Transcriptional Regulation Continued Flashcards
How is RNA isolated from a sample and converted to DNA? By the use of what?
By the use of a specific or random primer and RNA-Dependent-DNA Polymerase.
Describe Chromatin immunoprecipitation (ChIP)
Multi-step technique that detects the binding of a specific protein to a specific DNA element or multiple DNA elements in vivo.
Describe gel-mobility shift to analyze binding of transcription factors and histones to DNA.
Detects the binding of a specific protein to a specific DNA in vitro.
Describe the steps involved in ChIP with anti-RNA pol ll antibodies.
- Treat the living cells or tissues with a membrane-permeating cross-linker such as formaldehyde.
- Sonicate the cells to shear cellular chromatin to short fragments and add antibody to Pol ll.
- Immunoprecipitate to isolate Pol ll cross-linked to DNA.
- Reverse cross-linking, isolate DNA, and subject to massively parallel DNA sequencing.
In the pre initiation complex, the helicase activity of TFIIH locally ________ DNA at the initiation site.
Melts.
Mutations in the subunits of ______ have been identified as the cause of Xeroderma pigmenotsum.
TFIIH
Describe the symptoms of a patient with Xeroderma pigmenotsum.
The mutation inhibits DNA repair caused by exposure to UV light, so patients develop multiple skin lesions that develop into cancer.
Which transcription factor is involved in transcription-coupled DNA repair?
TFIIH
HIV (Humans Immune-deficiency Virus) has a mechanism to suppress CDK/CycT activity by a viral encoded protein called _____.
TAT
What is TAT (in HIV) responsible for?
Pauses RNA Pol II at its promoter (a weak promoter).
In HIV, upon stress, TAT releases ______ and the polymerase transcribes the virus.
CDK9/CycT
What two factors cause the pausing of the polymerase after initiation?
NELF and DSIF
Further phosphorylation of CTD by CDK9/CycT releases the polymerase: ________ begins
Elongation begins
What region is responsible for direct binding of RNA polymerase II to DNA, determining the site transcription initiation, and influence the frequency of transcriptional initiation?
The promoter region.
True or False? Promoter-proximal elements and enhancers are cell-type-specific?
True.
Describe transcription activators and repressors.
Modular proteins containing a single DNA-binding domain and one or a few activation or repression domains.
Multiple _____ regulatory elements are found close to the transcription initiation site, promoter-proximal.
Promoter.
______, distal regulatory elements, could be upstream or downstream of the initiation site.
Enhancers.
Describe the four main properties of promoters.
- Function with a short distance (several hundred bps from the transcription initiation site.
- Immediately upstream from the initiation site (for RNA Pol ll)
- Position DEPENDENT: Usually non-functional if moved.
- Orientation DEPENDENT: Drive transcription in one direction only.
Describe the four main properties of enhancers.
- Can function over a long distances (tens of kbps from the transcription initiation site).
- Can be upstream or downstream from the start or within introns.
- Position INDEPENDENT: Usually still functional when moved.
- Orientation INDEPENDENT: Function in ether normal or inverted orientation.
How do enhancers communicate with promoters?
Via bending DNA.
What kind of analysis can be used to identify independent DNA-binding domains and effector domains (activation or repression)?
Deletion analysis.
DNA domains have conserved _______ (structure folds).
Motifs
What is a motif found in DNA domains?
A frequently found sequence in proteins with a similar function.
What are two kinds of motifs found in DNA domains?
Zn++ finger and the Leu zipper.
In combinatorial binding, DNA-binding proteins work as dimers. Each monomer can recognize a specific DNA element, but cannot work on its own. What will it bind to?
It will bind to DNA only in a complex with a similar protein.
If you have three monomers, you can form dimers and have ____ combinations of these proteins. Adding an ____ of the three proteins increases possible combinations.
6, inhibitors
Protein _____ increases the complexity of DNA-binding specificity.
Dimerization.
