Gene Expression (I and II) Flashcards
What are the different components of a “gene unit”?
Insulator elements (i.e. barriers) on each end
Regulatory Elements
Proximal promoter region (-200 to +50)
Core promoter region (-40 to +40)
Coding region
True or false: Regulatory elements are orientation and distance dependent
FALSE
Regulatory elements are orientation independent and distance independent.
If the elements are inverted, no difference in effect is observed
If the elements are moved closer or farther away, no difference in effect is observed
How do regulatory elements interact with proteins?
Noncovalently
How can regulatory elements thousands of base pairs apart all work together to regulate transcription?
Loops bring the regulatory elements all near to each other.
A mediator complex (>25 proteins) transmits effects of regulatory elements to the RNA polymerase
What are the main features of regulatory elements?
Short (6-20 bp long) sequences of DNA that bind to transcription factors
Can be found up to 50 kB from start of transcription
Can be upstream or downstream
Each element can bind multiple transcription factors
What are the different ways that chromatin can be opened up to induce transcription?
A transcription factor binds to a regulatory site on the chromatin
The following mechanisms all work together to cause transcription
Chromatin remodeling complexes (like SWI/SNF) can unwind chromatin
Histone chaperones can remove nucleosomes and replace them with specialized histone protein (ex: CENP-A)
Histone-modifying enzymes can produce a specific pattern of histone modification
What do HAT and HDAC do?
HAT acetylates lysine residues, thus removing the positive charge present on the lysine side chain
HDAC deacetylates lysine residues
True or false: The methylation state of lysine residues alters the charge of the lysine side chain and therefore whch proteins can bind at that residue.
FALSE
Methylation of lysine does not alter the charge on the risidue, but it does change which proteins can bind and interact at that residue
How does acetylation lead to increased gene expression?
Acetylation opens up heterochromatin structure converting it to euchromatin which can be actively transcribed
1) It eliminates the ionic interactions between lysne (+) and DNA (-)
2) The acetyl group also provides a protein binding site
What modification can occur at arginine residues?
Methylation
Dimethyl arginines can be either asymmetrical or symmetrical
What is a chromodomain?
A protein domain that binds to methylated lysines and methylated arginines
What is a bromodomain?
A protein domain that binds to acetylated lysine groups
Describe the sequential process of writing and reading the histone code to regulate gene transcription.
1) Gene activator protein and HAT bind to regulatory element on DNA
2) HAT acetylates H3K9
3) Histone kinase phosphorylates H3S10
4) HAT acetylates H3K14
5) Chromatin remodelin complex binds 1 H3 and TFIID binds other H3 (Via bromomdomains)
6) Transcription machinery assembles leading to transcription
What are the common motifs of DNA binding proteins?
1) Helix-turn-helix
2) Helix-loop-helix
3) Zinc finger
4) bZIP
What are the different modules often found in transcription factors?
Activation domain
Dimerization domain
DNA binding domain
Which groove of DNA do transcription factors primarily bind to?
Transcription factors primarily bind to the major groove of the DNA double helix
Describe the helix-turn-helix motif
Composed of a recognition helix, which binds to the major groove of DNA, which has an edge specific to a particular nucleotide sequence, and a support helix
These motifs often dimerize with recognition helices 3.4 nm apart, allowing for binding at each turn of the helix
Dimers of this type always bind to palindromic DNA sequences
What is a pseudopalindrome?
A DNA sequence that is palindromic at the ends, but has a series of nucleotides in the middle (corresponding with the minor groove of the DNA helix) that are not palindromic.
Describe the helix-loop-helix motif
Can be homodimers or heterodimers
Composed of a recognition helix and a dimerization helix
The dimerization domain often is composed of leucine zuppers
Recognition helices from each monomer bind to adjacent turns of the major groove
Describe the zinc finger motif
A zinc atom holds the structure together
Four “ligand bonds” between the zinc atom and 2H/2C or 4C produce the characteristic shape
Distance between ligand bond residues in this structure is usually: 2or3bp—-12/13bp—-2or3bp
Often joins alpha helix (recognition helix) and beta sheet together
What gives zinc finger domains high affinity for specific DNA sequences?
Proteins typically have multiple zinc finger modules that recognize successive portions of the DNA sequence
What is a bZip motif?
Scissor shaped dimer joined by leucine zipper composed of basic amino acids
Bind to palindromic DNA sequences
What are some methods used to analyze regulatory elements in gene expression?
Footprinting with DNAaseI for transcription factor binding sites
Gel-mobility shift assay to show protein-DNA complexes
Chromatin immunoprecipitation (CHIP assay)
Reporter genes to assay regulatory elements in specific cells
Describe the process of footprinting with DNAseI
This method allows us to determine where regulatory element may exist within a particular gene unit
DNA binding proteins will protect the DNA beneath from cleavage by nuclease.
Following random cleavage and separation of nucleotides by electrophoresis, a footprint where no cleavage is observed will be visible
Describe a gel retardation experiment and what it shows us.
Used to examine protein-DNA or protein-RNA interactions
The basic idea is that DNA with proteins complexed onto it will move slower than those without proteins bound in a gel electrophoresis experiment.
Size and charge of the proteins will impact how the complex moves through the gel
Describe the process of chromatin immunoprecipitation (CHIP)
DNA in living cells has regulatory proteins bound to the gene regulatory elements
These proteins are cross-linked to the DNA with formaldehyde
Cells are lysed and the DNA is broken into small fragments
Antibodies for a specific regulatory protein (that is now cross-linked to its DNA fragment) are used to precipitate out the DNA of interest
The only DNA left following precipitation will be the DNA that the regulatory protein was bound to
Crosslinks can be removed (with acid) and the DNA can be amplified for further analysis
How do reporter genes work?
Reporter genes are used to study the role that enhancers play in gene regulation.
An enhancer of interest is inserted into a bacterial plasmid
This enhancer is used to enhance the activity of a reporter gene that is easy to assay (example: luciferase, beta-galactosidase)
If the reporter gene shows high expression, then the transcription factor was active in promoting the transcription of the gene of interest
What is combinatorial gene control?
Multiple different regulatory proteins can bind to an identical regulatory element in a gene.
The combination of proteins that bind to an element depends on the cell type or physiological state of the cell
Some combinations will activate and some will repress gene expression
Describe the different dimers that can form between Max, Myc and Mad
Max exists as a homodimer Max-Max
Max can also heterodimerize to form Myc-Max and Mad-Max
How do the different dimers of Max, Myc and Mad affect gene transcription for cell division and differentiation?
These dimers bind to E-box elements of genes required for cell division and determine whether a cell will proliferate or differentiate
Max-Max binds the E-box and silences transcription (this is the normal state)
Myc is produced when cells want to divide, so Myc-Max dimers bind and upregulate the genes for cell division causing transcription.
Mad is produced when a cell wants to differentiate, so the Mad-Max dimers bind and repress the transcription of differentiation genes.
What DNA binding motif are the Mad, Max and Myc dimers?
Helix-loop-helix
In which stage of the cell cycle is Myc expressed?
Myc is expresed in the G1 to S transition of the cell cycle.
When cells want to divide.
How does a kinase cascade lead to activation of transcription factors?
An extracellular signal (mitogen) activates a kinase.
This kinase then activates (terminal phosphate from ATP transferred onto the serine of a protein) another, which activates another … and so on.
The final kinase in the cascade is usually a nuclear kinase (MAPK) which is found on the nuclear membrane. When phosphorylated, the MAPK enters the nucleus and activates the transcription factor by phosphorylation
How are Fos and Jun activated?
Fos is phosphorylated by ERK (a MAPK protein) and Jun is phosphorylated by JNK (a MAPK protein)
Why is 1 X chromosome inactivated in females?
Dosage compensation: if no silencing occured, then expression of the genes found on the X-chromosome would be twice as high as that seen in males, which would be toxic
Describe how females are mosaic due to X-chromosome inactivation
In the early embryo, each cell contains an active paternal and maternal X-chromosome
One of the X-chromosomes is randomly condensed and inactivated
All progeny cells from here will have the same x-chromosome inactivated as the parent cells
This leads to a mosaic in which patches of cells express each x-chromosome (Calico cat example)
Describe how the globin gene cluser on chromosome 11 is regulated.
Different regulatory control proteins bind and induce opening of the globin gene at different positions depending on the stage of life.
This causes the different hemoglobin subunit types between conception, birth, and early childhood
True or false: If two alleles have the same nucleotide sequence, they will have the same inheritable genetic information.
FALSE: epigenetics
Modification of DNA and regulatory proteins can lead to different inheritable genetic information from identical nucleotide sequences
What are the 4 main types of epigenetic mechanisms?
1) Positive feedback transcription factor loops (self-activation)
2) DNA methylation
3) Acetylation and deacetylation of histones (interconvert between euchromatin and heterochromatin)
4) Histone phosphorylation and histone methylation
Describe a positive feedback transcription loop.
Protein A is not made because it is required for its own transcription
A transient signal turns on expression of protein A, which then promotes the synthesis of more protein A
When the cell divides, protein A will be present, so A will be produced in the progeny as well.
How does asymmetry within the embryo lead to cell differentiation?
In successive cell divisions, different regulatory proteins will exist in each daughter cell.
The presence of different combinations of transcription factors leads to cell differentiation.
After 2 divisions with 5 transcription factors, 8 cell types can form
With 25 regulator proteins, 10,000 cell types can form
What sequences are methylated in DNA?
CpG sequences
These are palindromic
Does methylation usually promote or inhibit gene expression?
Methylation usually inhibits expression of the gene
True or false: Methylation of cytosine does not interfere with the formation of H-bonds.
True
The NH2, H and carbonyl O of the cytosine are all still available to participate in hydrogen bonding
What enzyme ensures that methylation is transferred to progeny cells?
Maintenance methylases recognize hemi-methylated CpG’s following DNA replication and produces dimethylated CpG’s to match the parent DNA
How can a gene be completely turned off due to methylation?
If gene regulatory proteins are lost from the DNA, the gene is off but leaky (expressed at a basal rate)
The exposed DNA can be methylated, which prevents RNA polymerase from binding properly
Proteins recognize the methylated DNA and recruit enzyme remodeling complexes and histone deacetylases which condense the gene and turn it completely off
How do CG islands protect genes from methylation?
CG islands are found at the 5’ end of many housekeeping genes
They have repetitive CG sequences close together that actually protect the DNA from methylation rather than induce methylation like CpG bases found elsewhere
What is the role of maintenance acetylases?
They act to acetylate histone proteins of nucleosomes that are hemi-acetylated in newly formed progeny cells. Preserves the acetylation patern of the parental cells
What is genetic imprinting?
Certain genes are expressed in a parent-of-origin manner rather than following Mendelian genetic rules
A particular gene retains its methylation in either the sperm or the egg, but not both
Most genes lose their methylation during meiosis, but a small percentage do not
