Gene Expression Flashcards
What is a transcription factor?
Any biochemical factor involved in achieving appropriate transcription levels
What are gene-specific transcription factors?
Transcription factors that bind to specific gene.
Which TFs are there and what do they do?
1) Basal TFs: required for level of basal transcription from naked DNA template
2) General TFs: required for transcription of every protein-coding gene in vivo
3) Co-factors: required to achieve appropriate regulation but not in themselves required for specificity.
4) Gene-specific TFs: proteins that regulate transcription through direct binding of specific sequences.
What is the preinitiation complex and what does it consist of?
Complex of TFs to start transcription. There is stepwise assembly of PIC on core promoter.
Consists of at least RNA Pol II, TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH.
What is TFFID?
TFFID = TBP + TAFS TBP = TATA binding protein TAFS = TBP associated factors
What is DNA looping?
The formation of a DNA loop when a protein of complex of proteins simultaneously binds to two different sites on DNA.
DNA loop can be responsible for transcriptional repression or activation.
For example, activation by causing that enhancer comes closer to the promoter.
What is the general build-up of GSTFs?
1) DNA binding domain (DBD)
Generally bind to major groove and recognizes 4-8bp motifs through large number of individually weak intermolecular interactions.
2) Activation domain (AD) (Also called ligand binding domain (LBD) )
Binding site for other proteins. Possible two have more than 1 AD in 1 TF.
3) Dimerisation domain
Domain to form dimer with other TF
What happens when TF binds to other TF through dimerisation domain?
1) Increase affinity
2) Increased specificity
Partner can for example bind to minor groove to increase specificity (example: CTE)
3) Combinatorial control
4) Negative regulation (inactive partner)
Why do GSTFs bind mostly in the major groove?
1) More space
2) More specific chemical groups that allow better distinction between different base-pairs
What are position weight matrices?
Representation of the “frequency distribution” of bases at different positions.
GSTF can bin can bind at DNA that is closely related to the most preferred DNA sequence.
What determines the structure of the ligand binding domain and how can this influence the transcription?
- Ligand binding determines the LBD structure
1) If a ligand agonist binds then the conformation changes in such a way that it creates new places for protein-protein interactions and attracts co-factors. –> favours transcription.
2) If a ligand antagonist binds then the conformation changes in such a way that there is a poor protein-protein interaction or that interactions are even blocked. –> unfavorable for transcription.
What is a reporter assay and how does it work?
Test that investigates whether a protein can activate or repress expression of the gene using a reporter protein (example Luciferase).
1) Take piece of DNA with promoter region
2) Clone that part of DNA
3) Hook promoter region to reporter
4) Transfect cells with plasmid
3) Production of reporter protein shows whether protein activates or represses expression.
What is the limitation of the reporter assay?
Does not represent chromatin very well.
What is squelching?
Liganded transcriptional activator (for example neuronal receptors) can inhibit the expression of another gene by inhibit the activity of another liganded transcription activator. Probably since it the interaction between these activators disrupt the biochemical pathways.
How does a coregulator/cofactor work?
1) Cofactors is a non-DNA binding protein
2) Interacts with both gene specific and basal TFs (“bridging”)
3) Interacts ligand-dependently
4) May harbour enzymatic activity
Can be coactivator or corepressor.
Example: Coactivator of nuclear receptor interacts with TFs through short alpha-helical motifs
Explain the activation of the TFs Nuclear receptors.
When NR TF is inactive: 1) Histone deacethylation 2) Corepressor NCoR/SMRT + HDAC is bound to NR When NR TF becomes active: 3) Ligand binds to NR --> folding of NR changes and NCoR/SMRT falls off 4) Coactivator SRC1+CBP/p300 binds NR 5) Histone acetylation --> gene opens up 6) Activation
What does the nucleosome consist of and how does it look like?
Octamer of H2A, H2B, H3 and H4
DNA is wrapped around it twice
N-terminal tails protrude away from core particle
Why can histone exchange be useful?
(1) Variant of histone can have an altered function.
H2AZ: stabilizes octamer, but prevents oligomerization which leads to less condensation so more ‘chromatin transcription’
(2) Alternative to de-modification
Getting rid of modified tails can be done enzymatically but also by exchanging a histone.
What is a poised enhancer/gene?
An enhancer or gene that is in between active and repressed genes.
Name an example of one modification that activates and one the represses.
Activates: H3K27ac
Represses: H3K9me2/3
What is a writer and name an example of a writer
An enzyme that puts a modification on a histone tail.
Example: HAT
Often a writer can also be a reader
What is a eraser and name an example of a eraser
An enzyme that removes a modification of a histone tail. –> Eraser is often a complex of 2 enzumes
Example: HDAC
How does acetylation activates gene expression?
Lysine in the histone tail has a positive charge. This causes the tail to ‘stick’ to the DNA. If the lysine is acetylated then the positive charge will be neutralized causing the histone tail to stick out more and do not bind as tight to the DNA. This loosens up the wrapping of DNA around the nucleosomes.
What are readers?
Proteins that bind to the acetyl or methyl groups on the histone tails. Each protein needs a specific pattern of acetyl and methyl groups this forms kind of a barcode for the binding of proteins.
A reader can activate or repress other modifications.
Often a reader can also be a writer
