flexible domain between DNA binding and activation/repression domain Dimerization domain
Give an example of transcription factor activation via acetylation
One example is the hematopoietic transcription factor GATA1 that is acetylated at conserved lysines that are required for its stable association with chromatin. This demonstrates that acetylation of GATA1 is essential for Brd3 association with chromatin.
TF regulating through co-activators
Some transcription factors have different roles For example, TFIID binds around the TATA promotor box, which is essential, in itself, to recruiting RNA polymerase II Other transcription factors have a role in placing the TFIID into the right place around the TATA box. This recruitment and positioning of the TFIID is a major rate limiting step in transcription limitation. In the image, you can see the recruitment of lots of different TF into the right place. Assisting this, you have distal promotor elements and enhancers (which can be 5' or 3'of the transcriptional start site.
What is the TF responsible for regulating the beta globing gene at the LCR?
Such a tight control of alpha and beta globin chains is dependent on regulatory regions of DNA located either in proximity or at great distances from the globin genes in a region characterized by the presence of several DNAse I hypersensitive sites and known as the Locus Control Region (LCR). All these sequences exert stimulatory, inhibitory or more complex activities by interacting with transcription factors that bridge these regions of DNA to the RNA polymerase machinery. Some genes such as GATA-1 and 2 are master regulators that act on large numbers of genes at early stage of differentiation whereas others, like EKLF, stand on the lowest step and control only single or limited number of genes at late stages of differentiation.
What is an enhancer? How do enhancers act?
The enhancer is a short DNA sequence that influences the transcription of particular genes. The enhancer can change the rate of the transcription. It can locate in the vicinity of the gene. It is not necessary for it to locate near to the transcriptional unit of the gene. Enhancers mainly influence the activity of promoters of the genes. They always interact with the promoters in gene regulation. Enhancers can locate even within the introns of the genes!
Give an example of TF activation by post translational modification
The functions of NFkB and p53 are mediated through phosphorylation of transcription factors. MAP kinase is another example of this
Name a common promotor
The most famous promotor region is a TATA box. This is not the most common, but has a TATA base sequence. It is a section of non-coding DNA sequence, and is a cis-regulatory element.
What are transcription factors?
These are proteins that help turn specific genes on or off by binding to nearby DNA. They are proteins which control the rate of transcription for genetic information TF work alone or with other proteins to recruit RNA polymerase to specific genes You have activators, repressors, enhancers and silencers TFs interact with DNA binding sites via electrostatic Ven der Waals forces.
Where in the DNA molecule do TFs bind?
Transcription factors typically bind to the major groove of the DNA molecule Some can bind in the minor groove, but this is less common
Give the process of chromatin activation before transaction, and describe the role TFs have in this process.
Transcription factors, therefore, are intimately involved in chromatin re-modelling - long and short range.
What things recruit RNA pol?
Transcriptional factors do the recruiting of the RNA polymerase. These factors have activator and repressor sequences to attach into the promoter region and regulate the transcription.
What are co-activators and co-repressors?
You also co-activators, a protein that works with transcription factors to increase the rate of gene transcription co-repressor, a protein which works with transcription factors to decrease the rate of gene transcription
Name some different experimental methods to test for transcription factors.
You use biochemical methods for purification and interactions: Gel fractionation of nuclear protein extracts Biosynthesis and purification from bacteria/tissue culture expression systems Immunoprecipitation and co-immunoprecipitation Proteomics (mass spec) analysis of isolated complexes *You also have to do functional assays to test the function of transcription factors, this is learning how the TF turns the gene on or off:* • Analysis of test gene constructs • Cell transfection - transient/stable • Transgenic/ KO mice siRNA/miRNA knock down cells/mice
On TF's there are DNA binding domains. There are different types of these domains, which classify TFs into families. What are the names for the different types of domains?
Zinc finger ○ Eg. SP1 and GATA-1 ○ Involves the binding of Zn2+ ion by four AA ○ This forms a loop (finger) Basic leucine zipper, bZip ○ This is a basic AA DNA binding domain and leucine zipper dimerisation domain ○ A helical stretch of amino acids Basic helix-loop-helix ○ A common motif found on transcription factors ○ Two alpha helix ○ Connected to a flexible loop Homeodomain Typically, TFs exist as a family of proteins which have similar functions. For example, you have the GATA transcription family.
