Gene Expression 1

Question 1

What makes cells different and the same?

Answer 1

Each cell has the same genome, but they express different proteins.

Question 2

Name two post-transcriptional factors.

Answer 2

alternative splicing and post translational modification.

Question 3

Gene regulation requires these two things.

Answer 3

1. short stretches of DNA of defined sequence (recognitions sites for DNA binding proteins)
2. gene regulatory proteins (transcription factors that will bind and activate gene)

Question 4

Recognition sequence locations can be either __ or __.

Answer 4

proximal (base pairs away) or distal to first exon.

Question 5

Proteins recognize and bind to bases in what groove of the DNA?

Answer 5

Major

Question 6

When the protein binds to the complimentary DNA sequence, how many possible configurations of base pairs make contact with the protein?

Answer 6

4

Question 7

Within the 4 possible configurations that the protein binds to the DNA, what are the different elements of the motif?

Answer 7

H-bond donor
H-bond acceptor
Methyl group
Hydrogen atom.

Question 8

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

Answer 8

10-20

Question 9

What modules will a transcription factor have? What modules could a xcr factor have?

Answer 9

Will have: DNA binding module and activation module.

Could have: Dimerization module and/or regulatory module.

Question 10

How did they find the experimental evidence that transcription factors were modular?

Answer 10

Using two different plasmids, a reporter gene construct containing the gene of interest and a GAL4 binding site, and another experimental plasmid making GAL4 protein and DNA binding protein, a series of deletion mutants were tested.

Internal deletion mutants, with only the regions of the DNA containing the DNA-binding domain and the activation domain, produced fully functioning beta galactosidase activity when the complete domains of both regions were included. When the regions of the domains were not included in the plasmids, beta-galactosidase was either reduced or non-existent.

Question 11

Name the 4 DNA-binding domain structural motifs.

Answer 11

Helix-turn-helix
Zinc finger motif
Leucine zipper
Helix-loop-helix

Question 12

What is the simplest, most common DNA-binding motif? Describe its structure.

Answer 12

Helix-turn-helix.  Contains two alpha helices connected by a short chain of amino acids.  They are symmetric dimers. 
 The longer helix is the recognition module and the side chains of its amino acids binds to the DNA in the major groove.

Question 13

Describe the structure of the zinc finger domain.

Answer 13

Contains only one alpha helix. Zn finger domains found in tandem clusters and the multiple contact points stabilizes interaction with DNA.

Question 14

Describe the structure of the leucine zipper motif.

Answer 14

Two alpha helical domains, grabs DNA like clothespin. Contains a dimerization domain, activation domain, and DNA binding domain.

Leucine residues every 7 amino acids along the alpha helices in the dimerization domain.

Question 15

Describe the helix-loop-helix domain.

Answer 15

Contains a binding domain, activation domain, and a dimerization domain (like the leucine zipper).

Consists of a short alpha chain connected by a loop to a second longer alpha chain, either as a homodimer or heterodimer.

Question 16

A Zn finger transcription factor mutation leads to what disease?

Answer 16

Hereditary spherocytosis (HS), causing spherical and fragile RBCs. Can present clinically as hemolysis, anemia, and splenomegaly.

Question 17

HS has what pattern of inheritance?

HS effects Northern European descendants at what incidence rate?

Answer 17

Dominant.

1/2000.

Question 18

HS can be mutation in zinc finger protein gene ___. Normally, this protein binds to promoters of all ___ genes, turning them on.

The gene encodes how many zinc finger domains? A mutation in this gene results in what?

Answer 18

Klf1.

EMS.

3.

Mutation causes a non-functioning KLF1 Zn finger protein, and no EMS is made, leading to HS.

Question 19

Glutamic Acid in RER gets converted to ___, changing the configuration to ___, the mutant form of KLF1 resulting in no transcription.

Answer 19

Aspartic Acid.

RDR.

Question 20

This domain ___ of the abnormal KLF1 protein binds to the opposite strand, changing the binding configuration which leads to no transcription.

Answer 20

Domain 2.

Question 21

Describe the EMSA: electrophoretic mobility shift assay, used to ID xcr factors.

Answer 21

Free, radioactive DNA regulatory sequence fragments are mixed with protein extract and an electrophoretic gel is run. Proteins attached to DNA migrate according to size. A shift in the bands means you can then isolate the proteins to identify them.

Question 22

Describe Affinity Chromatography, used to identify xcr factors.

Answer 22

Broad: Wash total cell proteins in a column containing DNA. low-salt wash removes proteins that do not bind to DNA. medium-salt wash elutes many different DNA-binding proteins.

Specific: wash DNA-binding proteins with specific sequence (GGGCCC). medium-salt wash removes non-bonding proteins. high-salt wash elutes rare protein that binds specifically to sequence (GGGCCC).

Question 23

Describe CHIP, used to ID DNA binding sequences.

Answer 23

Chromatin Immuno-Precipitation (CHIP) is used when you know protein, but don’t know what region of DNA it binds to.

Cross-link proteins to DNA with formaldehyde, lyse cells, break DNA into small fragments, precipitate DNA using antibodies against gene regulatory protein, reverse formaldehyde cross-links, remove protein from DNA, amplify precipitated DNA by PCR.

Question 24

The gene control region contains what elements?

Answer 24

Includes DNA of the promoter (site of assembly of xcr factors and RNA Pol II) and regulatory sequences (binding regulatory proteins)

Question 25

Of the 21,000 human genes, this percentage encodes regulatory proteins.

Answer 25

9% (2,000 genes)

Question 26

Other gene regulatory proteins (activators or repressors) bind to regulatory sequences which can be located where?

Answer 26

Adjacent to the promoter, far upstream, or introns downstream of the promoter.

Question 27

The ___ serves as an intermediary between gene regulatory proteins and RNA Pol II

Answer 27

mediator.

Question 28

Describe the 4 ways DNA gets modified by Gene Activator proteins. These modifications favor xcr by increasing accessibility of ___ to proteins.

Answer 28

1. Nucleosome remodeling
2. Nucleosome removal
3. Histone replacement
4. Histone modification (acetylation loosens up histone, making them easier to remove).

DNA.

Question 29

What are the 6 ways that gene repressor proteins inhibit transcription?

Answer 29

1. Activator and repressor compete for the same binding site.
2. Both proteins bind to DNA but the repressor binds to and masks the activation domain of the activator protein, keeping it inactive.
3. The repressor binds to DNA, DNA folds back and repressor blocks assembly of general xcr factors.
4. The repressor recruits a chromatin remodeling complex, returning the promoter to the pre-xcr nucleosome state.
5. The repressor attracts a histone deacetylase to the promoter. Deacetylated histones are harder to remove in order to open up DNA.
6. The repressor attracts a histone methyl transferase which methylates histones. methylated histones bind to proteins, keeping chromatin in a transcriptionally silent form.

Question 30

Gene regulatory proteins come together to form ___ on DNA.

T/F: The same regulatory protein can be part of an activating complex or repressing complex.

Answer 30

Complexes.

True.

Question 31

Name the 7 ways gene regulatory proteins are activated.

Answer 31

1. Protein Synthesis.
2. Ligand Binding.
3. Covalent Modification (phosphorylation)
4. Addition of subunit.
5. Unmasking (inhibitor)
6. Nuclear Entry
7. Proteolysis (release from membrane)

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Question 32

Adult Hb is what type(s)?

Embryonic Hb is what type(s)? Fetal Hb is what type(s)?

Answer 32

Adult = alpha and beta.

Embryonic = zeta and epsilon.

Fetal = alpha and gamma.

Question 33

T/F: Beta-globin genes are arranged in 5’ to 3’ direction in the same sequence of activation and expression during development.

Answer 33

True.

Question 34

Sickle Cell Disease involves a sickle ___ globin. One possible cure could be what?

Answer 34

Sickle Beta Globin.

Convert back from Adult Hb to Fetal Hb would be a cure. Better understanding of beta-like globin gene regulation could facilitate the cure.