RR5: Proteins that regulate transcription-Activators Flashcards
Do transcriptions done by RNA pol 1 and 3 require the same elements as the one done by RNA pol 2?
No. RNA pol 2 requires TF elements, but transcription 1 and 3 have other required proteins to work.
Do transcription done by RNA pol 1 and 3 also require ATP?
NO. Only transcription done by RNA pol 2 is ATP-dependent. Pol 1 and 3 don’t need ATP to get from a Closed PIC to an open PIC
What could explain the RNA pol 2 to go in 2 different directions during transcription?
In genes that don’t have a TATA box, RNA pol 2 could go in 2 different directions during transcription. The TATA box is not necessary for transcription, but it helps transcribe in a single direction. In large promoter that don’t have a TATA box, there might be multiple start sites.
Why are promoter-proximal elements important?
They are recognized by specific DNA-binding proteins that act to influence transcription (enhance or inhibit). They are required for the activation of transcription.
How would we be able to know if a section is a proximal-promoter element essential for trasncription?
If the region interacts with proteins necessary for transcription, then we know it’s necessary. By putting that sequence in a gel, if the sequence moves faster, it means the protein is not interacting with the region, because the heavier, the slower they go on the gel.
What is the method used to know if a section of DNA is interacting with a protein?
Electrophoretic mobility shift assays (EMSA).
or
gel/band/mobility shift assays
How does ESMA or gel/band/mobility shift assays work?
Concept: DNA fragments will migrate in a different way through an electrophoretic field when it’s bound to a protein.
- Use a radiolabelled dsDNA segment as a probe
- Make the probe interact with nuclear subtract.
- If a protein in the nuclear subtract binds to a part of the probe, it will move differently on the gel.
- Creates a gel shift and a complex, so we can know which section is important for transcription for example.
But we still can’t know the exact sequence that is bound by the protein.
How can we make sure a given factor interacts with a cis-acting element and activates transcription?
- Generate a cDNA that corresponds to the protein we’re studying.
- Put that cDNA in a plasmid
- Create another plasmid with a reporter gene and the sequence that would bind to the protein we’re studying.
- Co-transfect the 2 plasmids (expression vectors) in a cell
- With the plasmid with the cDNA, the cell should be able to make the protein.
- The protein should then be able to interact with the other plasmid that has the sequence it would normally bind to.
- If it works, the protein will bind to the binding sequence and it should increase the transcription of the reporter gene.
- Observe if the reporter gene is expressed more than usual, if it is, you were right and that protein is binding to that sequence and it does play a role in increasing the efficiency of transcription.
What are transcription factors?
They can be either transcriptional activators or repressors.
They are DNA-binding proteins.
They can bind to proximal elements, enhancers or specific sequences that activate transcription.
They have multiple domains that have various functions.
They have domains made-up of helices.
The helices that interact with the DNA are called recognition helices.
What are recongition helices?
They are alpha-helical domains in transcription factors that interact with DNA to bind.
They interact with the nucleobases of the major groove of DNA.
How does the binding between recognition helices of transcription factors and the major groove of DNA happen?
Binding occurs through non-covalent interactions with the atoms in the bases.
Other amino acids within the recognition helices contribute to stabilizing the binding with DNA. The negative phosphates in the DNA backbone interact with the positive bases in the recognition helices.
What is GAL4?
It’s a transcription factor from yeast.
It has multiple domains.
What are the roles of GAL4?
Stimulate transcription with its activation domain.
Binds to UASgal with its DNA binding domain.
What is UASgal?
It’s an upstream activating region.
When GAL4 interacts with UASgal, the gene can activate transcription very effectively.
How precise does the interaction between GAL4 and UASgal need to be?
Very precise. GAL4 has a DNA-binding domain on the N-terminal region. When you remove 50 amino acids, it can no longer bind to UASgal.
And because it can’t interact, it can’t activate transcription.
