transcriptional control in eukaryotes

Question 1

What two characteristic features exist at common promoters in eukaryotes?

Answer 1

TATA box and CpG islands

Question 2

How can the methylation of cysteine affect promoters ability?

Answer 2

Methylated cysteine in CpG islands decreases transcription

Question 3

How can you map the transcriptional start site of a eukaryotic cell?

Answer 3

-viral reverse transcriptase can generated ssDNA from RNA template
-Anneal short primers to ssRNA
-Extend 5’-3’ by reverse transcriptase
primer extension products can be analysed by gel electrophoresis or by SDS PAGE

Question 4

Discuss deletion analysis of promoter regions

Answer 4

Use lacz or luciferase as reporter gene
assay cells for lacz activity
clone promoter of interest in front of the reporter gene
create promoter constructs with deletions to find out which regions are regulatory

Question 5

Discuss linker scanning mutagenesis

Answer 5

After deletion analysis, identify the specific sequences important for regulation
mutate promoter at different sites along the promoter and amplify by PCR. Which mutation sites affect the ability of translation

Question 6

Discuss the formation of the preinitiation complex at the promoter

Answer 6

• TATA box binding protein (TB) (in TFIID) binds the TATA box sequence in the minor groove of DNA
• TFIID binds TFIIB, and pol2 binds the TFIIDB complex.
• TFIIF binds.
• This cause TFIIE and TFIIH to bind, the helicase activity of TFIIH allows the dna to unwind ready for priming.

Question 7

What are regulons?

Answer 7

Regulons are genes with similar transcriptional profiles.

Question 8

Describe the effects of combinatorial control

Answer 8

The recruitment of different combinations of sTFS which interact with various enhancer regions can generate a variation in transcriptional response. This provides the complexity of transcriptional regulation.

Question 9

Discuss how a Yeast-2 Hybrid assay could determine protein-protein interactions

Answer 9

The distinct DNA binding domains and activation domains of the transcriptional activator Gal4 can be separated at the linker region and fused respectively to a bait and prey protein.
If the bait and prey interact, a functional Gal4 will be formed and this will induce expression of the Gal gene. A variety of reporter genes can be used to assay the expression of the Gal gene.

Question 10

Discuss the use of X-Gal in a yeast 2 hybrid assay

Answer 10

X-gal is a chromogenic substrate which can be used to demonstrate b-galactosidase activity. Lacz used as reporter gene will produce active b-galactosidase, causing the breakdown of X-gal leading to a blue product.

Question 11

Discuss the use of His3 in a yeast 2 hybrid assay

Answer 11

His 3 gene can be used to complement his3 mutants. An increased level of 3.AT inhibitor will allow selection for clones with a higher complemented phenotype (more expression of His3 gene)
This technique is useful for filtering out false positives

Question 12

Discuss the use of ade2 mutants in yeast 2 hybrid assay

Answer 12

ade2 mutants will develop a red pigment in yeast. Fuse Ade2 (wild type) as reporter gene to Gal site. If Gal is expressed, wild type Ade2 will suppress the mutant phenotype and no red pigment will be shown.

Question 13

What is Hmg1?

Answer 13

HMG1 is a protein which binds DNA in the minor groove promoting bending. Used in conjunction with other control elements at enhancer regions on DNA

Question 14

What is a specific example of the use of HMG1?

Answer 14

HMG1 assists in the enhancer complex b-interferon. This is a virally inducible complex, consisting of 3 distinct heterodimers, which cooperatively bind to adjacent regulatory elements.

Question 15

Briefly discuss how you can isolate specific transcription factors

Answer 15

Generate a haploid recessive mutant (cloned by complementation) - eg yeast gal- mutant cannot grow on galactose medium.
Clone wild type genes from the library through complementation.
Genes encoding factors essential for growth on galactose can be isolated from transformants with a complemented phenotype

Question 16

Describe the initiation and regulation of the lecoir pathway in yeast

Answer 16

Gal gene switch;
to utilize galactose as a substrate, yeast require gal2 permease, gal1 gal7 and gal10 structural gene expression.
This expression is regulated by transcriptional activator Gal4.
Gal3 senses galactose as a metabolite
Gal 3 binds to Gal80 (repressor protein) and promotes dissociation, reducing repression.

Question 17

Discuss the function of a gel shift assay, and briefly discuss the steps

Answer 17

A gel shift assay can be used to detect DNA protein binding activity.
-mix fractionated nuclear extract (contains DNA biding proteins) and DNA extract.
-Electropherese under non-denaturing conditions so that any protein dna complexes can remain stably bound.
Protein/DNA complexes will have a lower electropherecic mobility. (ie go slower)

Question 18

How can the gel shift assay be further refined?

Answer 18

Super-shift assay incorporates antibodies specific to binding proteins. If an antibody, protein, DNA complex forms, this will migrate even slower.

Question 19

Discuss a transfection assay of transcription factor activity

Answer 19

This assay occurs in vivo,
plasmids with cloned transcription factors and a reporter gene with cognate stf binding site and transformed into cells.
the amount of reporter mRNA is measured.
This allows mutational analysis of sTF

Question 20

What are the key features of a homeodomain fold in a DNA binding protein

Answer 20

Homeodomain fold is similar to the helix-turn-helix structure in bacterial repressors.
associated with morphogenesis
the genes encoding these proteins found in the hox cluster. Essential in anterior and posterior axis development - order of clusters reflects spatial expression in the embryo

Question 21

What are the key features of Zinc fingers in DNA binding proteins?

Answer 21

Zinc fingers comprise 2 beta sheet and one alpha helix, coordinating a central zinc ion.
The recognition helix binds a nucleotide triplet in target DNA via interactions with positions -1,2,3 and 6 in the helix.

Question 22

What is the difference between DNA binding of C2H2 and C4 zinc fingers?

Answer 22

C2H2 binds as a monomer, usually has multiple zinc fingers

C4 binds as dimers, found in nuclear receptors.

Question 23

What are the key features of Leucine Zippers?

Answer 23

These are coiled coil dimers, linked through parallel amphipathic helices.
Leucine residues at every 7th position. The Leu residues contribute to the hydrophobic interactions between the two helices.

Question 24

How do bZIPS mediate DNA binding?

Answer 24

Leucine zipper allows dimerization of the protein.
The region rich in basic residues allows DNA binding - both through interactions with the phosphate backbone and base specific interactions.
The extended alpha helices grip DNA at the adjacent major groove.

Question 25

Discuss the oestrogen binding to nuclear receptor

Answer 25

Conformational change occurs when oestrogen binds to its receptor, allowing interactions with coactivator.
This generates a hydrophobic pocket which can bind the amphipathic helix of coactivator.

Question 26

Briefly discuss the use of sTF CREB

Answer 26

CREB is phosphorylated by the catalytic subunit is protein kinase A.
When CREB is phosphorylated, it can bind to coactivator CBP.
Activated CREB also binds CRE(creb response elements) found upstream of genes which are dependent on cAMP