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Molecular Exam 3 > Control Of Gene Expression 2 > Flashcards

Control Of Gene Expression 2 Flashcards

1
Q

Cells differ in structure and function. What was the old train of thought regarding genes in cell differentiation?

How was this disproved?

What, then, makes cells different?

A

Cells lost genes, so that only certain proteins are expressed. Therefore, cells are able to differentiate between nerve and lymph for example.

Nuclei from skin cells isolated from adult frogs were implanted into a denucleated cell. These cells then formed a normal embryo. Clearly there was no loss of genes.

All cells contain the same genes, but they express different sets of proteins. Gene expression is responsible for cell differentiation. Regulatory proteins can be responsible for gene expression, along with environmental factors, transcription factors, etc.

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2
Q

Name and describe four protein limitations.

A
  1. Common: “Housekeeping proteins” (glucose metabolism)
  2. Specifically limited proteins (Hemaglobin)
  3. Average human cells express 30-60% of coding genes, but level of expression varies (fingerprint expression profiles/microarray/RNA sequencing)
  4. Post transcription factors like alternative splicing, post translational modification.
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3
Q

Gene Regulation Requires (2)

A
  1. Short stretches of DNA of defined sequence - recognition sites for DNA binding proteins - proximal or distal
  2. Gene regulatory proteins - transcription factors that will bind and activate gene
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4
Q

A gene regulatory ____ recognizes a specific __ ___.
The surface of the protein is extensively ____ to the surface of the DNA region to which it binds.
A series of contacts is made with the DNA involving __ possible configurations.

A

Protein, DNA sequence

Complementary

4

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5
Q

DNA Motif Recognition

A

Association of regulatory proteins with the major groove of DNA.
Proteins recognize and bind to bases in MG.
MG presents a specific face for each base pair. (IE- C is different than G is different than A is different than T).

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6
Q

The typical gene-regulatory protein and DNA interaction involves how many interactions?

A

10-20 interactions

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7
Q

What are the parts of a DNA transcription factor?

A

DNA-Binding Module

Dimerization Module

Activation Module

Regulatory Module

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8
Q

What parts are required in a DNA transcription factor and which are optional?

A

Req- DNA Binding Module & Activation Module

Opt- Dimerization Module & Regulatory Module

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9
Q

Name some DNA-Binding Domain Structural Motifs

A
  • Helix-turn-helix
  • zinc finger motif
  • leucine zipper
  • helix-loop-helix
  • Homeodomain
  • beta-sheet
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10
Q

Helix-Turn-Helix Domain

A

Simplest, most common DNA-Binding Motif

Two Alpha helices connected by a short chain of AA that make the turn at a fixed angle

Longer helix= recognition module, DNA binding mod fits into major groove

Side chains of AA recognize DNA motif

Symmetric diners: bind DNA as diners

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11
Q

Zinc Finger Domain

A

Different Types

DNA binding motif includes a Zn atom

So-named for the drawn out AA sequence that looks like finger projection (not the 3D version)

Binds to DNA Major Groove

Found in tandem cluster

Stabilizes interaction with DNA

Multiple contact points

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12
Q

Leucine Zipper Motif

A

Dimerizes through leucine zipper region (homo- hetero-)

Interactions between AA side chains

L residue every 7 AA down one side of alpha helix in dimerization domain forms the zipper structure

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13
Q

Helix-Loop-Helix Domain

A

Consists of a short alpha chain connected by a loop to a second longer chain

Can occur as homo- or heterodimers

3 domains or modules: binding domain, dimerization domain, activation domain

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14
Q

Homeodomain proteins

A
  • contain Homeodomain
  • is made of 3 alpha helices
  • helix-turn-helix motif
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15
Q

Beta Sheet DNA Recognition Proteins

A

2 stranded beta sheet

B sheets consist of B strands

Connected laterally by 2-3 backbone H bonds

Forms twisted, pleated sheet

Binds to major groove of DNA

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16
Q

What disease exemplifies the mutation of a Zn finger transcription factor leading to disease?

A

Hereditary Spherocytosis

Hemolytic anemia, spherical and fragile RBC that lose and release hemoglobin

Clinical Presentation: hemolysis anemia, splenomegaly

Mutations in genes for EMS (not enough protein)

Dominant inheritance

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17
Q

What is the function of the EMS?

A
  • confers durability and stability to RBCs during million passages in circulation
  • allows RBCs to pass through tight capillary spaces
  • prevents lysing from bumps and bruises of travel and osmosis
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18
Q

Kruppel-like factor 1

A

KLF1 is a zinc-finger protein gene that binds to promoters of all EMS genes and turns them on.

Mutation: non-functioning KLF1 Zn finger protein
(No EMS protein made)
Leads to HS
GAA to GAT/ Glu to Asp (exon 3)

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19
Q

What is the specific mutation of KLF1 that causes HS?

A

GAA to GAT
Glu to Asp
R..E..R to R..D..R

This defect results in less RNA made (no transcription) from target promoters of EMS genes and therefore HS.

20
Q

What is the impact of KLF1 binding to CACCC on the DNA?

A

Normally, the 3 finger zinc domain binds to the DNA without problems and leads to transcription of KLF1 mRNA.

In HS, the mutation causes the “D” in RDR (vs RER) to bind to the wrong strand of DNA, resulting in NO transcription. No RNA trans, no protein!

21
Q

What test detects for sequence-specific DNA binding proteins?

A

Gel Mobility shift assay, EMSA: electrophoretic mobility shift assay

Use of radioactive DNA from known promoter

  1. Mix radioactive DNA fragment with cell’s protein extract
  2. Run electrophoretic gel
  3. Proteins with DNA attached migrate according to size
  4. See shift of radioactive band when protein is bound to DNA
  5. Isolate protein to identify
22
Q

Affinity chromatography for IDing Transcription Factors

A

Isolate the DNA binding protein via purification of sequence specific binding proteins.

Start by broadly identifying any DNA binding protein, then get specific using 1 promoter recognition sequence to test presence.

2 step process. Broad —> specific

23
Q

CHIP: Chromatin Immuno-Precipitation

A

Technique allows identification of the sites in the genome that known regulatory protein binds to

Done in living cells

PCR product at end can by used to identify sequence

24
Q

Gene Control Region

A

Region of DNA involved in regulating and initiating transcription of a gene

Includes the promoter, where transcription factors and RNA polymerase II assembles

Regulatory sequences to which regulatory proteins bind to control rate of assembly process at the promoter

25
Q

Where on the gene control region do RNA polymerase, general transcription factors, and other gene regulatory proteins assemble?

A

Promoter
Promoter
Bind to regulatory sequences which can be adjacent, upstream,, or downstream to promoter

26
Q

Activation of Transcription

A

DNA looping and a mediator complex allow the gene reg proteins to interact with the proteins that assemble at the promoter

The mediator serves as an intermediary between gene regulatory proteins and RNA polymerase II.

27
Q

Gene Activator Proteins Modify DNA

A

Transcription regulators bind to DNA in nucleosomes with lower affinity than naked DNA (even thought they have a ^affinity for naked DNA)

The surface of the nucleotide recognition sequence may be facing inward if attached to nucleosome

4 ways to overcome

28
Q

What are the 4 ways to overcome Local Chromatin Structure

A

Nucleosome remodeling

Nucleosome removal

Histone replacement

Histone modification

29
Q

Nucleosome remodeling

A

Process that favors transcription by increasing availability of DNA to proteins

Nucleosome sliding along strand exposes areas allowing for transcription machinery to access DNA

30
Q

Nucleosome removal

A

Process that favors transcription by increasing availability of DNA to proteins

Transcription machinery assembles on nucleosome-free DNA

31
Q

Histone replacement

A

Histone variants allow greater access to nucleosomal DNA

Different histone types? Different conformations?

32
Q

Histone Modification

A

Specific patterns of Histone modification destabilize compact forms of chromatin and attract components of transcription machinery

Eg- acetylation of histones makes them easier to remove, giving access to DNA

33
Q

Competitive DNA Binding

A

Activator and Repressor compete for the same binding site

Their areas of attachment overlap

Form of Gene repressor proteins inhibiting transcription

34
Q

Masking of the activation surface

A

Both proteins bind to the DNA but the repressor binds to the activation domain of the activator protein

Form of Gene repressor proteins inhibiting transcription

Cancels each other out

Separate binding sites used

35
Q

Direct interaction with the general transcription factors

A

The repressor binds to DNA and blocks assembly of the general transcription factors (GTF unit)

Form of Gene repressor proteins inhibiting transcription

36
Q

Recruitment of Chromatin Remodeling Complexes

A

The repressor recruits a chromatin remodeling complex which returns the promoter to the pretranscriptional nucleosome state.

Form of Gene repressor proteins inhibiting transcription

37
Q

Recruitment of histone deacetylases

A

The repressor attracts a histone deacetylase to the promoter to make it harder to remove deacetylated histones and open up the DNA

Form of Gene repressor proteins inhibiting transcription

38
Q

Recruitment of histone methyl transferase

A

The repressor attracts a histone methyl transferase which methylated histones

These methylated histones are bound to proteins which act to maintain chromatin in a transcriptionally silent form

Form of Gene repressor proteins inhibiting transcription

39
Q

Gene Regulatory Proteins Assemble into Complexes on DNA (not when in solution alone)

A
  • depending on the composition of complexes, proteins can be either activating or repressing
  • the same protein can be part of an activating or repressing complex
  • regulation by committee
40
Q

How can Gene Regulatory proteins controlled?

A 
Synthesis
Ligand binding
Covalent modification-phosphorylation
Addition of subunit
Unmasking
Nuclear entry
Proteolysis
41
Q

What mammalian globin genes for hemoglobin are classified as alpha-globin like?

A

Zeta

Alpha

42
Q

What mammalian globin genes for hemoglobin are classified as beta-globin like?

A

Epsilon
Gamma
Delta
Beta

43
Q

Embryonic Hb includes

A

Zeta

Epsilon

44
Q

Fetal Hb includes

A

Alpha

Gamma

45
Q

Adult Hb includes

A

Alpha

Beta

46
Q

Hb switching

A

Embryonic to fetal to adult

Not clear how

47
Q

B-Globin Gene Expression

A

B-Globin are linearly arraigned and ordered 5’to3’ like their sequence of activation and expression during embryonic, fetal, and adult development.

Sickle B Globin can occur

Cure for SS could be reverting back to production of fetal Hb from Adult Hb

Molecular Exam 3 (5 decks)

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