Nov 4 Flashcards
What is a reporter gene, what are some examples?
They are gene that is expressed to produce some visible effect from a yeast two-hybrid assay. You can make it GFP so get green fluorescence for example, or can use media without histidine, so only when gene expressed then get cell growth
How would you conduct a yeast 2-hybrid assay?
You have prey protein attached to an activating domain, and a bait protein gene and DNA binding gene together so makes hybrid protein. If the prey binds to the bait, it has the activation domain with it so it will get transcription. You would try diff combinations of transcription factors to see which ones interact
Describe the helix-turn-helix motif for transcription factors.
It has 1 helix supporting and positioning another helix that is fitted in the major groove
What are homeodomains in term of transcription factors?
They have very conserved amino acid sequences for specific DNA sequences. They have 3 helices, 2 helices position another helix that fits with the major groove. One of the supporting helices can interact with the DNA too
How do zinc-containing domains in transcription factors work?
They have a helix with 2 histidines, and B sheet with 2 cys. These 4 AAs bind to 1 zinc atom between them and stabilizing the structure. The helix recognizes the DNA
How does the leucine-zipper motif work for transcription factors?
It looks like an X. It has a coiled-coil domain that has leucine residues in the top half of the X. Then the bottom of the X is made of 2 helices that bind to the major groove. This motif is 2 monomers coming together to make the X.
How does the helix-loop-helix motif work for transcription factors?
It is 2 monomers coming together to make a dimer. Each monomer has a short and long helix. The timer makes an X, with the long helices flanking the major groove on each side.
How to identify targets of a transcription factor?
You do chromatin immunoprecipitation (ChIP). It determines protein-DNA interactions.
1. Crosslink the TF to promotor with formaldehyde
2. Shear the DNA into fragments
3. Put antibody for a TP on agarose beads, then get the antibody off
4.Isolate the strands once get antibody and TF off
5. Can do PCR then sequence the strand
How does activator enhance transcription?
It binds to enhancer, has additive/synergystic effect. It can help recurit things directly and indirectly (through other proteins)
How do activators recruit nucleosome modifiers?
Th bind to promotor on strand on histone, They recruited histone acetyl transferase to open chromatin or uses chromatin remodelling complex that uses ATP to move nucleosomes to expose promotor (locally opens chromatin)
What do insulators do?
Enhancers can work from big distance, either up or down stream. If insulator bound to a site between enhancer and promotor, it blocks it from working. Sometimes it means that the same enhancer will go to a different promotor instead for diff gene.
How can activators work at such a long distance?
The DNA folds so the activator bound to the enhancer can interact with transcription factor and PIC through other proteins
What does the locus control region do?
It regulates expression of cluster of thenes. They turn on genes in specific order.
How are activators synergistic? Give example with histones
They can bind to enhancers cooperatively by coming in contact with one another, or both connect with a protein between them.
An example is only 1 activator can bind to promotor, then modifier recruited which opens up heterochromatin to reveal a second binding site for the second activator
