Transcriptional Regulation Continued

Question 1

How is RNA isolated from a sample and converted to DNA? By the use of what?

Answer 1

By the use of a specific or random primer and RNA-Dependent-DNA Polymerase.

Question 2

Describe Chromatin immunoprecipitation (ChIP)

Answer 2

Multi-step technique that detects the binding of a specific protein to a specific DNA element or multiple DNA elements in vivo.

Question 3

Describe gel-mobility shift to analyze binding of transcription factors and histones to DNA.

Answer 3

Detects the binding of a specific protein to a specific DNA in vitro.

Question 4

Describe the steps involved in ChIP with anti-RNA pol ll antibodies.

Answer 4

1. Treat the living cells or tissues with a membrane-permeating cross-linker such as formaldehyde.
2. Sonicate the cells to shear cellular chromatin to short fragments and add antibody to Pol ll.
3. Immunoprecipitate to isolate Pol ll cross-linked to DNA.
4. Reverse cross-linking, isolate DNA, and subject to massively parallel DNA sequencing.

Question 5

In the pre initiation complex, the helicase activity of TFIIH locally ________ DNA at the initiation site.

Answer 5

Melts.

Question 6

Mutations in the subunits of ______ have been identified as the cause of Xeroderma pigmenotsum.

Answer 6

TFIIH

Question 7

Describe the symptoms of a patient with Xeroderma pigmenotsum.

Answer 7

The mutation inhibits DNA repair caused by exposure to UV light, so patients develop multiple skin lesions that develop into cancer.

Question 8

Which transcription factor is involved in transcription-coupled DNA repair?

Answer 8

TFIIH

Question 9

HIV (Humans Immune-deficiency Virus) has a mechanism to suppress CDK/CycT activity by a viral encoded protein called _____.

Answer 9

TAT

Question 10

What is TAT (in HIV) responsible for?

Answer 10

Pauses RNA Pol II at its promoter (a weak promoter).

Question 11

In HIV, upon stress, TAT releases ______ and the polymerase transcribes the virus.

Answer 11

CDK9/CycT

Question 12

What two factors cause the pausing of the polymerase after initiation?

Answer 12

NELF and DSIF

Question 13

Further phosphorylation of CTD by CDK9/CycT releases the polymerase: ________ begins

Answer 13

Elongation begins

Question 14

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?

Answer 14

The promoter region.

Question 15

True or False? Promoter-proximal elements and enhancers are cell-type-specific?

Answer 15

True.

Question 16

Describe transcription activators and repressors.

Answer 16

Modular proteins containing a single DNA-binding domain and one or a few activation or repression domains.

Question 17

Multiple _____ regulatory elements are found close to the transcription initiation site, promoter-proximal.

Answer 17

Promoter.

Question 18

______, distal regulatory elements, could be upstream or downstream of the initiation site.

Answer 18

Enhancers.

Question 19

Describe the four main properties of promoters.

Answer 19

1. Function with a short distance (several hundred bps from the transcription initiation site.
2. Immediately upstream from the initiation site (for RNA Pol ll)
3. Position DEPENDENT: Usually non-functional if moved.
4. Orientation DEPENDENT: Drive transcription in one direction only.

Question 20

Describe the four main properties of enhancers.

Answer 20

1. Can function over a long distances (tens of kbps from the transcription initiation site).
2. Can be upstream or downstream from the start or within introns.
3. Position INDEPENDENT: Usually still functional when moved.
4. Orientation INDEPENDENT: Function in ether normal or inverted orientation.

Question 21

How do enhancers communicate with promoters?

Answer 21

Via bending DNA.

Question 22

What kind of analysis can be used to identify independent DNA-binding domains and effector domains (activation or repression)?

Answer 22

Deletion analysis.

Question 23

DNA domains have conserved _______ (structure folds).

Answer 23

Motifs

Question 24

What is a motif found in DNA domains?

Answer 24

A frequently found sequence in proteins with a similar function.

Question 25

What are two kinds of motifs found in DNA domains?

Answer 25

Zn++ finger and the Leu zipper.

Question 26

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?

Answer 26

It will bind to DNA only in a complex with a similar protein.

Question 27

If you have three monomers, you can form dimers and have ____ combinations of these proteins. Adding an ____ of the three proteins increases possible combinations.

Answer 27

6, inhibitors

Question 28

Protein _____ increases the complexity of DNA-binding specificity.

Answer 28

Dimerization.