Gene Regulation II Flashcards
Gene expression can be regulated indirectly through what?
altering chromatin
Most transcription regulating proteins bind what?
- most transcription regulating proteins are DNA binding proteins
- some regulatory proteins attach to the promoter via another DNA binding protein (i.e. piggyback)
What two major domains are transcription factors generally considered to possess?
- the DNA binding domain
- the transcription activating domain or the transcription repressing domain
What is the general structure of a transcription activating protein?
- transcription activating domain (TAD) is bound to sequence specific DNA binding DNA
- TAD interacts with transcription initiation complex stabilizing RNA polymerase at the promoter
*this model applies to both general promoter factors and to enhancer binding proteins (i.e. gene specific factors)
Non-binding activators structure
- transcription activating protein non-covalently interacts with sequence specific DNA binding protein
- the transcription activating protein (TAD) interacts with the transcription initiation complex stabilizing RNA polymerase at the promoter
What is the function of the DNA binding domain?
to locate the transcription factor to a specific location (or locations) in the genome
*binding of the TF will now influence the transcriptional activity of the adjacent gene
How is a DNA binding domain able to identify a specific DNA sequence?
proteins extend into helix and can feel the sequence of the bases in there
proteins mostly bind between major groove (as opposed to minor groove - this is explained in DNA structure ILM)
What is the most important protein structure for sequence specific DNA binding?
the alpha-helix
*it fits surprisingly nicely within the major groove of the DNA double helix
Individual amino acid side chains of the alpha helix (especially charged residues) can contact specific base pairs within the DNA. What does this allow for?
this allows for sequence specific interaction of protein with the DNA helix
TATA binds through the minor or major groove?
fill in
What DNA binding structure is found in homeodomain proteins?
helix-turn-helix DNA binding proteins
The helix-turn-helix DNA binding protein structure is found in what proteins?
homeodomain proteins
What is the most important part of the helix-turn-helix DNA binding proteins?
a short region of two alpha-helices separated by a short loop region
- one of the alpha helices fits in the major groove to contact the DNA bases
The specific amino acids in the alpha-helix make contact with specific nucleotides - what are these?
Serine with T
Arginine with G
Asparagine with A
Lysine with C/T
Homeodomain proteins usually form what?
dimers
- the spacing of the alpha-helices in the dimer allows exact positioning of the helices into consecutive major grooves
- in general, the proteins only make stable contact with DNA when in dimer form
If the two proteins forming the dimer are identical (i.e. a homodimer), the binding site is a/symmetrical?
symmetrical
- because the alpha-helices point in opposite directions, the site is inverse symmetrical
- if the two proteins forming the dimer are different (i.e. a heterodimer), then the binding site will not be symmetrical
What are some important homeodomain proteins?
- the Hox proteins (anterior/posterior patterning)
- Nkx2-5 (heart development)
- Pax6 (eye development)
What is the zinc finger?
a loop of protein sequence arranged around a zinc ion
*one side of the finger forms an alpha-helix that contacts the bases in the DNA major groove
What two residues in the peptide sequence interact with zinc to stabilize the structure?
- cysteine
- histidine
- cysteine and histidine residues in the protein interact with a single zinc ion per finger
Some DNA binding proteins contain multiple, consecutive zinc fingers. What does this allow for?
this allows the protein to make contact with extended regions of DNA leading to increased specificity
*the fingers are arranged to contact consecutive major grooves
What is the general structure of a single zinc finger?
- cysteine and histidine residues complex with the zinc ion
- residues 12-25 of the finger form an alpha-helical structure which contacts the major groove of DNA
What do Leucine zipper proteins contain?
these proteins contain an extended linear alpha-helical region
- one portion of the helix (the leucine zipper) is involved in protein-protein interactions that allow dimerization
What is the structure of the zipper region of leucine zipper proteins?
- the zipper region contains a Leu residue every 7 amino acids (every second turn of the helix)
- this lines them up on one face of the helix
What is the leucine zipper involved in?
the leucine zipper portion of the helix is involved in protein/protein interactions that allow dimerization
What occurs to permit stable dimerization of two zipper proteins (forming The Leucine Zipper)?
two linear helices now interact via hydrophobic interactions of leucine residues
What composes the leucine zipper - what are the functions of it’s two regions of the alpha helix?
- one portion of the helix is involved in protein/protein interactions that allow dimerization
- -> this region contains a leucine every 7 residues (one leucine every 2 turns of the helix)
- -> two linear helices now interact via hydrophobic interactions of leucine residues
- a second portion of the helix is involved in sequence specific DNA binding
- -> this region of the alpha helix makes contact with specific bases in the major groove of the DNA helix
- -> this structure is sometimes called the “clothes-peg”
The leucine zipper is a generic structure. What is required for interaction of two proteins?
both must possess a leucine zipper region
- this permits heterodimerization of leucine zipper proteins
- increases the possible binding specificities
ex:
- inverted blue sequences (two blue)
- inverted red sequences (two red)
- one blue, one red sequence
In most classes of DNA binding proteins, what is required for establishing sequence specific DNA binding?
an alpha-helix is the critical region for establishing sequence specific DNA binding
Basically, what is the function of the leucine zipper?
A leucine zipper, aka leucine scissors, is a common 3D structural motif in proteins. These motifs are usually found as part of a DNA-binding domain in various transcription factors, and are therefore involved in regulating gene expression.
The leucine zipper is a super-secondary structure that functions as a dimerization domain, and its presence generates adhesion forces in parallel alpha helices. A single leucine zipper consists of multiple leucine residues at approximately 7-residue intervals, which forms an amphipathic alpha helix with a hydrophobic region running along one side. This hydrophobic region provides an area for dimerization, allowing the motifs to “zip” together. Furthermore, the hydrophobic leucine region is absolutely required for DNA binding.
- Wiki
- Long story short, the leucine zipper motif will bind to a DNA binding motif in order to provide more specificity and help in binding an activator (primarily at the DNA binding domain).
Consider a protein that must homo-dimerize to bind DNA:
What happens if one copy of the gene for the protein making the TF is deleted?
One copy, can still dimerize, and make TF just fine, just only makes half as much
Consider a protein that must homo-dimerize to bind DNA:
What happens if one copy of the gene for the protein making the TF has a mutation that prevents DNA binding?
Good protein gene, Bad protein gene (one copy of each) makes: GG GB BG BB
GB, BG, BB all do not function, so you only get 25% that could work
Which is more likely to be deleterious:
only one copy of the protein gene
one copy of the protein gene has a mutation
mutation is worse
Unlike the DNA recognition domains of the protein, the region required for activation of transcription is _____ conserved.
not highly
- the transcription activation domains are completely interchangeable between different proteins
- TAD2 could still result in efficient transcription of gene 1, just like TAD1 would (transcription activation is the same, independent of the source of the transcription activating domain TAD)
What is one of the primary properties of the transcription activating domain?
the presence of an alpha helix with negatively charged (acidic) amino acids lined up along one surface
*further experiments have shown that additional protein domains capable of activating transcription also exist
In principle, any protein domain that can positively interact with the RNA polymerase II complex will _____ the preinitiation complex and ultimately _____ the rate of transcription.
stabilize
increase
Basically, any domain that stabilizes the pre-initiation complex can act as a transcription _____. (Any domain that interacts with proteins bound to the promoter region). In contrast, any domain that functions to destabilize the pre-initiation complex, will function as a transcription _____.
activator
initiatior
*Also, some transcription activators recruit histone modification enzymes
Mutations in any transcription factor can alter the expression of the genes that they regulate. What are the consequences of this in regards to downstream genes?
transcription factors regulate expression of more than one downstream gene and so effects will be observed in expression levels of multiple genes
NKX2-5 (homeodomain transcription factor)
What does this make it?
helix-turn-helix class
What is NKX2-5 necessary for? What are the effects of defects in NKX2-5?
- essential for embryonic heart development
- defects in human NKX2-5 are a common cause of congenital heart defects, especially atrial septal defects and hypoplastic left heart syndrome
- Tin Man
- hypoplastic left hearts have many serious defects; note that atrial septal defect is one of the errors; underdeveloped left ventricle
There are genes directly regulated by NKX2-5, so defects in NKX2-5 will have what effects on these proteins?
they will be disregulated, and the genes directly regulated by these will also in turn be disregulated
To what level are NKX2-5 mutations detected in heterozygotes?
all mutations are detected
“ter”
termination of protein
Waardenburg Syndrome
a group of hereditary conditions, mostly associated with defects of neural crest cells, but also inner ear development
*group of hereditary conditions characterized by deafness and partial albinism (pale skin, hair, and eye color)
What is a major cause of Waardenbur Syndrome?
defects in the SOX10 transcription factor
-loss of function of one copy of SOX10 leads to multiple developmental problems including abnormal pigmentation, intestinal defects, and deafness
What is Waardenburg Syndrome due to?
mutations in SOX10 (neural crest)***
- X = termination
- HMG domain is the binding domain
Transcription factors regulate a lot of proteins, and other transcription factors. Why is a mutation in a TF gene bad?
not only will it effect the transcription factor, it will cause defects or termination of the proteins and other transcription factors it regulates
How will a mutation in the enhancer sequence of a gene compare to a mutation in the transcription factor that binds that sequence.
fill in
We have Gene X, with transcription factor Y, no enhancer
how does this compare to Gene X, with no transcription factor Y
TFs still okay, just can’t upregulate
*
*there’s lots of gene expression being messed up because TF is not
THINK ABOUT
Complete loss of transcription factor activity is almost always lethal. What does this mean for the genes of human diseases related to transcription factor?
most human disease related to transcriptoin factor activity is associated with mutation of one copy of the gene
