Module 3: Genetic and Epigenetic Gene Regulation

Question 1

Describe promotor-protein interactions

Answer 1

-35 region:
- RNA pol binding
- sigma factor binds here

-10 region:
- A-T rich sequence

Question 2

Under what conditions is the Lac operon active?

Answer 2

1. cell needs energy
2. glucose not available
3. lactose is available

Question 3

Describe the stringent response

Answer 3

Under good nutrition:
- sigma D binds to RNApol
- 60-80% of genes transcribed, transcripts are very stable
- proteins involved in translation and other housekeeping functions

Under poor nutrition:
- Sigma S binds to RNApol
- housekeeping genes suppressed
- 10% of genes induced (stringent response genes)

Question 4

Describe the function and properties of alarmone

Answer 4

• aka ppGpp
• binds to RNA polymerase
• reduces affinity of RNApol for sigma D
• sigma S and other stress sigmas can now compete for binding
• produced by RelA and SpoT
• RelA (amino acid starvation)
• SpoT (glucose starvation)

Question 5

What happens if glucose is not available (catabolite repression)

Answer 5

• cAMP is a specific glucose starvation signal
• cAMP binds to CRP
• Lac -35 sequence does not match consencus
• CRP and sigma S promotes strong binding of holoenzyme –> facilitates transcription

Question 6

What happens if lactose is available?

Answer 6

• allolactose signals lactose availability
• allolactose binds to lac operon repressor –> prevents inhibition of lac operon transcription

Question 7

Regulation by peptide hormones

Answer 7

1. receptor protein is embedded in the membrane of the target cell
2. peptide hormone binds to the receptor
3. cytoplasmic protein signal is activated
4. transcription factor in the cytoplasm is activated and imported into the nucleus

Question 8

Regulation by steroid hormones

Answer 8

steroid hormones are hydrophobic
1. pass through the membrane
2. binds to cytoplasmic receptor
3. receptor and hormone enter nucleus and acts as a TF that regulates gene expression

Question 9

Describe induction of heat shock genes

Answer 9

1. heat stress causes phosphorylation of HSTF (pre-made, but inactive)
2. HSTF binds to HSE and helps RNA pol II to initiate transcription of heat shock genes
3. transcribes Hsp70 genes
4. Hsp70 controls:
   - restoration of damaged proteins
   - stabilisation of membranes
   - changes to gene expression

induction of response genes in 30 seconds
suppression of housekeeping genes in 300 seconds

Question 10

Longevity inducers

Answer 10

result in increased longevity, decreased growth and reproduction:
1. decreased temperature
2. decreased mitochondrial function
3. amino acid deficit
4. decreased insulin signalling (glucose deficit)
5. lack of food (dietary restriction)

Question 11

Structure of core promotor

Answer 11

• RNA pol II preinititiation complex assembly site
• 30-100 bp upstream of transcription start
• “TATA” box found in 25% of promotors

Question 12

Proximal element structure and function

Answer 12

• sites of binding for activator proteins
• immediately upstream of promotor
• lose activity if moved further upstream
• increase promotor activity

Question 13

Enhancer element function

Answer 13

• site of binding for specific transcription factors
• upstream of promotor and proximal control elements

Question 14

Describe the experimental analysis of beta glucuronidase

Answer 14

GUS = beta glucuronidase
if GUS expressed, cell turns blue

1. use a progressive 5’ deletion
   - this deletes successive upstream regulatory regions
   - if pollen grain continues to turn blue, core promotor is still in tact
   - when pollen grain is colourless, this is where the core promotor lies in the gene

Question 15

Describe basal transcription factors in gene regulation (core promotor protein)

Answer 15

Assembly:
- TFIID binds to TATA box and bends DNA sharply
- other basal factors assemble
- RNA Pol II holoenzyne binds to TFIID –> bends the DNA
- initiates DNA strand separation –> promotes ‘melting in’ of RNA pol II

Question 16

Proximal element binding proteins

Answer 16

Activator proteins:
- important for maximal expression of housekeeping or regulated genes

Question 17

Enhancer binding proteins

Answer 17

• sites of binding for specific TFs
• precise control of gene regultion
• activity of enhancer is mediated through proteins that bind to it

Question 18

Transcription factor structure

Answer 18

1. DNA binding domain (recognises DNA sequences)
2. dimerisation domain
3. activation domain (interacts with regulatory proteins)

Question 19

Promotor Bendin

Answer 19

Mediator complex:
- bends DNA of a regulatory region
- brings distal elements to the promotor

Question 20

H3K9 acetylation

Answer 20

gene active

Question 21

H3K9 methylation

Answer 21

gene inactive

Question 22

H3K4 methylation

Answer 22

enhances active state

Question 23

Methylation of repetitive DNA

Answer 23

1. heterochromatin
   - centromeric and telomeric DNA highly methylated
   - highly condensed
2. dispersed repeats
   - viruses and transposons
   - inactivated by methylation
   - become reactivated when host under stress

Question 24

DNA imprinting

Answer 24

• occurs in gametes
• genes imprinted in sperm are never imprinted in oocytes and visa versa
• genes are differentially inactivated by methylation in gametes
• imprinted genes observed as mutant phenotypes that depend on parent of origin
• if gene is imprinted - dominance = none

Question 25

Four types of DNA methylation

Answer 25

1. dynamic gene regulation - erased in early embryogenesis, re-established during lifetime 2. imprinting - erased and re-established during early gamete formation 3. suppression of transposons (by piRNA signalling) - maintained indefinitely - erased under stress 4. X chromosome inactivation - one X chromosome is inactivated in each cell (in females)

Question 26

Describe co-suppression

Answer 26

- When a normal gene and a transgene are both suppressed - suppression can spread to non-transgenic tissue --> RNA mediated - causes a loss of flower pigmentation - suppressed plant transgenes were methylated (transcription was inhibited) or transcripts were degraded

Question 27

siRNA

Answer 27

- externally introduced dsRNA - directs proteins to degrade homologous mRNA - signal can spread to other cells - also mediator of co-suppression

Question 28

miRNA

Answer 28

- natural cellular transcript - blocks tranlation

Question 29

piRNA

Answer 29

- transcript degradation or chromosome remodelling - suppresses transposons in the germline

Question 30

Describe RISC

Answer 30

1. a large dsRNA is diced into small double stranded interfering RNAs 2. small interfering RNAs and proteins assemble into ribonucleoprotein particles 3. small interfering RNA i a particle is unwound to produce an RNA-induced silencing complex 4. RISC targets a sequence in a mRNA that is complementary to the interfering RNA 5. RISC interfering RNA base pairs with its target in the mRNA 6. if perfectly base-paired, the mRNA is cleaved and degraded 7. if imperfectly base-paired, translation of mRNA is blocked