Lecture 18 - Regulation of transcriptional regultors, RNAPI and III, elongation and termination of transcript, ChIP Flashcards
What two categories of regulation are involved in regulating the regulators of eukaryotic gene expression?
- Regulated synthesis of transcription factors
- Regulation of transcription via transcription factor heirachy - typical of factors that regulate development
- regulation of splicing
- regulation of tranlsation - Regulted activity of transcription factors (when TF already present but held so that it doesn’t have access to DNA - rapid response)
-Activation by preotein-ligand or ptoein-protein interactions;
Ligand binding
Dissocation of an inhibitor proetin
Protein modificaton e.g. phosphorylation
cleavage of a precursor
What are the different activation mechanisms involved in regulating the regulators of gene expression?
Protein synthesis e.g. Homeo-protein
Phosphorylation e.g. HSTF
Dephosphorylation e.g. repressors
Ligand binding (transports TF into nucleus) e.g. Steroid receptors
Release inhibitor e.g. NF-KB
Change partner e.g. HLH (MyoD/ID)
Cleavage releasing active factor e.g. Sterol response
What is involved during the induction of heat shock genes?
-accompanied by the binding of a protein HSF to the HSE
How does HSF stimulate transcription upon heat-shock?
- HSF is present in unstressed cells
- it can activate transcription of heat shock genes even in the presence of inhibitors of protein syntheis
- a temperature dependent change from monomer to a trimer followed by phosphorylation that allows it to bind to the HSE act activate transcription
- this prevents constituative binding but constant presence in unstressed cells allows quick response
What is the process of activation of the HSF?
Disruption of a protein-protein complex and phosphorylation
- under conditions of no stress, HSF-1 interacts with the hsp90 protein
- under heat stress, this splits the interaction, releasing HSF-1 as a monomer
- this allows it to trimerise with 2 other HSF-1’s and bind to DNA at the HSE
- phosphorylation activates the bound trimer leading to transcriptional acitvation
What is the NFKB family of TF involved in?
- NFKB transcription factors involved in the immune response
- e..g viral infection
- therefore needs to be available quickly - not synthesised from scratch
How does NFKB regulate gene expression?
- NFKB normally stored in the cytoplasm where the NFKB dimer interact with IKB - an inhibitor of NKFB
- upon viral infection/induction of a signalling cascade, lKB is phosphorylated, releasing it from the NFKB dimer before being ubiquitinated and destroyed
- NFKB is now free to be impoted into the nucleus and be an activator of transcription
What is involved in the preinitiation complex (PIC)? What causes it to become the initation complex and enter the elongation phase?
PIC -TFIIA -TFIIB -TFIIE -RNA pol II -TFIIH -TFIIF -at the TATA box IC -ATP hydrolysis and loss of TFIIE -TFIIH phosphoryates the CTD then causes further DNA unwinding allowing the bubble to grow and RNAp to go into elongation phase
What are the features of the RNA pol II CTD?
- repeating heptapeptide sequences
- 52 repeats human
- 27 in yeast
- tyr-ser-pro-thr-ser-pro-ser
- target for phosphorylation
How does elongation begin?
- RNA pol II is phosphorylated at ser5 to initate enlongation (by TFIIH)
- RNA pol Ii is phosphorylated at ser2 after +50bp during elongation (by CTDK-1)
- RNAP makes short RNAs, but stalled at +10 - +12
What phosphorylation mechanism is required for the termination of elongation?
Removal of phosphorylation of RNA pol II at ser5 and ser2
When does the first essential pause of RNA pol II occur in transcription ? Why does it pause?
between +10 and +12 sites Obstacles: -nucleosomes -bound transcription factors -specific pause sites
What does phosphorylation of RNA pol II CTD by TFIIH and CTDK-1 promote?
RNAPII phosphorylation at ser5 and ser2 promotes interaction site for mediator, elongation factors, RNA processing proteins, transcription terminatior, chromatin modifiers etc.
What is the phosphorylation of RNA pol II CTD stripped by at elongation termination?
Phosphatase and Ser5P
-this recylcles RNA polymerase
What is the process of phosphorylation mechanisms invplved in elongation?
- RNAPII at the promoter is non phosphorylated [pre-initiating RNAPII]
- TFIIH phosphorylates ser5, melting begins, and the RNAPII moves off the promoter as the CTD is no longer bound [initiating RNAPII]
- RNAPII makes short sections of RNAs as melting continues and the Ser2 of the CTD is then phosphorylated by CTDK-1 [elongating RNAPII]
- After elongation, Ser5P and phosphotase dephosphorylate the CTD and elongation terminated [terminating RNAPII]
