Lecture 17 - Regulation of transcription

Question 1

How do transcription factors activate transcription?

Answer 1

• contacts with the basal transcription machinery

- chromatin modification

Question 2

How did scanning experiments identify the DNA elements (activators) in the β globin promoter?

Answer 2

• mutation of every base 100bp upstream of the start point revealed 3 sequence elements that were critical for transcription
  1. TATA box (~25-30)
  2. CAAT box (-75) [mutations have a strong affect on promoter strength]
  3. GC box (-90)

These are found in the promoters of many genes (mot necessarily in the same location

Question 3

Give examples of how promoters have mix and match modules

Answer 3

SV40 early promoter
-promoter in a viral gene
-6XGC box regulatory element, followed by TATA
Thymidine kinase promoter
-uses a combination of sequences, octamer, GC, CAAT, GC, TATA
Histone H2B promoter
-octamer, CAAT, CAAT, octamer, TATA

Dependent upon the different regulatory regions, get different recruitment of different complexes

Question 4

How are transcription factors expressed?

Answer 4

1. Constituatively
   -some TF binding sites are found in many promoters
   -e.g. GC box recognised by SP1
2. Inducible
   a-tissue specific TFs involved in developmental regulation
   -e.g. Erythroid specific GATA factor
   b-some promoters are directly inducible rather than developmentally programmed
   -e.g. heat shock

Question 5

What are the different modalities of transcriptional activators?

Answer 5

Activators have independent DNA binding and activation domains

• shown by domain swap experiments
  1. DNA binding domain
• Zn finger
• H-T-H
• Leucine zipper
  2. Activation domain
• Acidic e.g. VP16, Gal4
• Glu rich e.g. SP1
• Pro rich e.g. CTF

Question 6

What is the process of the domain swap experiments done to identify the different domains of the transcriptional activators?

Answer 6

-took yeast transcriptional activators, LexA and Gal4 - both of which have an activating domain and DNA binding domain
-took DNA binding domain from Gal4 and the the Gal4activating domain this would initatie transcription (same for LexA)
-took the activating domain of Gal4 and made a mutant by combining it to the DNA binding domain of LExA
FOUND;
-in the presence of Gal4 promoter transcription was not acitvated (no opportunity to bind to Gal4 binding domain)
-in the presence of the LexA promotor it would bind to the DNA and initate transcription

Key conclusion: the DNA binding domain and transcription activating domains of activator proteins are independent molecules

Question 7

What was the key conclusion from the domain swap experiments?

Answer 7

Key conclusion: the DNA binding domain and transcription activating domains of activator proteins are independent molecules

Question 8

In what manner do TF function?>

Answer 8

TF function cooperatively

• 1TA give 1 unit of transcription
• 2TA give 10 units of transcription
• 4TA give 500 units of transcription

Not completely proportionate - only require limited numbers of TA to amplify the signal

Question 9

What to TA bind to?

Answer 9

Promoters and enhancers

Question 10

What are enhancers?

Answer 10

• regulatory sequences that act at a distance (up to several thousand bases away
• lack promoter activity themselves
• dramatically enhance the activity of promoters
• can be located either upstream or downstream of the promoter
• can act in either orientation
• can be composed of multiple sequence elements
• interact with mediator protein
• can have an effect on chromatin structure and mediate accessibility

Question 11

What is involved in the regulation of transcription?

Answer 11

Basal factors: Required by RNA pol II to form the initiation complex at all promotors
-protein:protein interactions stabilise
-important to transmit signal
Activator: binds to the DNA sequence elements and stimulates transcription
Co-activators: factors that required for transcriptional activation but do not directly bind DNA
-can’t activate on their own

Question 12

What is an insulator?

Answer 12

A sequence that prevents an activating or inactivating effect passing from one side to another
-an insulastor between an enhancer and a promoter results in no enhancement of transcription

Can also prevent the spread of heterochromatin

Question 13

What might mediate the action of enhancers to be limited to particular promoters?

Answer 13

Insulators

-stops the enhancer, which can act many bp away from the promoter, from acting on many many promoters

Question 14

How do insulators prevent the spread of heterochromatin?

Answer 14

Heterochromatin, which is highly compact, can spread as a repressive modulation e.g. methylation, can spread.
This is prevented by insulators

Question 15

What is The Mediator?

Answer 15

• a large modular complex (21 proteins) that conveys regulatory signals from gene specific activators and repressors to the core transcriptionl machinery
• physically interacts with RNApol II CTD
• how it transmits the signal from the activator to the polymerase is unknown
• can interact independently of DNA

Question 16

What are the ways in which chromatin can be remodelled?

Answer 16

1. Histone modifying complexes can make covalent modifications to histone proteins e.g. (+) charged histone is metabolised by acetyl transferase
2. ATP-dependent remodelling: uses the energy of ATP to alter chromatin in a non-covelent way, relies on ATP hydrolysis

Can be split into ATP dependent and independent mechanisms

Question 17

What are the ATP dependent and ATP independent chromatin remodelling activities?

Answer 17

ATP dependent

1. SWI/SNF (switch in sugar pathway) {tend to be activators}
2. ISWI (imitation switch) {tends to be activators}
3. Mi2 (methylation)
   - CHD (chromo-helicase/ATPase-DNA binding) {represses}

ATP-independent

1. HAT (Histone acetyl transferases)
   - GCN5 coactivator (yeast)
   - PCAF (P300/CBP associated factor) homolog of the yeast GCN5
   - P300/CBP coactivator (interacts with many different activators, required for activation)
2. HDAC (histon deacetylases - represses gene expression)

Question 18

What is involved after acetylation (histone modification)?

Answer 18

• Acetylated Lys is a target site of proteins containing bromodomains (bind)
• bromodomain: protein moiety that binds to acetyllysine
• interaction results in Bromodomain proteins binding to a specific chromosomal site
• can slightly move histones out of the way or remove completely
• bromodomain containing proteins are activators

Question 19

What is involved in activator-directed hyperactelyation of histone N-terminal tails?

Answer 19

Results in a more open chromatin structure

• upstream activating sequence is the binding site for activatior DNA binding domain
• GCN4-DBD binds to UAS and activation of the domain recruits GCN5, allowing GCN5 to bind to hyperacetylate histone N-terminal tails

Also can act as landing pads for ATP dependent chromatin remodellers

Question 20

What are the features of ATP-dependent chromatin remodelling?

Answer 20

• changes in chromatin range from slight changes in the position of the nucleosome to complete removal of the nucleosome
• there are several chromatin remodelling complexes that use ATP hydrolysis
• these complexes are very large and have some common subunits
• can be divided into three groups based on the ATP subunit they posess
• likely that ATP-dependent remodellers and covelent modifiers work together to regulate gene expression