lecture 4

Question 1

TSS

Answer 1

Transcription Start Site

Question 2

TATA containing transcription initiates and proceeds in..

Answer 2

1 direction

Question 3

at other promotors (called CpG islands) transcription initiates in ____ but then the polymerase stalls and falls off DNA in ___ of the directions and the proceeds in the ___ other direction only

Answer 3

both
one
other

Question 4

graph shows transcription intensity over the analysed locus:

Answer 4

unidirectional initiation of RNA synthesis

Question 5

transcription peaks at _____. this means that RNA pol2 _________ in this region up to +200 bp region before elongating further

Answer 5

+50
initiates transcription and the pauses

Question 6

CpG rich in DNA contains _____ and are ___ to transcribe

Answer 6

less nucleosomes
easier

Question 7

at CpG islands transcriptions initiates at _____ within the island

Answer 7

any position

Question 8

transcription from CpG island initiates in ____ directions but proceeds in the direction of the ________ only

Answer 8

both
open reading frame (ORF)

Question 9

at CpG islands equal numbers of RNA polymerase initiation in both the ____ and _____ direction

Answer 9

sense
antisense

Question 10

sense transcripts:

Answer 10

pause before elongating further

Question 11

antisense transcripts:

Answer 11

pauses at the other end of the CpG island and do not continue

Question 12

Confirmation of the RNAseq by ChIP (4 steps)

Answer 12

1. treat living cells or tissues with a membrane permeating cross-linker such as formaldehyde
2. sonicate to shear cellular chromatin to short fragments and add antibody to pol 2
3. immunoprecipitate to isolate pol 2 cross–linked to DNA
4. reverse cross-linking, isolate DNA and subject to massively parallel DNA sequencing

Question 13

review slide 26

Question 14

explain how HIV is inhibited and expressed through TAR and Tat binding

Answer 14

the short end of RNA is synthesized before the polymerase is paused which then forms TAR and TAR holds the pol and inhibits Cdk9/CycT. HIV encodes protein Tat which binds to TAR. when cells are stressed they produce more Tat. when bound to TAR Cdk9/CycT is activated and releases the paused pol 2 and viral genes are fully expressed. HIV is dormant but are activated once cells are stressed slowly killing T-cells. patients die from lack of immunity