Control Of Gene Expression 1

Question 1

What makes cells different?

Answer 1

Structure and function

It was previously thought that cells lose some genes so only certain proteins are then expressed. However, if normal skin cells are isolated from a frog and the nucleus is implanted into a denucleated cell a normal embryo forms. This obviously shows that NO genes are lost.

Different cells all contain the same set of genes but express different sets of proteins. Cell differentiation of cells depends on gene expression.

Question 2

Different cell types -diff proteins

Answer 2

1-4

Question 3

Control of Gene Expression (6)

Answer 3

1) transcriptional control
2) RNA processing control
3) RNA transports and localization control
4) translation control
5) mRNA degradation control
6) protein activity control

Question 4

Gene regulation requires

Answer 4

1) short stretches of DNA of defined sequence - recognition sites for DNA binding proteins
2) Gene regulatory proteins - transcription factors that will bind and activate gene

Question 5

Steps of DNA Motif Recognition

Answer 5

• association of regulatory proteins with major groove
• proteins recognize and bind to bases in major groove
• major groove presents a specific face for each of the specific base pairs
• gene regulatory protein recognizes a specific sequence of DNA
• surface of protein is complementary to surface of DNA region to which it binds
• series of contacts is made with the DNA involving 4 possible configurations

Question 6

Four Base Pair Configurations in Motif Recognition

Answer 6

Blue- possible h bond donors
Red- possible h bond acceptors
Yellow- methyl group
White- h atom

Question 7

What are the mandatory parts of a DNA transcription factor? What are the non mandatory parts?

Answer 7

Every factor will have a DNA-Binding Molecule.

Dimerization module- form diners with other subunits
Activation module- turn on gene
Regulatory module- regulate transcription factor

Question 8

Evidence for trans factors being modular

Answer 8

Experiment

Question 9

What are the 4 kinds of DNA-binding domain structural motifs?

Answer 9

1) Helix-turn-Helix
2) Zinc Finger motif
3) Leucine Zipper
4) Helix-loop-Helix

(Also Homeodomain and Beta-sheet)

Question 10

Helix-Turn-Helix

Answer 10

• Simplest most common motif
• two alpha helices connected by short chain of amino acids that make the turn (turn is at fixed angle)
• longer helix = recognition module - DNA binding module - fits into major groove
• side chains of amino acids recognize DNA motif
• symmetric diners: ind DNA as dimers

Question 11

Zinc Finger Domain

Answer 11

• Includes Zinc atom
• when drawn out, the amino acid sequence looks like a finger projection
• 3D structure does not look like finger projection does not
• Binds to major groove of DNA
• Found in tandem clusters
• Stabilizes interactions with DNA
• Multiple contact points

Question 12

Leucine Zipper Motifs

Answer 12

• Two alpha helical DNA binding domains
• Grabs DNA like clothespin
• Activation domain overlaps dimer domain
• Interactions between hydrophobic amino acid side chains (leucine)
• Dimerizes through leucine zipper region (homo-/hetero-)
• One leucine residue every 7 AA down on side of alpha helix in domain forms the zipper structure

Question 13

Helix-Loop-Helix Domain

Answer 13

-Consists of a short alpha chain connected by a loop to a second longer alpha chain
-can occur as homodimers or heterodimers
Three domains or modules to this protein: DNA binding domain, dimerization domain, activation domain

Question 14

Homeodomain Proteins

Answer 14

• Contain Homeodomain which consists of 3 alpha helices

- helix-turn-helix motif

Question 15

Beta Sheet DNA Recognition Proteins

Answer 15

• 2 stranded beta sheet
• Beta sheet consists of beta strands
• connected laterally by two or three backbone h bonds
• forms twisted, pleated sheet
• binds to major groove of DNA

Question 16

What disease is an example of Zn finger transcription factor mutation?

Answer 16

Hereditary spherocytosis

Question 17

What are the clinical presentations of HS?

Answer 17

Hemolysis, anemia, splenomegaly

Symptoms range from mild to severe, eg- fatal anemia

Question 18

HS is caused by mutation in genes for the ____ ___ ___ of _BCs - not making enough protein.

Answer 18

Erythrocytes membrane skeleton

Red

Question 19

HS is ____ inherited.

The purpose of the EMS is:

Answer 19

Dominantly

-Conferring durability and stability to RBCs while they pass through tight capillary spaces and make 1/2 million passages in circulation.

Question 20

KLF1 Zinc Finger Protein
(Kruppel-like factor 1)

Mutation leads to…

Answer 20

Binds to promoters of all genes in EMS - turns them on

Non-functioning KLF1 Zn finger protein, no EMS made, leads to HS

Question 21

In HS, what is mutated and what is the result?

Answer 21

GAA —> GAT or Glu —> Asp or RER —> RDR
Domain 2 is mutated
No transcription occurs
Defective: makes less RNA from target promoters of EMS genes therefore less protein is available and HS develops

Question 22

Describe the identification of transcription factors using radioactive DNA.

Answer 22

• Use radioactive DNA from known promoter
• mix radioactive DNA fragment (regulatory DNA sequence) with protein extract from cell
• run electrophoretic gel
• Proteins with DNA attached migrate according to size
• see shift of radioactive band when protein is bound DNA
• isolate the protein to identify it

Allows Detection of sequence-specific DNA binding proteins

• Gel Mobility shift assay
• EMSA: electrophoretic mobility shift assay

Question 23

Describe the identification of transcription factors using affinity chromatography

Answer 23

Isolate the DNA binding protein
Purify the sequence specific binding proteins
Start Broad- identify any DNA binding protein, then Get Specific- use only 1 promoter recognition sequence

Question 24

Identification of a DNA binding sequence

Answer 24

CHIP: Chromatin Immuno-Precipitation

• technique allows identification of the sites in the genome that a known regulatory protein binds to
• done in living cells
• PCR product at end can be used to identify sequence

Question 25

Gene Control Region

Answer 25

- DNA region 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 promoter - 9% of human genes encode reg proteins - control of mammalian genes is extremely complex - few generalities can be made

Question 26

Gene Control Region ___ Polymerase and ___ ____ factors assemble at the promoter. Other gene regulatory proteins (___ or ___) bind to regulatory sequences which can be adjacent, far upstream or introns downstream of the promoter.

Answer 26

RNA General Transcription Repressors or activators

Question 27

Transcriptional Activation Model

Answer 27

DNA loops upon itself and a mediator allows the gene reg proteins to interact with the proteins assembled at the promoter The mediator serves as an intermediate between gene reg proteins and RNA polymerase II

Question 28

Nucleoside remodeling and histone removal favors transcription by increasing accessibility of ____ to ____.

Answer 28

DNA to proteins

Question 29

What are the 4 ways Proteins modify DNA (local chromatin structure)

Answer 29

Nucleosome remodeling Nucleosome removal Histone Replacement Histone modification (eg- acetylation makes it easier to remove histones)

Question 30

True or False: In competitive DNA binding activators and repressors have separate binding sites.

Answer 30

False, they compete for the same binding site

Question 31

In the masking form of Gene repression which protein binds to the DNA? How does this block activation?

Answer 31

Both proteins (activator and repressor) bind to DNA but the repressor binds to the activation domain of the activator protein, stopping activation.

Question 32

When direct interaction with the general transcription factors occurs...

Answer 32

The repressor binds to DNA and blocks assembly of general transcription factors

Question 33

Recruitment of chromatin remodeling complexes

Answer 33

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

Question 34

Recruitment of Histone deacetylases

Answer 34

The repressor attracts a histone deacetylase to the promoter - Harper to remove deacetylated histones and open up DNA

Question 35

Recruitment of histone methyl transferase

Answer 35

- 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

Question 36

Gene regulatory proteins assemble into complexes on DNA

Answer 36

In solution the proteins are separate floating. On DNA they assemble into complexes - depending on the composition of the complexes, proteins can be activating or repressing. - the same protein can be apart of an activating or repressing complex - regulation by committee (group effort)

Question 37

Name the 7 control processes of Gene reg proteins

Answer 37

``` A. Synthesis B. Ligand binding C. Covalent modification-phosphorylation D. Addition of a subunit E. Unmasking F. Nuclear Entry G. Proteolysis ```

Question 38

What are the alpha-globin chains

Answer 38

Zeta and alpha

Question 39

What are the beta-globin like chains

Answer 39

Epsilon, gamma, delta, beta

Question 40

Hemoglobin Switching

Answer 40

- 2 alpha type glob is and 2 beta type glob is - embryonic = zeta, epsilon - fetal = alpha, gamma - adult = alpha, beta - embryonic to fetal to adult (switching) - not clear how this occurs

Question 41

Globin Gene Expression

Answer 41

Beta-globin genes are arranged in linear fashion, in 5’ to 3’ direction in all stages of development (emb, fetal, adult)

Question 42

Beta Globin Gene Regulation

Answer 42

100 kB region containing five beta globin genes and locus control region (LCR) Far upstream in sequence but required for transcription Regulatory proteins bind to LCR Sickle cell disease - sickle beta globin - serious anemia (This is a cure) If make fetal Hb, not much needed to cure sickle cell -switch back from adult to fetal Hb is cure. Understanding of globin gene reg may allow the induction of fetal hemoglobin in sickle cell anemia cure