Week 4 - Gene Transcription Effect

Question 1

How do RNA polymerases work?

Answer 1

• all require many transcription factors
• to modify chromatin structure
• so polymerase can access DNA
• RNA is made from 5’ to 3’
• template followed is from 3’ to 5’

Question 2

What is the structure of a gene?

Answer 2

• promoter: DNA region RNA polymerase binds to initiate transcription
• startpoint: DNA position corresponding to the first base incorporated into the RNA (+1)
• terminator: DNA sequence that causes RNA polymerase to terminate transcription
• transcription unit: sequence between initiation and termination sites

Question 3

What are the roles of each of the transcription factors?

Answer 3

DABFEH: DAB For Every Human

• TFIID: binds to TATA element and deforms promoter DNA
• contains 1 TBP (Tata Binding Protein) and 14 TAFs subunits
• TFIIA: stabilises TBP and TFIID binding
• TFIIB: stabilises TFIID-promoter binding
• TFIIF: binds to RNA polymerase II and recruits it to the pre-initiation complex
• TFIIE: helps recruit TFIIH to promoter and required for promoter melting
• TFIIH: functions in transcription and DNA repair

Question 4

What is the transcription initiation process?

Answer 4

1. TBP bends TATA box around C-terminal domain of TFIIB
2. its N-terminal brings the complex to RNA polymerase II
3. TFIIB positions initiation site in polymerase’s active site
4. polymerase and TFIIB complex recruits TFIIE
5. new complex recruits TFIIH
6. TFIIH helicase activity unwinds DNA near initiation site
7. TFIIF captures non-template strand
8. template strand descends to active site

Question 5

How does elongation occur?

Answer 5

• TFIIH has cyclin-dependent protein kinase activity
• ser-5 phosphorylation (in RNA II) permits promoter clearance
• ser-2 and ser-7 phosphorylation during elongation
• dephosphorylated after each round
• elongation can be delayed
• to terminate it
• or in arrest for proofreading
• can be reactivated by TFIIS

Question 6

What are the properties of enhancers?

Answer 6

• can act at a distamce
• are orientation-independent
• can be positioned up/downstream of initiation site
• can be cell-type or tissue-specicific
• transcription activator proteins must bind for full gene expression
• turns on gene expression by interacting with PIC (Pre Initiation Complex) or promoting euchromatin

Question 7

What are the properties of silencers?

Answer 7

• mostly distance and orientation independent
• transcriptional repressors
• establish heterochromatin, block PIC formation, stop activators from binding

Question 8

What is the hypoxic response?

Answer 8

• HIF1 contains HIF-1α and HIF-1β
• HIF-1β in excess
• response of HIF1 depends on HIF-1α protein levels
• in well-oxygenated conditions HIF-1α bound by VHL (Von Hippel-Lindau) protein
• VHL recruits ubiquitin ligase
• ligase targets HIF-1α for degradation
• VHL binding dependent on proline hydroxylation in HIF-1α by PHD2 (Prolyl Hydroxylase)
• PHD2 uses O₂ as substrate

Question 9

What else impacts PHD activity?

Answer 9

• mutations in the TCA cycle components succinate dehydrogenase or fumarate hydratase
• accumulation of succinate and fumarate
• these metabolites inhibit PHD2 activity competitively
• PDK1 encodes PDH kinase 1
• phosphorylates and inactivates PDH
• thus inhibits pyruvate to acetyl coA conversion
• LDHA encodes lactate dehydrogenase A
• converts pyruvate to lactate

Question 10

What is the effect of KEAP1 on NRF2?

Answer 10

• a sensor of ROS
• contains highly reactive cysteines
• cysteine modification by electrophilic molecules
• prevent KEAP1 from targeting NRF2 for degradation
• NRF2 regulates expression of genes that contain an enhancer termed ARE (Antioxidant Response Element)

Question 11

What is the overall effect of activating NRF2?

Answer 11

increase in:
- mitochondrial membrane potential
- ATP levels
- respiration rate
- oxidative phosphorylation efficiency