RNA Synthesis LO

Question 1

What is the chemical reaction catalyzed by RNA polymerase?

Answer 1

RNA polymerase II catalyzes the creation of a new phoshodiester bond between the 3’OH end of the growing RNA and 5’ phosphate of the incoming nucleotide.

Question 2

Why is the chemical reaction catalyzed by RNA unidirectional?

Answer 2

Because the 3’ OH group of the RNA strand acts as a nucleophile and attacks the phosphoanhydride bond by linking the alpha and beta phosphates of the incoming nucleotide, RNA can only grow in the 3’ direction, thus is unidirectional.

Question 3

Distinguish the five steps in the transcription cycle common to bacteria and eukaryotic RNA polymerases.

Answer 3

• Initiation
  1) RNA polymerase binds the promoter sequence in the ds DNA.
  2) Polymerase melts duplex DNA near the Txn start site to create transcription bubble.
  3) Polymerase catalyzes the reactions linking the first two nucleoside triphosphates.
• Elongation
  4) RNA polymerase moves down the DNA template strand in a 3’ to 5’ direction (synthesizing in a 5’ to 3’ direction) and melts DNA as it goes, adding nucleosidestriphosphates.
• Termination
  5) At the transcription stop site, RNA polymerase releases the completed RNA and dissociates from the DNA.

Question 4

Name the four cellular RNA polymerases.

Answer 4

1. RNA polymerase I
2. RNA polymerase II
3. RNA polymerase III
4. Mitochondrial (related to viral polymerases)

Question 5

Function of RNA Polymerase I

Answer 5

Transcribes ribosomal RNA (18S, 28S, 5.8S)

Question 6

Function of RNA Polymerase II

Answer 6

Catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA (these regulate gene activity)

Question 7

RNA Polymerase III

Answer 7

Transcribes DNA to synthesize ribosomal 5S rRNA (part of a large ribosomal unit), tRNA (involved in translation), and other small RNAs, such as U6snRNA (involved in splicosome), and 7SK RNA (activates transcription).

Question 8

Define promoter

Answer 8

A promoter is a control element of the DNA where transcription factors bind that is involved in transcription initiation.

Question 9

TATA Box

Answer 9

• Common promoter in humans is the TATA box: Contains the sequence 5’-TATAAA-3’ and is usually followed by three or more adenine bases and is generally located about 30 bp pairs upstream of the transcription initiation site. This promoter determine the transcription start site and directs binding of RNA polymerase II to DNA. This is where general transcription factors also bind.

Question 10

Promoter Proximal Elements

Answer 10

• A promoter in humans. Generally located within 200 base pairs upstream of the transcription start site. Approximately 20bp long and are involved with the regulation of transcription.

Question 11

How does alpha-amanitin block transcription?

Answer 11

• Because alpha-amanitin is a non-competitive inhibitor of RNA polymerase II, it binds to an allosteric site on RNA polymerase, and prevents transcription from occuring. It specifically binds to a bridge helix of RNA polymerase (a bridge helix is the alpha helix that connects the upper and lower “jaws”of the enzyme) and blocks the RNA chain elongation by preventing translocation of the RNA polymerase. Shrooms.

Question 12

How does Rifampcin block transcription?

Answer 12

• Rifampcin is a broad spectrum antibiotic that is commonly used to treat Tuberculosis and leprosy (mycobacterium infections). It binds the beta subunit of bacterial RNA polymerase selectively. Once bound, it blocks the RNA exit channel, preventing elongation of the mRNA.

Question 13

Components of the RNA polymerase II pre-initation complex at TATA Box

Answer 13

1. Binding proteins (TBP) binds to the TATA box on the minor groove of the DNA and helps direct assembly of the pre-initiation complex (PIC) over the promoter.
   - Binding of TBP creates a 90 degree bend in the DNA, contained in TFIID.
2. TFIIH is a large multiunit protein complex with several functions
   - XPD and XPB, (DNA helicases)
   - CDK7 (kinase that phosphorylates the C-terminal domain of RNA pol II) –> this modification allows the polymerase to release from the promoter.
3. General Transcription Factors
   - TFIIA, TFIIB, TFIIE, TFIIF
4. Mediators (multiprotein complex that functions as a transcriptional coactivator in all eukaryotes)

Question 14

Function of TFIIA

Answer 14

Stabilizes TBP to DNA

Question 15

Function of TFIIB

Answer 15

Serves as a bridge between TFIID, which is responsible for initially recognizing the promoter and RNA pol II.

Question 16

Function of TFIIE

Answer 16

Is thought to be involved in DNA melting at the promoter.

Question 17

Function of TFIIF

Answer 17

Assists in the wrapping of DNA around the pre-initiation complex.

Question 18

What are three clinical syndromes caused by mutations in the TFIIH subunit?

Answer 18

Mutations in XPB, XPD, and p44 causes:

- Xeroderma Pigmentosum, Cockaynes Syndrome, and Trichothiodystrophy.

Question 19

Xeroderma Pigmentosum

Answer 19

Autosomal recessive disease where mutations in the DNA repair mechanisms of TFIIH subunits results in an inability to repair thymine dimers caused by UV radiation. This results in an abundance of basal cell carcinomas and other skin malignancies. Other symptoms include: eyes that are painfully sensitive to light, blistering or freckling from minimal sun exposure, severe sunburn, and more.

Question 20

Cockaynes Syndrome

Answer 20

Another autosomal recessive disease characterized by defects in DNA repair elements of TFIIH. Common symptoms include: growth failure, impaired nervous system development, photosensitivity, and premature aging.

Question 21

Trichothiodystrophy

Answer 21

Group of autosomal recessive disorders characterized by photosensitivity, brittle sulfur deficient hair, ichthyosis, developmental defects, and impaired nervous system. Again, resulting from loss of function of DNA repair mechanisms of TFIIH subunits.