Flashcards in Control Of Gene Expression I Deck (62):
True or false: cells are different because some cells have lost genes so that they only express certain proteins.
Each cell has the same genome. How is it that differentiation in cells is present?
Differentiation depends on changes in gene expression.
A typeical human cell expresses how much of its genes?
30 - 60%.
The level of gene expression varies.
What post-transcriptional factors affect gene expression?
What does gene regulation require?
Short stretches of DNA of defined sequence - recognition sites for DNA binding proteins
Gene regulatory proteins - transcription factors that will bind and activate genes.
Where can recognition sequences be located?
Proximal (base pairs away) or distal to the first exon
What are the steps in DNA motif recognition?
Association of regulatory proteins with major groove.
Proteins recognize and bind to bases in major groove.
Major groove presents a specific face for each of the specific base pairs.
What occurs during motif recogition?
A gene regulatory protein recognizes a specific DNA sequence.
The surface of the protein is extensively complementary to the surface of the DNA region to which it binds.
A series of contacts is made with the DNA involving 4 possible configurrations.
Where do gene regulatory proteins read?
Outside of the DNA helix.
A typical gene regulatory protein DNA interaction involves 10 - 20 interactions.
What are the parts of the DNA transcription factor?
DNA-binding molecule, dimerization module, activation module, regulatory module.
True or false: not every transcription factor will have all modules.
A transcription factor has what type of module?
DNA binding module
What is the function of the dimerization module?
It forms a dimer with other protein subunits.
What is the function of an activation module?
It turns on a gene
What is the function of a regulatory module?
It regulates a transcription factor.
What does the transcription factor do?
Binds to promoter DNA and activates it.
What is the evidence for transcription factors being modular?
There are two plasmids: a reporter gene construct (DNA target) and experimental plasmids (making transcription factor)
What are the four DNA-binding domain structural motifs?
Zinc finger motif
What is the structure of helix-turn-helix?
Two alpha helices connected by short chains of amino acids that make the "turn" turned at a fixed angle.
What part of the helix-turn-helix fits into the major groove?
The longer helix. It is a DNA binding module.
The side chains of amino acids recognize DNA motif, and symmetric dimers bind DNA as dimers.
What metal is part of the zinc finger domain?
What does the zinc finger domain bind to?
A major groove of DNA
How many domains are in the zinc finger domain?
How many binding domains are in the leucine zipper motif?
Two alpha helical DNA binding domains.
How do leucine zipper motifs grab DNA?
Like a clothespin.
Leucine residues every 7 amino acids form a zipper structure.
What are the three domains present in the helix-loop-helix domains?
DNA binding domain
What is hereditary spherocytosis?
A hemolytic anemia characterized by fragile red blood cells that lyse and release hemoglobin.
It is caused by mutations in genes for the erythrocyte membrane skeleton of red blood cells not making enough protein.
Hereditary spherocytosis is causd by what transcription factor?
Zn finger transcription factor
What is the significance of the erythrocyte membrane skeleton?
It confers properties of durability and stability to red blood cells.
A mutation in what gene can cause hemolytic spherocytosis?
Klf1 (Krueppel-like factor 1)
Klf1 zinc finger protein binds to promoters of all genes in the EMS and turns them on.
What does the gene Klf1 encode?
3 zinc finger domains.
What occurs when there is a non-functioning Klf1 Zn finger protein?
No EMS protein is made, which leads to hemolytic spherocytosis.
What is the mutation in hemolytic spherocytosis?
GAA to GAT or Glu to Asp in exon 3 (zinc finger domain 2)
What are the wild type and mutant forms of KLF1?
RER (arg-glu-asp) wild type DNA binding motif
RDR (arg-asp-arg) mutant form.
What does a defective Klf1 gene cause?
Less RNS is made from the target promoters of EMS genes, therefore less protein is present and hemolytic spherocytosis develops.
What occurs when normal KLF1 binds to DNA?
DNA unwinds, which causes transcription.
The HS KLF1 Zn finger domain binds to the opposite strand so DNA cannot unwind, and transcription does not occur.
How are transcription factors identified?
Electrophoretic mobility shift assay.
What is the process of EMSA (electrophoretic mobility shift assay)?
Radioactive DNA from a known promoter is used. It is mixed with protein extracts from a cell.
Electrophoresis is run, and proteins with DNA attached migrate according to size.
A shift of the radioactive band is present when the protein is bound to DNA, and the protein is isolated and identified.
What is the process of affinity chromatography?
Isolate a DNA binding protein, and then purify sequence-specific binding proteins.
Start broadly and identify any DNA binding protein, and then get specific by using only one promote recognition sequence.
What is CHIP (chromatin immuno-precipitation)?
A technique that allows for identification of sites in the genome that a known regulatory protein beinds to.
It is done in living cells.
PCR products at the end can be used to identify a sequence.
What is a gene control region?
A DNA region involved in regulating and initiating transcription of a gene.
What does the gene control region include?
A promoter (where transcription factors and RNA polymerase II assemble)
And regulatory sequencs to which the regulatory proteins bind to control the rate of assembly process at the promoter.
Where do RNA polymerase and general transcription factors assemble?
At a promoter.
Where do gene regulatory proteins (activators or repressors) bind to?
Regulatory sequences which can be adjacent, far upstream or in introns downstream of the promoter.
What allows gene regulatory proteins to interact with the proteins that assemble at the promoter?
DNA looping and a mediator complex.
In transcription, what is the function of a mediator?
It serves as an intermediary between gene regulatory proteins and RNA polymerase II.
What do nucleosome remodeling and histone removal favor?
Transcription by increasing accessibility of DNA to proteins.
What is the function of gene repressor proteins?
They inhibit transcription in different ways.
They compete for the same binding site with activators.
Where do gene repressor proteins bind?
The repressor binds to the activation domain of the activator protein.
After a gene repressor protein binds to DNA, what does it block?
The assembly of general transcription factors.
How do gene repressor proteins affect chromatin?
The repressor recruits a chromatin remodeling complex which returns the promoter to the pre-transcriptional nucleosome state.
Gene repressor proteins attract histone deacetylase to the promoter. What effect does this have on histones?
It is harder to remove deacetylated histones and open up DNA.
Gene repressor proteins attract histone methyl transferases, which methylate histones. What is the function of the methylated histones?
They are bound to proteins which act to maintain chromatin in a transcriptionally silent form.
Depending on the composition of complexes, proteins can either be ___ or ___.
Activating or repressing.
The same protein can b part of an activation or repressing complex.
How are gene regulatory proteins controlled?
Addition of subunit
What are the alpha-like chains of globins?
What are the beta-like chains of globin genes?
How are globin genes arranged?
In a linear fashion.
They are ordered in the 5' to 3' direction in the same sequence of activation and expression during embryonic, fetal and adult development.
What is the beta globin gene regulation?
A 100 kb region containing five beta globin genes and locus control region.
It is located far upstream in sequence, but is required for transcription.
What do regulatory proteins bind to?
Locus control region (LCR)
The understanding of globin gene regulation may allow for what?
The induction of fetal hemoglobin in sickle cell anemia.