MBB 267 Week 8: Mitchel 2

Question 1

What are the common features of transcription between eukaryotes and prokaryotes?

Answer 1

Transcription in prokaryotes and eukaryotes shares common features; initiation factors (s factor; gTFs respectively) target RNA polymerase to promoter regions.

Question 2

Difference between eukaryotic and prokaryotic transcription

Answer 2

Differences

• In prokaryotes, regulatory proteins directly target the polymerase.
• In eukaryotes, regulatory proteins impact polymerase indirectly via the mediator complex and by altering chromatin structure. Specific transcription factors (sTFs) can activate or repress transcription.

Question 3

What do functionally linked genes have in common?

Answer 3

Functionally linked eukaryotic genes are coordinately expressed under different experimental conditions. Groups of genes showing similar transcription profiles are called regulons. Coordinated regulation is typically achieved through the activity of sTFs that recognize promoter-proximal elements upstream of each gene of the regulon.

Question 4

How does the GAL gene switch work?

Answer 4

Galactose is converted to glucose in the absence of glucose, this is by a series of reactions that constitute to the lelior pathway

• The enzymes Gal1 , Gal7 and Gal10 are used to convert galactose to glucose
• Gal2 brings in galactose, Gal3 is a sensor protein = binds galactose
• Gal4 is used to promote the transcription of Gal genes by binding to UAS
• Gal80 is a repressor protein which binds to Gal4
• -If Gal3 binds to a galactose, then Gal80 will bind to it, preventing Gal80 from binding to to repress the production of the Gal promoter via Gal4. If Gal3 does not bind galactose, then Gal80 represses Gal4 by binding to

Question 5

What is the modular structure of transcription factors?

Answer 5

sTFs typically have a DNA-binding domain and one or more activation (or repression) domains, separated by flexible regions.

Question 6

How are protein interactions identified?

Answer 6

Yeast 2-hybrid assay was used to identify and analyse protein-protein interactions. The distinct activation domain and DNA-binding domains of Gal4 are fused to bait and prey proteins.
-If the prey and bait proteins interact, then a functional Gal4 protein is constituted, meaning that those domains bind to each other.

Question 7

What are the typical reporter genes used in hybrid assays?

Answer 7

Include

• HIS3
• ADE2
• LacZ

Question 8

How are protein interactions analysed using LacZ?

Answer 8

LacZ shows a chromogenic substrate that is used to demonstrate beta-galactosidase expression which can be seen by a change of colour from white to blue.

Question 9

How are protein interactions analysed using ADE2?

Answer 9

Yeast ade2 mutants develop a red pigmentation. ADE2 expression suppresses this phenotype.

Question 10

How are protein interactions analysed using HIS3?

Answer 10

HIS3 expression is used to complement a his3 auxotrophic allele. Increasing concentrations of the inhibitor 3- aminotriazole (3-AT) allow selection of clones that express higher levels of His3.

Question 11

How do transcription factors function?

Answer 11

Transcription factors typically function as homo- or heterodimers.

• Some heterodimers recognise the same DNA sequence – target genes are responsive to distinct combinations of activation domains under different conditions.
• Other heterodimers recognise different DNA sequences, increasing the number of potential targets.

Question 12

How is transcriptional control by repressors and activators achieved?

Answer 12

would interact with their cofactors. Transcriptional repressors are found bound to the promoter regions of actively transcribed genes as well as silenced genes. The combination of transcriptional activators and transcriptional repressors allows the expression of genes to be finely tuned rather than have a binary on/off response.