Lecture 9: Control of Gene Expression in Eukaryotes: Part II and III

Question 1

Describe overview of the ways that transcription can be stimulated by activators

Answer 1

Activation by recruitment
- activation domain interacts with one or more component(s) of the basal transcriptional machinery and stabilizes its binding to the template DNA
Activation by conformational change
- activation domain induces a conformational change in a component of the basal transcriptional machinery to stimulate RNAP II to initiate transcription
Activation by altering chromatin structure
- activation domain recruits chromatin remodelling/modifying proteins (coactivators) to modulate chromatin structure around promoters

Question 2

Recall the different activation domains and the factors that they contain

Answer 2

• acidic region- yeast GCN4, GAL4, nuclear receptos
• glutamine-rich regions- SP1, Oct1,AP2
• Proline-rich domain-CTF/NF1, AP2, c-jun,Oct2

Question 3

Explain how activators interacting with TFIID can stimulate transcription

Answer 3

• Enhances the binding of TFIID to TATA box-> improve rate of PIC assembly
• > Interaction is through TBP or one or more of TAFs
• Activators alters conformation of TFIID -> stimulating its activity
  = increasing TFIID ability to recruit other PIC components
  =enhancing ability to stimulate transcription

Question 4

Explain the different of activation by recruitment via binding to TFIID in TATA-less promotors

Answer 4

• Different activators target different TAFs
• Different cell types can have cell type-specific TAFs
• TAFs -critical intermediaries between activators and the basal transcription complex

Question 5

Recall the different activate domains of activators and their TAFs

Answer 5

Acidic->TAFII31
Glutamine-rich ->TAFII110
Proline-rich-> TAFII55

Question 6

Explain how activators interacting with TFIIB can stimulate transcription

Answer 6

• Allows stimulation of assembly of PIC/activity of basal transcriptional complex after TFIID has bound
• TFIIB interacts with acidic activators
• Activators can stimulate binding of TFIIB to the promoter and can also alter its configuration when bound-> improve its ability to recruit other components of the PIC – TFIIF+RNAPII

Question 7

Explain how activators interacting with RNAP II can stimulate transcription

Answer 7

through the mediator complex

• CTD of RNA pol II interacts with the mediator complex ->required for the response to transcriptional activators
• Mediator forms a molecular bridge between activators and RNA pol II

Question 8

Describe the mediator complex

Answer 8

• Approx. 30 subunits
• Made up of 3 subcomplexes: the head, middle and tail
• Head interacts with RNA pol II CTD
• Tail interacts with activators
• Different classes of activators interact with different mediator subunits

Question 9

State the principle that governs the function of all activators

Answer 9

DNA-binding domain determines specificity for the target promoter or enhancer

Question 10

State the responsibility of the DNA-binding domain of activators

Answer 10

making transcription-activating domain close/local to basal apparatus/transcription machinery

Question 11

Explain how activators can work directly and indirectly

Answer 11

• An activator that works directly-> has DNA-binding domain +activating domain within the same protein that affects transcription directly
• An activator that doesn’t have activating domain ->work by binding to a co-activator that has an activating domain
• OR activator can have activator domain which doesn’t act directly on transcription
• > acts through the recruitment of co-activators

Question 12

Explain how co-activators interact with DNA

Answer 12

• A co-activator doesn’t bind DNA directly
• > interacts with a DNA-bound transcriptional activator = transmit the signal to the basal transcription complex
• > e.g. CREB-binding protein (CBP/p300) is co-activator -recruited to DNA by the DNA-binding transcription factor partner CREB
• CREB-binding protein (CBP) is a histone lysine acetyltransferase
• Co-activators mediates transcriptional activation -> via a number of other DNA-binding transcription factors partners involved in a variety of signalling pathways

Question 13

Explain how GNC4 stimulates transcription

Answer 13

• GCN4 = transcriptional activator-> binds to upstream enhancer (UAS) -via DNA-binding domain
• GCN4 activation domain-> interacts with GCN5 HAT protein complex (co-activator)
  = allows access of PIC to DNA + transcriptional start sites =by opening up chromatin

Question 14

Describe how HAT stimulate transcription

Answer 14

• HATs recruited to promoters as co-activators by activators (don’t have active domain)
• Activate transcription -> by opening up/ ‘loosening’ chromatin OR by recruitment of the basal transcriptional machinery

Question 15

Explain how readers of Histone Lysine Acetylation can stimulate transcription

Answer 15

• Acetyl-lysine proteins recognised by binding protein family that contains bromodomains (BD in TAFII250)
• TAFII250 = part of TFIID
• > transcription is promoted by recruitment of the PIC

Question 16

Describe what weaken acetylated histones do

Answer 16

• Facilitate access of an activator to DNA
• Recruitment of activators that recognise and bind to acetyl-lysine
• Promote nucleosome displacement from promoter in an energy-dependent process

Question 17

Explain the properties of remodelling complexes

Answer 17

• All remodelling complexes -> contain related ATPase catalytic subunit
• ATPase catalytic subunit grouped into subfamilies
   Have more closely related ATPase subunits
• Remodelling complexes-> alter, slide, or displace nucleosomes
• Some remodelling complexes-> exchange 1 histone for another in a nucleosome

Question 18

Explain the activity of remodelling complexes as co activators

Answer 18

• A remodelling complex= co-activator
• > doesn’t have specificity for any particular target site
• > MUST be recruited by activator
• recruited to promoters by sequence-specific factor (=activator)
• Factor released once remodelling complex bound
• Transcriptional activation -> involves nucleosome displacement at the promoter

Question 19

Explain how SWI-SNF Chromatin Remodelling Complex works to stimulate transcription

Answer 19

• SWI-SNF -> acts to alter chromatin structure of these genes
  = facilitating subsequent activation by exposing DNA- binding site for other transcription factors to bind (‘Y’) (activator= ‘X’)

Question 20

Explain the general path that can be taken for activation of transcription

Answer 20

• Enhancer has activator bound
• co-activators recruited
• acetylation of Lys residues
• histone eviction OR partial PIC
• recruitment of TFs OR histone eviction + promotor escape

Question 21

Explain how transcription activation is synergistic

Answer 21

• > when one activator binds to one enhancer= effect is less
• > when both activators bind to their enhancer= combined effect
• Different activators can recruit different co-activators-> stimulate transcription