Cellular Signalling and Pol II Cis-Regulation Flashcards
What is cis-regulation? Give some examples.
This is defined by the use of sections of non-coding DNA that regulate the rate of transcription.
This can be promoter proximal elements or cis-regulatory modules that are further from the TSS such as enhancers, silencers, insulators and tethering elements.
How long are enhancers?
A few hundred base pairs
How do enhancers work?
The enhancer binds specific transcription factors that can be activators or repressors.
These can remodel the chromatin to clear the promoter region and allow for the DNA to bend so that the enhancer lies antiparallel. They also come into direct contact with the PIC via the mediator and GTFs as well as other co-activators.
How do TFs bind to enhancers and why is this useful?
Multiple TFs bind cooperatively - hence in a very varied way.
This allows for great fine-tuning of expression as the various co-operative models allow for sharp temporal variation in the amount of bound TF without a change in the local concentration of that TF or an increase in its (generally low) specificity for the DNA section. w
What affects whether or not two TFs bound to different sites on an enhancer can interact?
the relative order, spacing and orientation of the different binding sequences on the enhancer - AKA the motif positioning or grammar
What things can affect enhancer motif positioning?
if the DNA binding sequence is palindromic the TF may bind in the wrong direction, preventing interaction with the other TF.
The sites for binding TFs may be interrupted by another binding motif for another TF.
If the binding sites for two co-operative TFs is too close or too far away then the helical nature of the DNA may mean that they are sticking out of different sides of the strand and so not in position to interact.
What are the three main models of enhancer activity, and what do these aim to explain?
The enhanceosome model, the billboard model and the TF collective model.
These account for the seemingly counter-productive motif grammar that allows enhancers to be arranged in an ineffective manner.
What is the enhanceosome model?
The enhanceosome model suggests that in fact these seeming issues with the grammar in fact act as a scaffold for all of the necessary TFs to be recruited to form a higher-order protein that forms an interface through which transcription is regulated.
What is the Billboard Model?
The billboard model states that the transcription factors bind in to the sites in patterns so that while they may not always directly interact with one another, only specific combinations of sites may be occupied and so transcription can be regulated depending on which pattern is used.
What is the TF collective model?
The TF collective model suggests that regardless of which TFs are able to bind they will be able to recruit the remaining TFs that have been unable to bind through protein-protein interaction due to improper motif grammar. This allows for variable patterns of TF bindings that can be used to regulate transcription.
What is CREB Extracellular Signalling
cAMP Response Element Binding signalling
This is a method of using extracellular signals to directly regulate transcription of a wide variety of genes in metabolism, cell structure and neurotransmission simultaneously using their common cAMP Response Element (CRE).
What is the delay between signalling and CREB response?
30 minutes to an hour
What initiates CREB signalling?
The signalling process is initiated when the agonist, often hormones such as adrenaline, bind to the relevant GPCR causing the signal to be transferred through the membrane to the heterotrimeric G protein.
How does the CREB GPCR transmit the signal into the cell?
When the GPCR is activated the Guanine nucleotide Exchange Factor (GEF) activates the G protein by replacing the GDP bound to the α subunit with a GTP.
What is the structure of a G-protein pre-activation?
The G protein is made up of an α, β and a γ subunit. The alpha and gamma subunits bind into the membrane.
What happens when the G-protein is activated? What effect does this have?
Activation allows the G protein to dissociate from the GPCR receptor domain and itself split into two active and still membrane bound components; the βγ complex and the GTP bound α subunit.
The two components may have different effects allowing the one signal to have multiple effects already, but the main focus is the α subunit.
What is the role of the activated G-protein alpha subunit?
Once activated by GTP, the α protein itself activated the membrane-bound enzyme adenylate cyclase
What is the role of adenylate cyclase in CREB signalling?
It converts ATP to cAMP.
What is the role of cAMP in CREB signalling? How does it do this?
cAMP activates PKA.
Prior to signal activation the two catalytic subunits of PKA are inhibited by the regulatory subunit. When four cAMP bind to this it releases the activated catalytic domains allowing them to travel to the nucleus.
What is the role of PKA in CREB signalling?
Once in the nucleus the PKA phosphorylates the, often already DNA bound, cAMP Response Element Binding Protein (CREB protein) on its Ser-133 residue.
What is the binding habit and structure of the CRE Binding Protein?
The dimeric CREB protein binds by its leucine zipper domain to the CRE, a palindromic sequence never more than 250bp away from the promoter.
The CREB protein has a central regulatory domain and an N-terminal activation domain where the Ser-133 is found.
What happens when PKA activates CREB?
When the CREB is phosphoactivated by PKA its conformation change allows for the recruitment of a large protein called CBP/p300.
What is the role of CBP/p300 in CREB signalling?
This effects the actual regulation by directly interfacing with the Basal Transcription Complex via TFIIE and TFIID. It is also involved in chromatin remodelling to allow for easier PIC/Pol II recruitment.
