What do embryonic stem cells differentiate to when low levels of retinoic acid are present?
Cardiomyocytes
What do embryonic stem cells differentiate to when high retinoic acid levels are present?
Neuronal Cells
What do marker genes do?
Indicate what cell types are present.
What method can we use to quantify mRNA expression?
real time RT(Reverse transcriptase) - PCR(Polymerase chain reaction)
What are the first two steps of real time RT-PCR?
- Culture cells and treat them according to your experimental protocol
- Purify RNA from the cells.
What is the 3rd step to real time RT-PCR?
Reverse transcriptase reaction to synthesise cDNA from RNA.
What is the 4th step to real time RT-PCR?
PCR
What is a reference gene?
A gene that doesn’t change during the experiment. Aims to normalize possible variations.
What are transcription factors?
Proteins that bind to specific DNA sequences, regulate transcription by recruiting co-activators and RNA polymerase II.
What are enhancers?
Short regions of DNA that can be 100s of kb away from the promotor, thought to loop over and interact with factors bound at the promotor.
What sort of structure do transcription factors have?
Modular. They have an activation domain and a DNA binding domain.
The TATA box recruits TFIID. What does TFIID consist of?
TATA-Binding Protein (TBP) and TBP-associated factors
What does TBP induce?
Sharp bend in DNA
What is the pre-initiation complex?
a massive, multi-protein assembly that forms on eukaryotic promoters to position RNA polymerase II (Pol II) at the transcription start site, unwind DNA, and initiate transcription
What complex is particularly present in the pre-initiation complex?
Mediator complex
- acts as a bridge between TFs, basal transcription factors and the C-terminal domain of RNA pol II.
What does assembly of the pre-initiation complex allow?
Phosphorylation of the polymerase C-terminal domain.
What does C-terminal domain phosphorylation allow?
The RNA pol II to clear the promoter and initiate transcription.
How can we regulate transcription factor activity via conformation altering?
Conformational change can alter: DNA binding ability, protein interaction ability, nuclear localisation.
You can regulate transcription factor activity by altering the __________ level.
Expression
How can you regulate transcription factor activity in regards to the DNA binding site?
Alter the accessibility of the DNA binding site
What does MAPK stand for?
Mitogen activated protein kinase
State the 3 steps of a MAPK pathway
- Growth factors bind to cell surface receptors
- Receptor activation sets up cascades of protein phosphorylation inside the cell, amplifying the signal.
- At the bottom of the cascade, transcription factors get phosphorylated and activated, driving transcription.
What can MAPK signalling drive?
Proliferation or stress responses.
What disease are mutations that constitutively activate MAPK signalling common in?
Cancer
