EX3 - Control of Gene Expression 1 Flashcards
What are two other domains? VERY low testability
Homeodomains Proteins
- Contain homeodomain
- Homeodomain is three alpha helices
- helix-turn-helix motif
Beta sheet DNA recongition Proteins
- 2 stranded beta sheet
- beta sheets consist of beta strands
- connected laterally by two or three backbone hydrogen bonds
- forms twisted, pleated sheet
- binds to major groobe of DNA
What is the Klf1 DNA binding motif and its relation to HS?
Normal = (ARG-GLU-ARG) ; R_ _- E_ _ - R_ _ -
HS = (ARG-ASP-ARG) ; R_ _ - D_ _ - R_ _-
Defective: makes less RNA from target promoters of EMS genes – therefore less protein and HS develops
HS KLF1 Zn finger domain 2 (RDR instead of RER) binds to the opposite strand – change in binding so no transcription!!
No RNA – no protein
Where on the DNA do DNA motif recongition sites lie?
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 is gel mobility assay “EMSA”?
EMSA - eletrophorectic mobility assay
used for SEQUENCE SPECFIC DNA BINDING PROTEIN ID
- radioactive DNA from promoter is mixed with protein extract from cell. Different proteins of different sizes binds to DNA. CHANGING THE MOLECULAR WIEGHT
- Run eletrophoretic gel
- Proteins with DNA migrate according to size
- see shift of radi active band when protein is bound to DNA
- Isolate protein to identify
How do gene activator Proteins work?
Transcription regulators binds to DNA in nucleosomes. THey bind to DNA in ucleosome with loweraffinity than naked DNA.
Surface of nucleotide recognition sequnce may be facing inward when attached to nucleosome
Nucleosome remodeling and histone removal favors transcription by increasing the acessibility of DNA to Proteins
How are ZN finger transcription factor and hereditary spherocytosis (HS) related?
- Hereditary spherocytosis (HS) = spherical and fragile RBC that lyse and release hemoglobins
- EMS protein that can due to a mutation in zinc finger protein gene Klf1 (kruppel like factor)
- KLF1 zinc finger protein binds to promoters of all gene sin EMS and turns them on
- 3 zinc finger domains
Mutation: Non-functioning KLF1 Zn finger protein
no EMS protein made
Leads to HS
What is regulation by commitee
Depending the composition of the complexes, the protein can either be an activator or a repressor
What is the Helix loop helix domain?
Consists of a shot alpha chain connected by a loop to a second longer alpha chain
can occur as homodimers or heterodimers
Three domains or module: DNA binding domain, Dimerization domain, Activation domain
What is the model for transcriptional activation?
DNA loopng and a mediator complex allow the gene regulatory ptroyeins to interact with the proteins that assemble at the promoter
The mediator serves as an intermediary between gene regularotry proteins and RNA polymerase II
What does gene regulation require?
Short stretches of DNA of defined sequences (recongition sites for DNA binding proteins)
Gene regulatory proteins - (transcription factors that will binds and activate gene )
Recognition sequences for regulatory proteins
Ex GATA1 : TGATAG
What are the different types of proteins and factors that cause cells to be different
- Common Proteins
- House keeping proteins
- ex: glucose metabolism
- House keeping proteins
- Specifically limited proteins
- Ex: Hemoglobin
- Level of gene expression
- Ex: finger print expression profiles, micro RNAs , or RNA seq.
- Post transcription modifications
How does this Gene Repressor protein work?
The repressor attracts histone deactylase to the promoter ( B/c harder to remove deacytlated histones and open up DNA )
How does this Gene Repressor protein work?
The repressors binds to DNA and blocks assembly of general transcription factors
What is hemoglobin switching?
Switching of type of hemoglobin proteins from embryo -> fetal -> adult
Embryonic Hb = zeta epilson -
Fetal Hb = alpha gamma -
Adult Hb = alpha beta
NO idea how it occurs
However can be used to cure sickle cell anemia b/c beta globin is affected . switching back from adult to fetal hemoglobin could work as cure
How are gene proteins controlled
A. Synthesis
B. Ligand binding
C. Covalent modification phosphotlyation
D. Additon of subunit
E. Unmasking
F. Nuclear entry
G. Proteolysis
How does this Gene Repressor protein work?
both proteins bind to DNA but the repressor binds to the activation domain of the activator protein
What is the Helix turn Helix Motif
Simplest most common DNA binding motif
Two alpha helixes connect by short chain of amino acids that make the turn (at a fixed angle)
Longer helix = recognition module - DNA binding module fits into major groove
side chains of amino acids recognize DNA motifs and symmetric dimers binds DNA as dimers
What is the Leucine Zipper Motif?
- two alpha helical DNA binding domain
- Grabs DNA clothespin
- activation domain overlaps dimer domain
- Interactions between hydrophobic AA side chains (leucines)
- Dimerizes through leucine zipper region (homo- / hetero)
- Interaction between hydrophobic AA (leucines)
- Leucines residue every 7 amino acids down one side of alpha helix in dimerization domain forms zipper structure
What was the evidence for transcription factors being modular
Using the UAS(GAL) [an activation sequence upstream of tata and B- lactosidase (lacZ)] was bound to GAL4 made in a plasmid. GAL4 acts a transcription factor binding to DNA to activate B lactosidase production. THe Protein made in an wildtype and a multiude of mutant forms to finds out if the transcription factors work modularally. BAsed on how transcription of beta Lactosidase was affect on the portion of GAL4 present show evidence of transcription factors being modular
What are the parts of a DNA transcripiton factor?
Not every transcription factor will have all modules
A Transcription factor will have A DNA binding module
It could have a dimerization module (forms dimer with other protein subunits)
It will have an activation module (turn on gene)
Could have regulatory module (regulates transcription factor )
What is CHIP?
CHIP = Chromatin immuno Precipitation
Allows identification of sites in the genome that a known regulatory protein binds to
Done in living cells
pcr product at end can be used to identigy sequence
How does this Gene Repressor protein work?
Competitive DNA Binding
Activator and repressor compete for the same binding site
How does the gene control region of a typical eukaryotic gene work ?
Gene control region: DNA Region involved in regulating and initating transcription of a gene
Includes the promotor where transcripton factors and RNA polymerase 2 assembles
and Regulatory sequences (activators and repressors) to which regulatory protein binds to control the rate of assembly process at the promoter (adjacent, upstream, or downstream)
What is the zinc finger domain?
- DNA binding motif includes ZN atom
- Left: drawn out AA sequence and cartoon looks like finger proejction - hence the name
- Right: however 3D structure looks nothing like a finger projections
- Binds to major groove of DNA
- Related to KFL1 mutation in HS
- Zinc finger domains found in tandom clusters
- stablize interaction with DNA
- multiple contact points
What is affinity Chomatography?
Isolate DNA binding protein
Purification of sequence specific binding proteins
How does this Gene Repressor protein work?
The repressor recruits a chromatin remodeling complex which returns the promotor to the pre-transcriptional nucleosome state
How does this Gene Repressor protein work?
The repressor attracts a histone deacetylase to the promotor - harder to remove deacetylated histones and open up DNA
How does this Gene Repressor protein work?
The repressor attracts hisotne methyl transferase which methylates histones.
These methylated histone sare bound to proteins which act to maintain chromatin in transcriptionally silent form
What makes cells differnet
Cells contain the same genomes (and genes )
the expressed different sets of protein which can cause differentiation
