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Molecular Biology Test II - Jourdan > Control of Gene Expression 1: Transcriptional Controls > Flashcards

Control of Gene Expression 1: Transcriptional Controls Flashcards

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0
Q

What makes a cell different?

A

There are the same amount of genes (~25,000) but they express different sets of proteins

Each cell has the same genome - differentiation in cells depends on changes in gene expression

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1
Q

What is an experiment that proves there is no loss of genes in cell differentiation?

A

Take a skin cell from a frog and remove its nucleus.
Take an unfertilized egg and destroy its nucleus by UV light
Inject the nucleus from the skin cell into the empty egg and a normal embryo will form. A tadpole will be produced

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2
Q

What is RNA Seq?

A

Technology that uses the capabilities of next-generation sequencing to reveal a snapshot of RNA presence and quantity from a genome at a given moment in time

Can look at the different populations of RNA

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3
Q

What gene using RNA seq, showed that many cells including embryonic stem, liver, muscle etc expressed it?

A

Beta-actin gene

The higher the read the more it is expressed. How many times it sequences the RNA shows that relative expressiveness

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4
Q

Proteins that are common among different cell types are called what?

A

Housekeeping proteins

e.g glucose metabolism - required by many cells

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5
Q

Proteins that are only found in a few select cell types are called what?

A

Specifically limited proteins

e.g hemoglobin

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6
Q

A typical human cell expresses how much of its 25,000 genes?

A

30-60%

But level of gene expression varies

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7
Q

Different cell types expressing different genes and level of gene expression can be used in what procedures?

A

Fingerprint expression profiles
microarrays
or RNA Seq

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8
Q

What are factors that can change gene expression post transcription?

A

Alternative splicing (dystrophin gene), post translational modification

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9
Q

What does gene regulation require?

A

Short stretches of DNA of defined sequence (recognition sites for DNA binding proteins)

and gene recognition sequences for regulatory proteins

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10
Q

What is the master control protein for the production of red blood cells? What is its recognition sequence?

A

GATA1

TGATAG

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11
Q

Where can recognition sequences be relative to first exon?

A

Proximal or distal (e.g 50 kb away)

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12
Q

In DNA motif recognition regulatory proteins associate with what?

A

major groove

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13
Q

What do proteins recognize and bind to in major groove?

A

bases: major groove presents a specific face for each of the specific base pairs

CTGA

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14
Q

A gene regulatory protein recognizes a specific ______

A

DNA sequence

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15
Q

The surface of the regulatory protein is extensively _________ to the surface of the DNA region to which it binds

A

complementary

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16
Q

A series of contacts is made with the DNA involving ___ possible configurations

A

4

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17
Q

In the possible configurations of base pairs what does Blue stand for?

A

Possible hydrogen bond donors

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18
Q

In the possible configurations of base pairs what does Red stand for?

A

Possible hydrogen bond acceptors

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19
Q

In the possible configurations of base pairs what does the yellow stand for?

A

methyl groups

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20
Q

In the possible configurations of base pairs what does the white stand for?

A

Hydrogen atom

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21
Q

Gene regulatory proteins read the ____ of the DNA helix

A

outside

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22
Q

What does a typical gene regulatory protein-DNA interaction involve?

A

10-20 interactions

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23
Q

Sequence specific transcription factors are considered to be what?

A

modular: domains that have specific jobs to do

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24
What are the four possible modules of a DNA transcription factor?
DNA-Binding module Dimerization module Activation Module Regulatory Module
25
(Every/not every) transcription factor will have all modules
Not every
26
A transcription factor will have what 2 modules?
DNA-binding module | and activation module
27
What two modules COULD (optional) transcription factors possibly have?
Dimerization and regulatory modules
28
What experiment provided evidence for transcription factors being modular? ESSAY
Yeast study Two plasmids: reporter gene construct (DNA target) It is going to report of a promotor and gene is active (gene is the lacZ gene) and experimental plasmid (making transcription factor) that binds to UAS=upstream activation sequence Take a full protein- this protein has this one has two modules. How do we find that out? Create plasmids that have deletions and see if previous actions are not taking place. Example of a result: If DNA-binding domain is deleted then Binding to UAS will not happen. as a result then there will be no GAL4 protein activity
29
What are the four most common DNA-binding domain structural motifs?
Helix-turn-helix Zinc finger motif Leucine zipper Helix-loop-helix *also homeodomain and beta-sheet
30
What is the simplest most common DNA-binding motif?
Helix-turn-helix
31
Describe Helix-turn-helix
1. two alpha helices connected by a short chain of amino acids that make the "turn" -turned at fixed angle
32
Which helix of helix-turn-helix is the DNA binding module?
The longer helix, it fits into major groove | The side chains of amino acids recognize DNA motif
33
Most of the time Helix-turn-helix motifs bind as what?
Symmetric dimers: bind DNA as dimers
34
Describe the Zinc finger domain
Binds to major groove of DNA DNA binding motif includes Zn atom If you draw out amino acid sequences, the cartoon looks like a finger projection-hence the name 3D structure looks NOTHING like a finger projection
35
What can Zn finger domains be found in?
Tandem clusters - This allows multiple contact points and thus, This stabilizes the contact/interaction it has with DNA
36
What domains does a leucine zipper motif have?
Dimerization domain Activation Domain and DNA binding domain
37
Describe Leucine zipper motif structure and how it binds to DNA
Two alpha helical DNA binding domains Dimerizes through leucine zipper region (homo-/hetero-) Activation domain overlaps dimer domain Grabs DNA like a clothespin
38
What forms the zipper in leucine zipper motif?
Interactions between hydrophobic amino acid side chains (leucines) Leucine residue every 7 amino acids down one side of alpha helix dimerization domain: forms zipper
39
What does a helix-loop-helix domain consist of?
A short alpha chain connected by a loop to a second longer alpha chain
40
Describe Helix-loop-helix domain
Can occur as homodimers or hetero | Three domains or moddules to this protein: DNA binding, dimerization, and activation domain
41
What is hereditary spherocytosis?
A hemolytic anemia characterized by spherical and fragile red blood cells that lyse and release hemoglobin
42
What are the clinical presentations of someone with hereditary spherocytosis?
Hemolysis, anemia, and splenomegaly(enlarged spleen-red blood cell producing factory)
43
The HS clinical presentation ranges from what?
mild to severe anemia and can be fatal
44
What is HS caused by?
By mutation in genes for the erythrocyte membrane skeleton of red blood cells Dominantly inherited
45
Describe the erythrocyte membrane skeleton (EMS)
EMS: Cytoskeleton structure is made up of spectrin --Long chain; alpha and beta subunits --heterodimers->heterotetramers --connected to membrane by ankyrin (anchor symbol)
46
Why is the structure of EMS important?
Confers property of durability and stability to RBCs .5 million passages in circulation and tight capillary spaces can beat up a erythrocyte so it needs to be durable and stable to last in the blood for 120 days
47
What is the most common hereditary anemia ini people of Northern European Descent?
Hereditary Spheryocytosis 1/2000
48
Have most of the genes for HS been identified?
Yes
49
What EMS mutation is 63% responsible for HS?
Spectrin/ankyrin defect
50
What EMS mutation is the cause of 22% of HS cases?
Band 3 defect
51
What EMS mutation is responsible for 3% of HS cases? | what is responsible for 2%?
Protein 4.2 defect 2%-other defects
52
What is the percentage of patients that have no known defect for HS?
10% Dr. Whites rant: This should be disturbing. Very common genetic disease and 1 out of 10 patients we have no idea why they have it
53
What can the cause of HS of the 10% group of unknown defect possibly be?
A mutation in zing finger protein gene Klf1 (Kruppel-like factor 1)
54
The Klf1 gene has how many exons and encodes what?
3 exons and encodes 3 zinc finger domains
55
What does Klf1 zinc finger protein bind to?
Binds to promotoers of all genes in EMS-it turns them on
56
The mutation for gene Klf1 can be found where? What is defective
A single nucleotide change of GAA to GAT. This changed the Glu to Asp in exon 3: zinc finger domain 2) A whole CH2 group is missing defective: makes less RNA from target promotor
57
The Klf1 mutation is unique to HS how?
It is not found in any other life form with Klf1 protein - only HS carried nucleotide change
58
Where is the Klf1 DNA binding motif?
RER (Arg-Glu-Arg) DNA binding motif conserved in Klf1 from C. elegans to man
59
What does RER bind to?
CAACC DNA recognition sequence
60
What is the mutant form of RER?
RDR | Arg-Asp-Arg
61
In a normal Klf1, what does R (Arg) interact with?
E (Glu acid) in RER to stabilize interaction with C in the major groove of the CACCC recognition site
62
In HS Klf1 mutation what does the D (Asp acid) in RDR mutants alter?
The interaction of Arg with E (Glu acid) Now D in the 2nd Zn finger interacts with the G nucleotide of the opposite strand rahter than the C so DNA cannot unwind - no transcription can then occur
63
Explain Gel mobility shift assay or EMSA: electrophoretic mobility shift assay
Use: take DNA and see what proteins bind to target promotor regions Mix radioactive DNA fragment with protein extract from cell Run electrophoretic gel See shift of radioactive band when protein is bound to DNA Depending on size of Protein attached will determine the distance the DNA will travel. The smaller the further it goes
64
What is affinity chromatography?
Use: isolate DNA binding protein; purification of sequence specific binding proteins Step 1: Find binding proteins Have a column with matrix containing DNA of many different sequences Add low-salt wash to remove proteins that dont bind Use medium-salt wash to remove binding proteins Step 2: find specific binding protein Column with matrix only containing specific DNA sequences Medium salt wash to remove those not specific to sequences High salt wash elutes rare protein that specifically binds
65
What does CHIP: Chromatin Immuno Precipitation allow?
This technique allows identification of the sites in the genome that a regulatory protein binds -done in living cells This helps find the specific sequence a known binding protein will bind to. Uses formaldehyde to glue protein to sequence Lyse cells Break DNA into small fragments Precipitate using Antibodies against protein PCR product at end can be used to screen a microarray gene chip
66
What is the DNA region involved in regulating and initiating transcription of a gene?
Gene control region
67
What does a gene control region include?
Promoter: where transcription factors and RNA polymerase II assembles & Regulatory sequences to which regulatory proteins bind to controll rate of assembly process at the promoter
68
Out of the 25,000 human genes,, how many encode gene regulatory proteins ?
8% or 2,000 genes
69
What is the encode project?
Figure out: What does the genome do | Exceedingly complex network of controls governing the expression of mammalian genes
70
What are a few generalities that can be mentioned about the Gene control region?
RNA polymerase and general transcription factors assemble at the promoter Other gene regulatory proteins (activators or repressors) bind to regulatory sequences which can be adjacent, far upstream or in introns downstream of the promoter
71
What do gene activator proteins modify?
Local chromatin structure
72
What favor transcription initiation by increasing accessibility of DNA to other proteins?
Nucleosomes remodeling and histone removal
73
What are some proteins involved in modifying local chromatin structure?
Chromatin remodeling complex-nucleosomes looser Histone chaperone protein-remove nucleosome structure Histone chaperone-allows DNA to relax Histone-modifying enzyme-histone acetylation allows easier removal of histones
74
What is the order of events leading to transcription initiation of specific gene?
Gene activator protein binds to chromatin + chromatin remodeling complex Chromatin is remodeled Then histone modification enzymes covalently modify histones The other activator proteins bind to gene reg region Next, mediator, general transcription factors, and RNA poly assemble the pre-initiation complex at the promotor Transcription!
75
Regulatory proteins include gene repressors. What is their role?
Inhibit transcription in different ways 1. activator and repressor compete for same binding site 2. Both proteins bind to DNA but the repressor binds to the activation domain of the activator protein 3. The repressor binds to DNA and blocks assembly of general transcription factors (direct interactions with general TF) 4. The repressor recruits a chromatin remodeling complex which returns the promoter to the pre-transcriptional nucleosome state 5. Repressor attracts a histone deacetylase to the promotor - harder to remove deacetylated histones 6. Repressor attracts a histone methyl transferase - methylated histones bound to proteins which act to maintain chromatin in silent form
76
Depending on their composition, the gene regulatory proteins assembled into complexes on DNA can be either what?
Activating or repressing (they are schizophrenic) The same protein can be part of an activating or repressing complex. Regulation by "committee"
77
How are gene regulatory proteins controlled?
1. synthesis - make them or not 2. Ligand binding - that can make the DNA reg protein but needs a ligand 3. Covalent modification-phosphorylation 4. Addition of subunit 5. Unmasking-use an inhibitor that is removed by phosphorylation 6. Nuclear entry - inhibitory protein that does not allow passage 7. Proteolysis - released from membrane - cut and free to do as it pleases
78
What are the alpha-like chains of the globin genes?
Zeta and alpha
79
What are the beta-like chains of globin chains?
epsilon gamma delta beta
80
What is the order of hemoglobin switching from embryo-> adult? How does this process happen?
Embryonic Hb = zeta and epsilon Fetal = alpha and gamma Adult = alpha and beta -not fully clear how hemoglobin switching occurs - lots of research being done to understand Sickle cells disease. Fetal globin can be used to replace the sickle cell human Hb. Be a cure.
81
What is interesting about the expression of globin genes during human development?
Globin genes are arranged in linear fashion Ordered in the 5' to 3' direction in the same sequence of activation and expression during embryonic, fetal, and adult development
82
How big is the region containing five globin genes and locus control region (LCR)?
100kb
83
Where is the LCR for globin genes? What binds to it?
Far upstream in sequence but required for transcription Regulatory proteins bind to LCR
84
The understanding of globin gene regulation may allow what?
The induction of fetal hemoglobin in sickle cell anemia

Molecular Biology Test II - Jourdan (9 decks)

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