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What do different DNA elements do?
they react at different places
What experiment was done regarding radioactive DNA fragments?
A gel shift mobillity assay was performed to identify genes in the promoter to identify the transcription factors that bind to them
How do you perform a gel mobility shift assay?
Run gel: isolate the promoter; radioactive band
take the DNA promoter region adn extract DNA from the nucleus; put the promoter with the DNA
You have to have enough DNA so that each protein can be bound
Run under non-denaturing conditions= no SDS
How can you modify the gel shift mobility assay?
By mixing DNA, you can do chromatography to isolate/ fractionate the bands
You'll get a fraction number from chromotography
What is yet another method?
affinity chromatography that allows isolation of one single transcription factor
1) Attached to beads are random DNA sequences; mash up cells and solubilize proteins--> elute to separate nonbinding proteins from binding proteins
2) Now, to find the TF that binds just to our sequence of interest, we do the same but just with the DNA sequence of interest; only proteins that bind to this stick; the rest are eluted
What experiment allows you to start with a protein and find the sequence it binds to?
1) Offer many DNA sequence options; float them together; let them intermingle to see which it bidns to
2) Use a gel, column, and several assays
Explain how to form a DNA footprint.
Specific promoter: Where in the promoter does it bind?
- Create a footprint
- Entire promoter sequence, label it, mix protein, let bind, chop up via nuclease;
Only the region where proteins are not bound= limited nuclease digestion
What is an alignment?
- Line up and see the homology; look for conserved regions
- If region is highly conserved in promoter, mutations that occurred were detrimental and killed off the organism
What is a CHIP assay?
- DNA fragment and protein= X-linked
- nuclease--> immunoprecipitate
- remove cross link
When no repressor is present, what happens?
What happens when there is enough tryptophan present?
Trp binds to repressor, allowing it to bind to operator--> no transcription
When it binds, it causes a conformation change in the recognition helix and allows it to bind
bound repressor protein prevents transcription: ligand turns it on
Ligand binds to allow regulatory protein to bind to DNA: removal of ligand switches gene on by removing repressor
Addition of ligand switches gene off by removing activator protein
Removal of ligand switches gene off by removing activator protein
Proteins can be __ or __.
cAMP binds to CAP--> CAP binds to activator binding site
genes responsible for catabolism of lactose only active when there's no glucose but lactose is present
Explain the four conditions of the lac operon.
Glucose present; lactose present= Operon off because CAMP not bound
Glucose present; lactose absent= Operon off both because the Lac repressor bound and because CAP not bound
Glucose and lactose both absent: operon off because Lac repressor bound
No glucose; lactose present: cAMP is a sign that energy levels are low; binds to CAP, which binds to the promoter--> operon on
What is DNA looping?
Even though in double helix, it can loop around, allowing different regions of DNA to be in contact with each other
optimal distance is 500 nucleotide pairs
- Less than 500= region not flexible enough
- More than 500= probability decreases with increasing distance
Proteins are tethered to teh same DNA molecule
What does the enhancer's location provide?
Because it is so far upstream, the enhancer allows the looping effect
Different proteins allow what?
different bindign capabilities and thus regulation
ex: sigma factors
- Viruses enter the cell with their own sigma factors.
- They use the host sigma factor, which allows transcription of the virus' own sigma factor. This then allows transcription of the virus' own sigma factor--> synthesis of its genes
How do eukaryotes perform transcription?
They have a mediator that is able to scoop up all of the things needed for transcription to occur; bring them all down; accumulate everything at the start site
Explain the experiment that was performed to determine the structure of the activator.
Using the Gal4 protein, which is teh gene that turns on activation of transcription of the galactokinase
The DNA binding and transcription-activating domains are inactive
A functional activator can be reconstituted from the C-terminal portion of the yeast Gal4 protein if it is attached to teh DNA binding domain of a bacterial gene regulatory proteni (Lex A) by genetic engineering.
When the resulting hybrid protein is produced, it activates transcription from yeast genes
it is normally responsible for activating the transcription of yeast genes that code for the enzymes that convert galactose to glucose.
A chimeric gene regulatory protein, produced by genetic engineering techniques, requires a LexA recognition sequence to activate transcription. In the experiment, the control region for one of the genes controlled by LexA was fused to the LacZ gene, which codes for Beta-galactosidase, which is simple to detect since it acts as a reporter gene
What else can activators do?
they can bind chromatin remodeling complexes that unravel DNA
Bring in the histone chaperone to rearrange histones
Attract histone modification enzymes to modify the tails
What is the initial modification?
- A gene activator protein binds to DNA packaged into chromatin and first attracts a histone acetyl transferase to acetylate lysine 8 of histone H4.
- Next, a histone kinase, attracted by the gene activator protein, phosphorylates serine 10 of histone H3, but can only do so after lysine 9 has been acetylated.
- The serine modification then signals teh histone acetyl transferase to acetylate position k14 of histone H3.
- At this point, the histone code for transcripton initiation, set into motion by the binding of the gene activator protein, has ben written.
- The final reading of the code occurs when the general TF TFIID and the chromatin remodeling complex SWI/SNF bind, both of which strongly promote teh subsequent steps of transcription initiation.
TFIID and SWI/SNF both recognize acetylated histone tails through a bromodomain, a protein domain specialized to read the particular mark on histones.
What is an interferon?
- It is a signaling molecule
- When a cell is infected, it signals other molecules to signal others to look out
What are teh places where transcription can be affected?
- Gene activator protein binds to chromatin
- chromatin remodeling complexChromatin remodeling
- histone modification enzymesCovalent histone modification
- other activator proteinsAdditional activator proteins bound to gene regulatory region
- Mediator/ general TF/ RNA polymeraseAssembly of pre-initiation complex at the promoter
- other gene activator proteins; rearrangement of proteins in the pre-initation complexTranscription initiation
What are the six ways in which eucaryotic gene repressor proteins operate?
1) gene activator proteins and gene repressor proteins compete for binding to the same regulatory DNA sequence
2) Both proteins can bind DNA, but the repressor binds to the activation domain of the activator protein, thereby preventing it from carrying out its activation functions. In a variation of this strategy, the repressor binds tightly to the activator without having to be bound to DNA directly
3) The repressor blocks assembly of the general TFs; some also act at late stages in transcription initiation by preventing the release of the RNA polymerase from the general transcription factors
4) The repressor recruits a chromatin remodeling complex which returns the nucleosomal state of the promoter region to its pre-transcriptional form
5) The repressor attracts a histone deacetylase to the promoter. Histone acetylation can stimulate transcription initiation and the repressor simply reverses this modification
6) the repressor attracts a histone methyl transferase which modifies certain positions on histones which, in turn, are bound by proteins that maintain the chromatin in a transcriptionally silent form