f. LEC 3 + 4 Flashcards
What are the 7 GTFs associated w/ RNA pol II?
TFIIA, TFIIB, TFIID, TFIIE, TFIIH, TFIIF, TBP
T or F - all TATA-less promoters are CpG islands
F - There are some TATA-less promoters that are not rich in just Cs and Gs
Name 3 features that make CpG islands special compared to most promoters
- place, where the TATA box would be, is instead rich in Cs and Gs
- transcription initiation is bidirectional
- they transcribe continuously and at a low rate
T or F - CpG islands initiate and elongate in both directions
F - they only initiate bidirectionally
What is the significance of GTFs?
to help load the RNA pol II and help w/ transcription regulation
a) What does the peak represent compared to the plateau
b) what type of promoter is being used
c) Is this bidirectional or uni-direction?
d) how many pol II’s are present?
e) What does the noise represent?
f) ChIP or RNAseq?
a) peak = initiation phase, plateau = elongation phase
b) TATA or TATA-less promoter
c) unidirectional
d) 1
e) the amount of RNA transcribed by the RNA pol II
f) RNAseq?
CpG islands continuously transcribe at a low rate. What feature allows it to do this?
less nucleosome = DNA is more open making it easier to transcribe continuously
T or F - CpG islands don’t have to initiate transcription at the start site
T - they can initiate at any position w/in its ‘island’
a) What does the peak represent compared to the plateau
b) what type of promoter is being used
c) Is this bidirectional or uni-direction?
d) how many pol II’s are present?
e) What does the noise represent?
f) ChIP or RNAseq?
a) peak = initiation phase + plateau = elongation phase
b) CpG islands
c) bidirectional
d) 2
e) the amount of RNA transcribed
f) RNAseq?
Name + Describe the 2 types of graphs that are used as evidence of the bi-directionality of GpC islands.
- RNAseq = RNA synthesized over the region
- Chromatin immunoprecipitation (ChIP) = the binding of RNA pol II to the DNA using anti-RNApol II antibodies
a) What does the peak represent?
b) what type of promoter is being used
c) Is this bidirectional or uni-directional?
d) how many pol II’s are present?
e) ChIP or RNAseq?
a) initiation phase = the binding of a pol II
b) CpG islands
c) bidirectional
d) 2
e) ChIP
a) What does the peak represent?
b) what type of promoter is being used
c) Is this bidirectional or uni-directional?
d) how many pol II’s are present?
e) ChIP or RNAseq?
a) the binding of the Pol II
b) TATA or TATA-less
c) unidirectional
d) 1
e) ChIP
What are the components that makeup TFIID?
- TBP = TATA-binding proteins
- TAFs = TBP associated factors
What GTFs make up the core PIC? What is its purpose?
a) preinitiation complex = TFIID + TFIIA + TFIIB + TFIIF + TBP
b) loads the pol II onto the DNA at the right spot
What GTFs make up the closed PIC? Which one is the most significant?
- Core PIC = TFIID + TFIIA + TFIIB + TFIIF
- closed = TFIIE + TFIIH (most)
TFIIH is known as a multiprotein complex. What does this mean?
This means it has multiple domains;
1. Helicase DNA = unwinds the DNA so transcription can take place
2. kinase activity = phosphorylates the CTD to release the RNA pol II from the promoter sequence
Why is the TFIIH GTF so important?
It facilitates the transition from the initiation phase to the elongation phase
What are the 2 elongation factors that pause elongation? Why do they do this?
a) NELF + DSIF
b) check to see if transcription is ready to begin
After NELF + DSIF cause the attend of elongation what restarts the phase?
CDK9/CycT or PTEFb = a kinase that phosphorylates the CTD and the NELF causing the NELF to fall off which restarts elongation
What are the 7 steps to amplifying DNA using the massive parallel DNA sequencing technique?
- Fragmentation: fragment DNA
- Primers: ligate linkers (primers onto DNA) onto each side of the fragment
- Fix to plate: denature and anneal one of the linkers to a complimentary link that is stuck on a plastic plate
- Get target DNA: Synthesize the fragment (including the other linker that is not annealed)
- Get rid of original DNA: denature and wash away the old fragment of DNA (the synthesized strand contains the target DNA)
- Horseshoe: Form a horseshoe shape w/ the Fragment by annealing the other linker to a complimentary primer that is fixed to the plastic plate
- PCR: Synthesize and amplify the DNA using PCR
Describe the 6 steps associated w/ the dNTP fluorescent part of Massive parallel DNA sequencing
- Cut, denature and wash away one DNA strand from the horseshoe
- anneal a new primer onto the end of the DNA that is not fixed to the plastic plate.
- Then Wash fluorescently labeled dNTPs (each labeled using a differ colour) to see if it binds (if it’s complimentary to the DNA strand)
- once the dNTP is bound use fluorescent imaging to determine the type of dNTP
- Remove the fluorophore
- repeat steps 3 to 5 until who genome is sequenced
What are the 2 types of libraries involved in putting all the dNTPs in the correct sequence wrt massive parallel sequencing? Which one is easier?
- aligned sequencing = where you chop up the fragments at the beginning and keep them in the same order. So by the end, all you need to do is put them together to get the sequence
- random sequencing = Fragmentation is random for this usually causing some overlap due to there being multiple genomes in the sample. This overlap allows for a computer to sequence the DNA. (easier)
We know many ways to sequence DNA but how is RNA sequenced?
Due to RNA being so unstable compared to DNA, we convert the RNA to DNA using an RNA-Dependent-DNA Polymerase. This allows us to sequence RNA no differently then DNA.
Why is massive Parallel sequencing named the way it is?
- massive = generates billions of reads for a genome within a short period of time
- parallel = the Genome a fixed parallelly to the plastic plate during the amplification phase and it organized parallelly during the sequencing phase
