L22 Gene regulation by transcription initiation 2

Question 1

Gal utilisation in yeast

Answer 1

see onenote

Gal non-utilising mutants
- gal- 
- structural genes: GAL1,2,7,10, MEL1 
- regulatory genes:
GAL4,3,11,80

Mutations in these genes only affected one function

These structural genes identified by mutagenesis, affected one of these steps in galactose metabolism

Question 2

Galactose regulon

Answer 2

see onenote

• Utilisation and adaptation
• ChIP

Galactose also affected other genes

FUR4 involved in bringing uracil into the cell, UDP would be depleted if there was a mutation in FUR4. UDP required for core reaction of galactose metabolism.

Much bigger regulon of genes controlled by galactose

Question 3

Gal utilisation - regulatory genes

Answer 3

see onenote

GAL4,3,11,80 = affected many of the genes required for gal utilisation

Question 4

GAL4

Answer 4

gal4- = recessive, non-inducible, common

gal4c = dominant, constitutive, rare

positively-binding regulator

DNA binding, activate gene expression

Question 5

GAL80

Answer 5

gal80- = recessive,
constitutive, common

gal80s = dominant, non-inducible, rare

negatively-acting regulator

block gene expression in absence of inducer

Question 6

Double mutants

Answer 6

see onenote

gal4- gal80- = non-inducible, Gal4 required for activation, Gal80 acts through Gal4

gal4c, gal80- = constitutive, gal80 acts through gal4

gal4c, gal80s = constitutive, gal80 acts through gal4, gal4c blocks gal80s inhibition

gal4 downstream of gal80

Question 7

Mutant phenotypes

Answer 7

see onenote

gal4- = no activator
GAL4c = no interaction with gal80
gal80- = no repressor
GAL80s = hyperactive repressor 

gal4 and gal80 physically interact in the nucleus

Question 8

GAL1,10,7

Answer 8

see onenote

Gal1 and gal10 share regulatory regions

UAS can consists of multiple binding sites, not common, need a number of TF to turn on high level of gene expression

Question 9

GAL4 binding

Answer 9

• binds as a dimer

Each gal4 binding sequence consists of a generalised sequence: CGG at one end and CCG at the other end with any 11 nucleotides in the middle

Question 10

GAL3

Answer 10

see onenote

• signalling molecule that binds galactose
• binds gal80 when bound to galactose, relieves gal80 mediated inhibition
• cytoplasmic localisation

BUT gal3 cytoplasmic but gal80 is nucleo

gal3- = recessive, slower induction, common

GAL3-
- Induction still happened but expression of gal genes activated at a much slower rate, not reduced induction just slower

Question 11

GAL11

Answer 11

see onenote

Gal11 is a component of the mediator complex, bridge potential binding sites e.g. enhancers with promoter of gene and RNA pol

interacts with gal4 directly

gal11- = recessive, induced reduction, common

GAL11P

GAL11-
- Absolute level of induction was lower e.g. from 1-800, was 1-400

Question 12

Two models of regulation of Gal4 by Gal80 and Gal3

Answer 12

see onenote

Question 13

Gal utilisation overview

Answer 13

see onenote slides

GAL4 always sitting on UAS and GAL80 controls it, GAL3 sits in cytoplasm
GAL3 controls GAL80

SAGA complex, regulatory of many genes

Question 14

Regulatory proteins

Answer 14

see onenote

DBD proteins can:

• recruit proteins for repression as well as activation
• recruit proteins to control transcription initiation ie. co-activator or co-repressor

Glucose represses GAL gene expression

• glucose results in Mig1 binding
• recruitment of Tup1-Ssn6 co-repressor

Question 15

Regulatory proteins - synergy between activators

Answer 15

see onenote

applies to binding DNA as well as activation of transcription by recruitment of RNA pol

Question 16

Negative control mechanism

Answer 16

see onenote

1. masking the activation complex
2. competitive DNA binding
3. direct interaction with general TF

Question 17

Regulatory proteins - modular in function

Answer 17

see onenote

• DBD recruit AD to DNA
• AD recruit TAFs, mediator or other factors
• these in turn, recruit RNA pol2 complex

Question 18

Mediator

Answer 18

multi subunit complex that associated with transcription apparatus

Question 19

Regulatory proteins - chromatin

Answer 19

see onenote slides

• DBD proteins can recruit chromatin modifying complexes to control transcription initiation

Enzymes that modify histones, modifying chromatin state: gene more/less accessible to being transcribed

Question 20

Regulatory proteins - modular in structure

Answer 20

see onenote slides

• DNA binding domain can be separated from other domains such as activation

Question 21

Yeast two hybrid assay

Answer 21

see onenote

BAIT = DBD with target protein

PREY = transcription AD with binding partner

if successful => reporter protein produced

Question 22

Regulatory genes are not species specific

Answer 22

see onenote

Gal4-UASg system in Drosophila

Core transcription machinery conserved in all organisms