Eukaryotic Transcription Chapter 26

Question 1

Which RNAP is resistant to a-amanitin (toxin produced by mushroom)?

Answer 1

RNAP I (RNAP II/III 50% inhibition at 0.02ug/mL and 20 ug/mL respectively)

Question 2

Which polymerase is Very sensitive to actinomycin D?

Answer 2

RNAP I (RNAP II/III are slightly sensitive)

Question 3

Where are the three RNAPs located?

Answer 3

I - nucleolus II/II Nucleoplasm

Question 4

RNAP I products

Answer 4

Pre-rRNA

Question 5

RNAP II products

Answer 5

pre-mRNA and some snRNAs

Question 6

RNAP III products

Answer 6

tRNA, rRNA, snRNA

Question 7

CTD

Answer 7

Cyan; alternating b-turn subunit; in RNAP II; hydrophillic and phosphorylatable; important for transcription, splicing, termination and capping of RNA (recruit proteins) and RNA processing ; when CTD is not phosphorylated transcription occurs.

Question 8

TFIIH

Answer 8

Transcription factor needed for RNAP II; includes 5’ to 3’ and 3’ to 5’ helicases and is involved in nucleotide excision repair. Has three subunit Cdk7/MO15 + Cyclin H + MAT1 kinase complex that phosphorylates Ser5 of the CTD during transcription initiation

Question 9

Does RNAP have repair mechanisms?

Answer 9

Yes. 1) Stalls just like DNAP allows polymerase to do reverse reaction to fix (pyrophosphate should be there) 2) polymeration stalls causes RNAP to let RNA fall off and endonucleases peform activity.

Question 10

Housekeeping genes in Eukaryotes

Answer 10

Typically do not have TATA boxes to initiate transcription. CpG islands, upstream from start site. Methylation of CpG islands can stop transcription

Question 11

TATA box

Answer 11

In Eukaryotes (-31 to -26 region) Conserved region.

Question 12

BRE

Answer 12

In Eukaryotes (-37 to -32 regions) Recognized by TFIIB

Question 13

Pre-initiation complex

Answer 13

Platform of transcription factors on promoter sequence to attract RNAP. (TBP-TAFs; TFIID, TFIIB, and TFIIA (platform) TFIIF brings RNAP aids in recruiting TFIIH and TFIIE

Question 14

Process of RNAPII Attraction

Answer 14

1) TBP + TAFs bind to TATA box 2) TBP-TAFs form TFIID 3) binding of TBP and associated factors causes binding of TFIIB on BRE

Question 15

TAFs

Answer 15

Tata box associated factor

Question 16

TBP

Answer 16

Tata box binding protein; uses b-sheets to interact with DNA, binds minor groove of DNA and bends it, has aromatic AAs,

Question 17

TFIID

Answer 17

Transcription factor, RNAP II, D;

Question 18

TFIIB’s role

Answer 18

Binds to BRE and determines the orientation of the RNA polymerase has loop (similar to sigma region 4-3 in prokaryotes) sits in RNAP in eukaryotes

Question 19

TFIIA

Answer 19

Stabilizes PIC complex. primarily TBP with the TATA box.

Question 20

TFIIF

Answer 20

Associated with the RNAP and recruits RNAP to the pre-initiation complex. After TFIIF action, TFIIH and TFIIE are recruited.

Question 21

Mediator complex

Answer 21

20-21 protein complex; interacts with RNAP II and specific transcription factors to change conformation of RNAP II to initiate transcription.

Question 22

TFIIE

Answer 22

needed to aid TFIIH to do its activity.

Question 23

TFIIH

Answer 23

1) Helicase activity (in both directions, opens up polymerase) 2) TFIIH phosphorylates ser resides, specifically serine 5, on CTD tails of RNAP II causing the recruitment of capping enzymes.

Question 24

Phosphorylation patterns of CTD

Answer 24

1) CTD must be dephosphorylated to initiate transcription 2) TFIIH (CDK7) with the aid of TFIIE phosphorylates CTD at Ser 5 which aids in capping of RNA 3) TFIIE/H disassociate with CTD, phosphorylates Ser 2 causes recruitment of negative elongation factors (via phosphorylation) and splicing enzymes.

Question 25

What is responsible for phosphorylating Ser 2?

Answer 25

P-TEFb which has CDK9 activity.

Question 26

Ser 5 phosphatase

Answer 26

removes phosphate from ser 5 so that polyadenylation factors and termination factors can associate with the tail.