4. RNA polymerase, transcription and transcription initiation

Question 1

What are the three main processes involving RNAs?

Answer 1

RNA involving processes:
1) transcription (initiation and regulation)
2) processing
3) degradation

Question 2

What is the machinery of transcription?

Answer 2

RNA pol II - transcribes coding + nc RNAs - conserved between domains of life - core 5 subunits

3 RNA pol types:
- RNA pol I: rRNA genes
- RNA pol II: protein coding genes into RNA
- RNA pol III: tRNA genes

Question 3

What recruits RNA pol II for transcription?

Answer 3

RNA pol II recruited by TF - TFIID binds at core promoter:
- helps to position RNA pol II at promoter region
- pulls ds DNA strands apart
- helps to release RNA pol II to start elongation

Question 4

What are the factors regulating transcription?

Answer 4

Transcription factors (TFs):
general TFs (GTFs) - for transcription initiation
+ activors (enhancers) / repressors (regulatory TFs when + how much to transcribe - open / close chromatin)

Question 5

What are the methods for studying transcription?

Answer 5

• Footprinting (DNase assay)
• EMSA (protein+ DNA size differences)
• ChIP (Ab)

Question 6

What are the main steps of transcription process?

Answer 6

Transcription steps:
1) recognize TSS (transcription start site) + get RNA pol II into position
2) Start transcribing moving along DNA
3) Elongation
3) Transcription termination (stop codon)

Question 7

What is the structure of TFIID?

Answer 7

TFIID - TF for RNA pol II recruitment at transcription promoter - 2 subunits:
- TBP: TATA binding protein - binds TAT box in minor groove - induces DNA kink
- TAF: main body

Question 8

What is the sequence of events in transcription initiation?

Answer 8

Transcription initiation by GTFs:
1) TFIID binds TATA box at core promoter - recruits TFIIA + TFIIB (BRE element)
2) RNA Pol II binds TFIIF - stabilizes interaction between RNA pol II and others (TBP at TATA) - attracts TFIIE
3) TFIIE attracts and regulates TFIIH
4) TFIIH unwinds DNA + ph RNA pol II tail -> (DNA helicase, ATPase and kinase activity
5) RNA pol II starts elongation when tail gets ph

Question 9

Explain transcriptional elongation process

Answer 9

RNA pol II disengages from GTFs (TFIID) - acquires other proteins for transcription - released GTFs can initiate another round of transcription

Question 10

Function of enhancers

Answer 10

Enhancers bind to distal elements + to GTF cluster -> transcription

Question 11

What are the modes of action of repressors?

Answer 11

Transcriptional repressors work to supress transcription by:
- competitive DNA binding with activators
- blocking bound activator surface
- directly interacting and supressing GTFs (initiate transcription)
- recruit chromatin remodellers that promote close chromatin
- recrecruit deacetylases for histones -> close chromatin
- recruit methyl transferases for histones -> close chromatin

Question 12

What is the exact structure of PIC?

Answer 12

PIC - pre-initiation complex:
- GTFs
- mediator complex
- RNA pol II
- activator (enhancer) bound at distal elements

Question 13

What is an enhanceosome?

Answer 13

Enhanceosome - enhancer bound transcription initiation complex: enhancer + general Poll II machinery + RNA pol II

Question 14

What can be investigated in studying transcription? What methods can be used for that?

Answer 14

What region is bound by TF - footprinting + ChIP
Whar complex is formed by TF binding - EMSA

Question 15

Explain footprinting method

Answer 15

Footprinting DNase assay:
- amplify DNA
- label DNA
- gel electrophoresis for sample 1 (reference)
- bind specific protein to labelled DNA
- digest using DNase / hydroxyl radical - won’t digest protein protected regions
- gel electrophoresis for sample 2 (bands that missing - where protected by bound protein)

Question 16

Explain EMSA method

Answer 16

EMSA - protein binding to DNA assay:
- purify protein of interest
- introduce complimentary DNA
- introduce Ab for the protein
- load and run gel -> compare distances travelled by different samples (DNA only, DNA+protein, DNA+protein+Ab)

Question 17

Explain ChIP method

Answer 17

Chromatin immunoprecipittaion (ChIP) - pulling out protein:
- crosslink TFs to bound DNA sequences
- lyse cells
- cut DNA into small fragments
- Ab for specific TFs - separate from mixture
- reverse crosslink - remove all proteins from extracted DNA - identify DNA sequence => DNA corresponding to TF position will be purified + identified

Question 18

Lecture summary

Answer 18

Question 19

Quiz 1

Answer 19

Question 20

Quiz 2

Answer 20

Question 21

Quiz 3

Answer 21