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]
What is the differemce in the rates of transcription in vivo compared to in vitro?
In vivo -1200-2000 nucl/min In vitro -100-300 nucl/min -frequent pausing and arrests -even with chromatin free DNA
Suggests something is missing from in vitro studies
What is missing from in vitro studies on the rate of transcription compared to in vivo rates?
Large number of proteins and protein complexes that regulate elongation of transcription
- supress pausing
- promote pausing
- oversome trancription arrest
- enable elongation through chromatin
Give some examples of RNA pol II elongation factors that supress pausing/arrest
- TFIIH
- PTEFb (CTD kinase
- SII (cleaves bits of DNA interacting with RNA pol)
Give an example of RNA pol II elongation factors that promote pausing/arrest
-NELF (binds early during RNA elongation)
Give examples of RNA pol II elongation factors that enable elongation through chromatin
- Elongator (HAT associated with PolII)
- Spt6 (Histone chaperon - replaces removed histones)
What are the processes or arrest and pause during transcriptional elongation?
Arrest
-irreversible backsliding 7-14 nucleotides
Parusing
-back tracking 2-4 nucleotides
As a consequence RNA pol II spends a long time not synthesising RNA
Give a detailed outline of elongation with the mecanisms involved in pausing/arrest
- RNAPII initiates transcription and is phosphorylatede o ser5 of its CTD by TFIIH
- RNApol II moves off the promoter site and beings elongation and DNA melting, producing the nascent transcript
- DSIF binds to RNAPII soon after initiation and recruits NELF. NELF is a pausing protein and as long as it is interacting with RNApolII it cannot move
- Capping enzymes join the complex via interactions with the ser5-modified CTD and DSIF. A cap structure is added to the 5’ end of the nascent RNA
- P-TEFb is recruited by the capping enzyme, phosphorylates ser2 of the CTD and DSIF. This leads to DSIF and NLEF being removed from the complex
- meaing that the repressive action of NELF is neutralised and RNAPII resumes elongation
What are teh many levels at which transcription regulation can occur?
- Depression of chromatin (activators target remodellers and histone modifiers)
- Binds of more activators, co activators and loss of repression
- Targeting GTF (TFIID and TFIIB)
- Formation of PIC
- Isomerize from closed to open complex
- Overcome the abortive initiation to elongate
- transcribe through nucleosomes
- Supress pausing and arrest
- Temination
What are the features of transcription termination in eukaryotes?
- Enzymatic
- Termination sequence signal
- Nuclease cuts phosphodiester bond in RNA
Very little time spend on assembly
What mechanism of transcription does RNA pol I employ?
RNA pol I
-terminated by a mechanism that requires a polymerase specific termination factor which binds downstream of the transcription unit (DNA binding protein)
What mechanism of transcription does RNA pol II employ?
RNA pol II
- terminated in a region 0.5-2kb beyond the poly (A) addition site
- termination is coupled to the process that cleaves and polyadenylates the 3’ end of transcript
What mechanism of transcription does RNA pol III employ?
RNA pol III
-terminated after polymerising a series of U residues
What are the two types of RNA pol III promoters?
Interagenic (promoter within the coding seq of a gene) -class I and II -e.g. VAI/tRNA or 5SrDNA Extragenic (in the 5' flanking region) -class III -U6
What is the first TF to bind to an 5SrDNA RNA polymerase III promoter?
TFIIIA
- contains 9 ZF domains and binds to DNA
- recriuts TFIIIC (massive complex of lots of different proteins)
What is the sequence of TF binding to the 5SrDNA RNA pol III promoter site?
- TFIIIA recruits TFIIIC
- TFIIA/TFIIC complex recruits TFIIIB
These together recruit RNAPIII
What is the sequence of TF binding to the VA1 RNA RNA pol III promoter?
- TFIIIC is rectuired to the promoter elements on its own without TFIIIA
- TFIIIB (made up of TAFs and TBP) is recruited by TFIIIC
- complex of TFIIIC/B recruits RNA pol III to the promoter
What are the features of RNA pol I promoter elements?
- nucleosomes are not present in the transcribed regions of the nucleolous where rRNA is transcribed
- UPE recruits UPF which then recruits SL1 (TAFs and TBP) which recruits RNApol I to the promoter elements)
What are the different names of the TBP universal transcription factor and assocaited TAFs in RNA pol II, III and I promoters?
RNA pol II -TBP + TAFs = TFIID RNA pol III -TBP + TAFs = TFIIB RNA pol I -TBP+TAFs = SL1
