Lecture 1 White "Gene Expression 1" Flashcards
What are the two things that gene regulation requires?
- short stretches of DNA of defined sequence- recognition sites for DNA binding proteins
- Gene regulatory proteins; transcription factors that will bind and activate
True or false: DNA binding motifs are only proximal to the first exon
False: They can be proximal or distal to the first exon
Describe how a DNA motif is recognized
The regulatory proteins associate with the major groove and bind to the bases
The surface of the protein is complementary to the surface of the DNA to which it binds (4 possible configurations)
What are the four possible configurations of the base pairs in DNA motif recognition?
- possible H bond donors
- possible H bond acceptors
- methyl groups
- hydrogen atoms
How many interactions does a typical protein-DNA interaction involve?
10-20 interactions
Which modules will a transcription factor be sure it has in its sequence?
DNA binding module
Activation module
Which modules MIGHT a transcription factor have but is not necessarily required?
Dimerization module
Regulatory module
Describe the basics of the Gal4 protein experiment.
What was it testing?
What did it do (molecular level)
- It was testing to see if the transcription factors were in fact, modular; wanted to map out the binding and activation domains of the yeast Gal4 protein
- Difference mutant GAL4 proteins were tested to see which combinations allowed the binding to the UAS and the galactosidase activity
What are the 4 DNA binding domain structural motifs?
- Helix turn helix
- Zinc finger motif
- Leucine zipper
- Helix loop helix
Describe the helix turn helix motif
simplest and most common
2 alpha helices connected by amino acids that turn at a fixed angle
Which module is the recognition module in the helix turn helix motif?
The longer helix, fits into the major groove of the DNA; side chains recognize the DNA motif
Describe the zinc finger domain
binding motif includes a Zn
binds to the major groove of the DNA
found in tandem clusters
there are usually multiple contact points
Why is the zinc finger domain called the finger domain?
Because when you draw it out with the amino acid ish, apparently it looks like a finger
Leucine zipper motif
- two alpha helical binding domain
- grabs the DNA like a clothespin
- activation and dimer domains overlap
- interactions between hydrophobic side chains
What helps to from the zipper structure in the Leucine zipper motif?
There is a leucine residue every 7 amino acids down the side of the alpha helix
Describe the helix loop helix domain
- short alpha chain connected to a longer alpha chain
- homo or heterodimers
What are the 3 domains that are commonly found in the helix loop helix domain?
DNA binding domain
dimerization domain
activation domain
Describe homeodomain proteins
- contain homodomain
- homodomain is three alpha helices
- helix-turn-helix motif
Describe the beta sheet DNA recognition proteins
- 2 stranded beta sheets with beta strands
- connected by backbone of hydrogen bonds
- twisted pleated sheet
- binds to the major groove
True or false: Hereditary Spherocytosis is dominantly inherited
TRUE
What does the KLF1 zinc finger domain do?
binds to all of the genes in the EMS and turns them on
What is the DNA binding motif that is critical for amino acids to bind to DNA that is found in the wild type?
RER
Arg, Glu, Arg
What is the mutant form of the RER motif that leads to no transcription?
RDR
How does the mutant form of the DNA binding motif KLF1 (RDR) result in HS?
makes less RNA from the target promoters of the EMS genes resulting in less protein and HS
The Zn finger binds to the opposite strand from the target and there is no transcription, therefore there is no protein
Describe how you identify transcription factors
Use gel mobility shift assay and EMSA
- use a radioactive DNA from a known promoter
- mix the radioactive fragment with protein extract from the cell
- run the gel
- proteins will migrate according to the size
- there is a shift in the band if the radioactive DNA bound to the protein
- isolate the protein and identify via affinity chromatography, isolation, and purification
Define CHIP
Chromatin Immuno-Precipitation
When is CHIP useful? and what is the process
allows the identification of the sites in the genome that a known regulatory protein is bound to
- LIVING CELLS
- PCR the end product to identify the sequence
Define the gene control region of a eukaryotic gene
the DNA region that is involved in regulating and initiating the transcription of a gene
Define the promoter
Where the transcription factors and RNA polymerase 2 assembles
What controls the rate of the assembly process at the promoter?
The regulatory sequences to which the regulatory proteins bind
How are the gene regulatory proteins able to interact with the proteins at the promoter?
There is a DNA looping and a mediator complex. The mediator is the intermediary between the gene regulatory proteins and the RNA polymerase 2
What do the transcription regulators bind to?
DNA in nucleosomes (higher affinity to the naked DNA)
Why do the transcription factors bind to the naked DNA with a higher affinity?
Because the surface of the nucleotide recognition sequence may be facing inward when it is bound to the nucleosome, so the naked DNA is more accessible
What are the 4 ways that the gene activator proteins are able to affect the local chromatin structure?
- nucleosome remodeling
- nucleosome removal 3. histone replacement
- histone modification
Describe nucleosome remodeling
The nucleosome sliding allows for the access of transcription machinery to the DNA
Describe the nucleosome removal
The transcript machinery assembles on the nucleosome free DNA
Describe the histone replacement
the histone variants allow greater access to the nucleosomal DNA
Describe the histone modification
There are specific patterns of histone modification that destabilize the compact forms of the chromatin and attract components of transcription machinery
What are the 6 ways that a gene repressor is able to inhibit transcription?
- Compete with the activator
- Masking the activation site
- direct interaction with the general transcription factors
- recruitment of the chromatin remodeling complexes
- recruitment of histone deacetylases
- recruitment of histone methyl transferase
Describe the competitive DNA binding
Activator and repressor compete for the same binding site
Describe the masking of the activation site
Both of the proteins bind to the DNA but the repressor binds to the activation domain of the activator proteins
Describe the direct interaction with the general transcription factors
The repressor binds to DNA and blocks the assembly of the general transcription factors
Describe the recruitment of the chromatin remodeling complexes
The repressor recruits a chromatin remodeling complex which returns the promoter to the pre-transcriptional nucleosome state
Describe the recruitment of the histone deacetylases
The repressor attracts a histone deacetylase to the promoter- harder to remove acetylated histones and open up the DNA
Describe the recruitment of histone methyl transferase
The repressor attracts a histone methyl transferase which methylates histones
These methylated histones are bound to proteins which act to maintain chromatin in a transcriptionally silent form
True or false: The same protein can be a part of an activating or repressing complex
TRUE
What are the 7 ways in which the Gene regulatory proteins can be controlled?
- synthesis
- ligand binding
- covalent modification phosphorylation
- addition of a subunit
- unmasking
- nuclear entry
- proteolysis
What are the alpha globin like chains?
zeta
alpha
What are the Beta globin like chains?
Beta
Epsilon
Gamma
Delta
Define hemoglobin switching
embryonic to fetal to adult
How are the beta globin genes arranged?
In a linear fashion; ordered in a 5’ to 3’ direction in the same sequence of activation and expression during embryonic, fetal, and adult development
Describe the Beta globulin gene
It is a 100 kb region that contains 5 beta globin genes and a locus control region
far upstream in sequence
