Term
| What was the Muller streptavidin experiment and what did it suggest? |
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Definition
Cut DNA between a working enhancer and the target -> enhancer stops working
Use biotin and streptavidin to bind the two fragments again -> enhancer works
Suggests looping or oozing model correct |
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Term
| What are the three possible methods of enhancer function? |
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Definition
Oozing - proteins interact forming a protein bridge, eventually linking with the ore promoter
Looping - DNA loops to bring enhancer in direct contact with promoter
Tracking- a factor interacts with the enhancer and tracks along DNA to the core promoter |
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Term
| What 3C experiment suggests chromosome looping? |
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Definition
Create ligation sites around the promoter and enhancer
Use formaldehyde to cross link interacting regions (such as the enhancer and promoter)
Cut cross-linked DNA
Dilute then ligate the DNA
Perform PCR using primers against the two sequences, if the reaction produces a product -> suggests interaction |
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Term
| What do activators target? |
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Definition
| Multiple possible targets, including TFIID, TFIIB and TFIIH |
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Term
| What is the structure of Gcn4 and what does it do? |
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Definition
| Gcn4 has 2 activator domains (1 on N term 1-100 and a central one next to it 100-134) and separate DNA binding domain (further downstream 209-281) containing bZip proteins – forms a dimer and binds DNA. Activator domains interact with multiple targets. |
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Term
| How does the androgen receptor activate the PSA, and what mechanism would this fall under? |
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Definition
| P160 co activator binds to the enhancer, the P300/CBP co-activator then binds to that. The androgen hormone receptor complex is on the proximal promoter, so more P160 and P300/CBP binds to the original ones and the DNA loops to create contact between the co-activators and the receptor complex with the help of mediators. The RNAp2 then associates at the enhancer, and tracks along the DNA around the loop towards the promoters. |
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Term
| Why does acetylation of lysines promote gene expression? |
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Definition
| Acetylation removes the positive charge, reducing the attraction between the histone and the DNA, loosening the chromatin structure and allowing RNAP and other proteins access. |
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Term
| What are the four main histones? |
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Definition
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Term
| What do histone tails do? |
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Definition
| Wrap around DNA and can be modified to control chromatin structure |
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Term
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Definition
| Seals the nucleosome and mediates higher order structure |
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Term
| What are the three general methods of co-activator interaction? |
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Definition
| Loosening chromatin, moving nucleosomes relative to DNA, making contact between activator and transcripion complex |
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Term
| What acetlyations lysines on the histone tail, and what is its opposite? |
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Definition
HATs - histone acetyltransferases
HDACs - histone deacetylases |
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Term
| Which lysines can be acetylated on H3? |
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Definition
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Term
| Which lysines can be acetylated on H4? |
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Definition
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Term
| What is the yeast equivalent to HATs and how does it bind to DNA? |
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Definition
| Gcn5, its bromodomain recognises and binds to acetylated histones |
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Term
| What is the main mammalian co-activator? |
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Definition
| CBP (CREB binding proteins) can be targeted by many activators |
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Term
| How does the SAGA complex work? |
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Definition
| Gcn4 binds to DNA, the complex binds to Gcn4 via Tra1 (part of SAGA), which activates the rest of the SAGA complex including Gcn5 - which does the chromatin modification |
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