Eukaryotic Transcription Factors

Question 1

What is metazoa

Answer 1

A kingdom of eukaryota, also known as animals

Question 2

How do environmental cues affect eukaryotic and prokaryotic gene expression?

Answer 2

Lead to genes being differentially expressed during cell growth. Transcription is regulated by different factors

Question 3

What are promotors comprised of?

Answer 3

TATA box which is 30nt 5’ of the transcription start site (TSS).
mRNA binds to the TATA box and targets transcription factors to the promotor regions

Question 4

Where can constitutively expressed genes be transcribed from?

Answer 4

CpG islands. These are susceptible to methylation so are downregulated.

Question 5

What do mammalian genes typically comprise of?

Answer 5

Distal enhancers which can be up- or downstream of the promotor. These can be many kb away from the TATA box, so are difficult to map.
Promotor proximal elements which are close to the TATA box

Question 6

What do yeast genes typically comprise of?

Answer 6

Upstream activating/repression sequences (UAS/ URS) and a TATA box.

Question 7

How is the transcriptional start site (TSS) of a gene found?

Answer 7

Viral reverse transcriptase generates ssDNA from an RNA template. This uses DNA primers which extend 5’ to 3’ with viral RT
Primer extension products are analysed by southern blotting

Question 8

Outline deletion analysis of cells

Answer 8

1. Cell extracts are assayed for lacZ and luciferase activity
2. Deletions are then done on its plasmids
3. The plasmid is transfected into cells and activation of lacZ or luciferase are measured to find how critical the sequence is for gene expression

Question 9

Outline linker scanning mutagenesis for thymidase kinase gene

Answer 9

Proteins which recognise RNA polymerase activity bind to the TATA box and two promotor-proximal regions. This is used to find short regions in the promotor responsible for transcriptional control

Question 10

Outline enhancer trapping

Answer 10

Enhancers are defined by localisation
1. When weak promotors are integrated close to an enhancer, upregulation takes place. Reporter genes with weak promotors are randomly integrated into a genome with transposons
2. Clones showing upregulated expression are isolated and insertions sites are isolated and sequenced.
3. This can be used o determine the sequence of the enhancer

Question 11

How do sTFs regulated transcription in eukaryotes?

Answer 11

sTFs can up- or downregulate gTFs (general transcription factors) and the mediator complex. It does this by altering the chromatin structure of the mediator complex.

Question 12

What are regulons?

Answer 12

Groups of genes showing similar transcription profiles. (operons but in eukaryotes)

Question 13

What coordinates gene transcription?

Answer 13

sTFs that recognise promotor-proximal elements

Question 13

What coordinates gene transcription?

Answer 13

sTFs that recognise promotor-proximal elements

Question 14

Explain how presence of galactose in yeast leads to its conversion to glucose-6-phosphate

Answer 14

Galactose is converted to glucose-6-phosphate via the Leloir pathway.
1. Galactose binds to Gal3 which subsequently binds to a Gal80 dimer
2. The Gal80 dimer splits and one Gal80 enters the nucleus and switches on the activation domain.
3. Gal4 (a sTF) is activated by this and switches on the upstream activating sequence

Question 15

Explain how presence of galactose in yeast leads to its conversion to glucose-6-phosphate

Answer 15

Galactose is converted to glucose-6-phosphate via the Leloir pathway.
1. Galactose binds to Gal3 (gal sensor) which subsequently binds to a Gal80 dimer
2. The Gal80 dimer splits and one Gal80 enters the nucleus and switches on the activation domain.
3. Gal4 (a sTF) is then activated and binds to the upstream activating sequence of the GAL gene, initiating the Leloir pathway

Question 16

Why are yeast two-hybrid analysis done?

Answer 16

Analyse protein interactions

Question 17

What is gal4?

Answer 17

specific transcription factor of the upstream activating sequence (UAS)

Question 18

Outline yeast two-hybrid analysis

Answer 18

A reporter gene (e.g Gal4/His3/lacZ) is reconstituted when the bait and prey proteins of the DNA binding protein and activation domain interact. A positive result shows the gene being activated

Question 19

What is the antibiotic that HIS3 gives resistance to?

Answer 19

3-AT (3-aminotriazole)

Question 20

Why is combinational control important?

Answer 20

Allows greater variety of transcription. Transcription factors can act as homodimer or heterodimers to recognise multiple sequences at once.

Question 21

Why is gene expression obtained at a range of values as opposed to binary?

Answer 21

Transcriptional repressors and activators work together to finely tune gene expression levels of actively transcribed genes.

Question 22

What is the eukaryotic homeodomain fold comparable to in bacteria?

Answer 22

Bacterial repressors have a helix-turn-helix pattern. This interacts with the major groove of DNA.

Question 23

Which genes have a key role in anterior-posterior development?

Answer 23

Hox genes which encode homeodomain proteins

Question 24

What is the organisation of Hox genes like?

Answer 24

Clustered. Their order correlates with spatial and temporal expression. Homeodomain proteins function as dimers with the recognition helixes interacting on the opposite side of a helical turn in DNA

Question 25

Explain the structure of leucine zipper proteins

Answer 25

bZip proteins are a sTF family
Coiled dimers linked through parallel amphipathic α-helicies which contain Leu at every 7th position.
The α-helices are rich in basic residues so DNA can bind to the phosphate groups in adjacent major grooves

Question 26

What is the most common DNA binding fold in human cells?

Answer 26

zinc-finger proteins. Can mediate DNA/RNA and protein interactions

Question 27

Explain zinc-finger protein structure

Answer 27

β, β, α protein fold is centred around a central zinc ion.
A recognition helix binds to nucleotide triplets by its residues -1, 2, 3 and 6

Question 28

What combinations of zinc finger protein are there?

Answer 28

C4, zinc finger folds bind DNA as monomers
C2H2 zinc fingers bind as dimers and are found in nuclear receptors

Question 29

How do the sTFs NFAT and AP-1 bind to DNA?

Answer 29

The both have weak binding activity to a 15nt sequence. Recognition sites in DNA have to be spaced out specifically
A heterodimer is formed by weak interactions between NFAT, AP-1 and DNA.

Question 30

Why do RNA binding proteins have synergistic specificity?

Answer 30

Most RBPs have multiple copies of structural domains which allow this specificity

Question 31

The RNA recognition motif (RRM) is a common eukaryotic domain. Describe its structure

Answer 31

Found in multiple copies or with other RNA binding domains
Interacts with 3 or 4 residues
2 aromatic side chains stack between hydrophobic residues in central β sheets and bases
They can also stack between hydrogen bonds of basic residues and phosphodiester bonds

Question 32

What are gel shift assays used for?

Answer 32

Detecting nucleic acid binding activity. Done with 32P labelled DNA or RNA and protein

Question 33

Give an example of an activator/coactivator interaction

Answer 33

In a GPCR response, the sTF, CREB, is phosphorylated by protein kinase A. This allows CREB to bind to its cofactor, CREB binding protein.

Question 34

Give an example of an activator/coactivator interaction where nuclear receptors act as a sTF

Answer 34

Oestrogen binds to nuclear receptors, causing coactivators to bind to the ligand binding domain. This leads to gene transcription.