Control Of Gene Expression 2 Flashcards
Cells differ in structure and function. What was the old train of thought regarding genes in cell differentiation?
How was this disproved?
What, then, makes cells different?
Cells lost genes, so that only certain proteins are expressed. Therefore, cells are able to differentiate between nerve and lymph for example.
Nuclei from skin cells isolated from adult frogs were implanted into a denucleated cell. These cells then formed a normal embryo. Clearly there was no loss of genes.
All cells contain the same genes, but they express different sets of proteins. Gene expression is responsible for cell differentiation. Regulatory proteins can be responsible for gene expression, along with environmental factors, transcription factors, etc.
Name and describe four protein limitations.
- Common: “Housekeeping proteins” (glucose metabolism)
- Specifically limited proteins (Hemaglobin)
- Average human cells express 30-60% of coding genes, but level of expression varies (fingerprint expression profiles/microarray/RNA sequencing)
- Post transcription factors like alternative splicing, post translational modification.
Gene Regulation Requires (2)
- Short stretches of DNA of defined sequence - recognition sites for DNA binding proteins - proximal or distal
- Gene regulatory proteins - transcription factors that will bind and activate gene
A gene regulatory ____ recognizes a specific __ ___.
The surface of the protein is extensively ____ to the surface of the DNA region to which it binds.
A series of contacts is made with the DNA involving __ possible configurations.
Protein, DNA sequence
Complementary
4
DNA Motif Recognition
Association of regulatory proteins with the major groove of DNA.
Proteins recognize and bind to bases in MG.
MG presents a specific face for each base pair. (IE- C is different than G is different than A is different than T).
The typical gene-regulatory protein and DNA interaction involves how many interactions?
10-20 interactions
What are the parts of a DNA transcription factor?
DNA-Binding Module
Dimerization Module
Activation Module
Regulatory Module
What parts are required in a DNA transcription factor and which are optional?
Req- DNA Binding Module & Activation Module
Opt- Dimerization Module & Regulatory Module
Name some DNA-Binding Domain Structural Motifs
- Helix-turn-helix
- zinc finger motif
- leucine zipper
- helix-loop-helix
- Homeodomain
- beta-sheet
Helix-Turn-Helix Domain
Simplest, most common DNA-Binding Motif
Two Alpha helices connected by a short chain of AA that make the turn at a fixed angle
Longer helix= recognition module, DNA binding mod fits into major groove
Side chains of AA recognize DNA motif
Symmetric diners: bind DNA as diners
Zinc Finger Domain
Different Types
DNA binding motif includes a Zn atom
So-named for the drawn out AA sequence that looks like finger projection (not the 3D version)
Binds to DNA Major Groove
Found in tandem cluster
Stabilizes interaction with DNA
Multiple contact points
Leucine Zipper Motif
Dimerizes through leucine zipper region (homo- hetero-)
Interactions between AA side chains
L residue every 7 AA down one side of alpha helix in dimerization domain forms the zipper structure
Helix-Loop-Helix Domain
Consists of a short alpha chain connected by a loop to a second longer chain
Can occur as homo- or heterodimers
3 domains or modules: binding domain, dimerization domain, activation domain
Homeodomain proteins
- contain Homeodomain
- is made of 3 alpha helices
- helix-turn-helix motif
Beta Sheet DNA Recognition Proteins
2 stranded beta sheet
B sheets consist of B strands
Connected laterally by 2-3 backbone H bonds
Forms twisted, pleated sheet
Binds to major groove of DNA
What disease exemplifies the mutation of a Zn finger transcription factor leading to disease?
Hereditary Spherocytosis
Hemolytic anemia, spherical and fragile RBC that lose and release hemoglobin
Clinical Presentation: hemolysis anemia, splenomegaly
Mutations in genes for EMS (not enough protein)
Dominant inheritance
What is the function of the EMS?
- confers durability and stability to RBCs during million passages in circulation
- allows RBCs to pass through tight capillary spaces
- prevents lysing from bumps and bruises of travel and osmosis
Kruppel-like factor 1
KLF1 is a zinc-finger protein gene that binds to promoters of all EMS genes and turns them on.
Mutation: non-functioning KLF1 Zn finger protein
(No EMS protein made)
Leads to HS
GAA to GAT/ Glu to Asp (exon 3)
What is the specific mutation of KLF1 that causes HS?
GAA to GAT
Glu to Asp
R..E..R to R..D..R
This defect results in less RNA made (no transcription) from target promoters of EMS genes and therefore HS.
What is the impact of KLF1 binding to CACCC on the DNA?
Normally, the 3 finger zinc domain binds to the DNA without problems and leads to transcription of KLF1 mRNA.
In HS, the mutation causes the “D” in RDR (vs RER) to bind to the wrong strand of DNA, resulting in NO transcription. No RNA trans, no protein!
What test detects for sequence-specific DNA binding proteins?
Gel Mobility shift assay, EMSA: electrophoretic mobility shift assay
Use of radioactive DNA from known promoter
- Mix radioactive DNA fragment with cell’s protein extract
- Run electrophoretic gel
- Proteins with DNA attached migrate according to size
- See shift of radioactive band when protein is bound to DNA
- Isolate protein to identify
Affinity chromatography for IDing Transcription Factors
Isolate the DNA binding protein via purification of sequence specific binding proteins.
Start by broadly identifying any DNA binding protein, then get specific using 1 promoter recognition sequence to test presence.
2 step process. Broad —> specific
CHIP: Chromatin Immuno-Precipitation
Technique allows identification of the sites in the genome that known regulatory protein binds to
Done in living cells
PCR product at end can by used to identify sequence
Gene Control Region
Region of DNA involved in regulating and initiating transcription of a gene
Includes the promoter, where transcription factors and RNA polymerase II assembles
Regulatory sequences to which regulatory proteins bind to control rate of assembly process at the promoter
