Mechanisms and Control of Gene Transcription II Flashcards
1
Q
Polymerases in eukaryotes
A
- 3 types, distinguished by sensitivity to a-aminitin
- Pol I = rRNA
- Pol II = mRNA
- Pol III = tRNA + snRNA
2
Q
Promoter
A
- Core promoter = minimal portion of promoter needed for initiation at TSSS
- 34bp us of TSS = bs for POL
- Core promoter binds general TF specific for each type of polymerase
- Pol I promoter = UCE, core promoter, pppA/G
3
Q
Pol I TF
A
- UCR of Pol I binds UBF1 (removes nucleosome)
- Core promoter binds 4 proteins inc RRN3
- TBP interacts w/ TAF 63 + 110
- SL1 recognises promoter + recruits Pol1a
4
Q
Pol III promoter
A
- Unusual = promoter lies ds of TSS
- URS = us of TSS
- Core promoter = +55 and +80
- TFIIIC recognises core promoter w/ zinc fingers
- TFIIIB also binds
- RNA pol III recruited by TFIIIC/B
5
Q
Pol II promoter
A
- Has 6 more proteins: TFIIB,D,A,F,D + H
- Multiple different elements, x conserved, act in combination
- Tata box, BREu/d, Initiator elements, ds elements like DCE
- TBP binds minor groove + crates bend, associates w/ TAFII
- TFIIB binds DNA, TAF2 binds initiator, TAF9+6 interact w/ DPE
- TFIID recruitment = essential
6
Q
Conserved factors in initiation
A
- TATA binding protein
- Only acts at small no. of promoters transcribed by RNA Pol II
- Pol II = SL1, hRRN3 + UBP3
- Pol III = TFiiB, BRF, TFIIIC
- Pol I = TBP + TFP
7
Q
Eukaryotic RNA Pol III TF vs σ70
A
- TBP + σ bend DNA
- TFIIB controls start site selection vs σ
- TFIIE melts DNA vs σ
- Non-template strand captured by TFIIF vs σ domain 2
- Template strand interacts w/ TFIIB finger vs σ domain 3
- TFIIB competes w/ RNA of >10bp for saddle, structural Δ in sigma promotes release
8
Q
Types of promoter transcribed by Pol II
A
- Distinct types of core promoter for transcription initiation by RNA Pol II
- Yeast = 1 type, conserved TSS, also found in mammals, has TATA, Inr, MTE, DPE x CGI
- Type II = drives expression of house keeping genes, 70%, dispersed, have CGI, x TATA
- Type III = developmentally regulated promoter, mixed
- Vertebrates = mainly focused (either single TSS or distinct cluster of start sites) or dispersed (several start sites over 50-100nt, typically found in CPG islands)
- S cerevisiae = just focused, d melanogaster = focus + dispersed or mixed
9
Q
CPG islands
A
- Dinucleotide C followed by G = often methylated at 5 of Cys after DNA synthesis
- Similar to bacteria
- 20% of expected frequency as methylated lys is deam to T
- CpG islands = clusters of C/G dinucleotides that are not methylated, found at promoters
- Methylation prevent TF/promote TF binding
- Have ↑ frequency of bs for TFs just us of TSS
10
Q
Methods for studying promoter/enhancer
A
- Confirm promoter
- Either destroy by deletion of mutation (CRIPSR cas9) + see effect on gene expression
- Make a chimeric gene e.g. to see if HSE really HSE make chimeric gene w/ HSE added to gene w/o HSE
- Or could fuse promoter/enhancer to ‘reporter gene’
- Cor element alone x enough to give ↑ levels of expression of reporter gene, promoter/reporter gene hybrid is introduced into cells + activity of promoter assessed
11
Q
Promoter + enhancer for genes = modular organisation
A
- Human hsp70 gene promoter = modular structure
- Can mix and match regulatory element that binds TF w/ core element
- Enhancers have regulatory element, activates expression from promoter in response to signals
- Enhancers activate a promoter when placed up to 100- bp from promoter
12
Q
How do enhancers work?
A
- Using SV40 DNA
- Has enhancer, core promoter, x promoter regulatory elements
- CG box binds TF SPI, gives promoter w/ low level of expression
- Need enhancer, us of promoter
- C, B + part of A = 72bp repeat, 1 = enough, have 2
- A, B + C contain individual enhansons
- Experiment (fuse TATA box to reporter gene, plate w/ ↑ cells, transvect TATA + reporter in, 72bp repeat gives enhancer + cells survive, then use just C, some survive, take + grow w/ G418, see duplicated C, same w/ A + B
- Showed 2 protoenhancer needed
- Enhancers often composed of same sequence of elements found in promoter e.g. AP1 bs
- SV40 has multiple TF, bs mutually exclusive
13
Q
Superenhancer
A
- Control a gene cluster on chromatin loop anchored by CTCF DNA binding protein at each end
- Interact w/ Mediator
- E.g. in a or B globin loci, confer tissue + stage specific global mRNA expression
- Resembles an enhancer, 5/6 DNAse hypersensitive sites
- MFine tune gene expression
- Evidence (mRNA spiced + placed in human B global gene into mouse, took larger parts until cloned region of 60kB + found global genes expressed where should be)
14
Q
Transcription factors
A
- Binds to promoters + enhancers
- Proteins bind to DNA, especially in chromatin, creates DNAse I hypersensitive sites bends DNA
- ‘Footprint’ next to DNA hypersensitive site, protein binding means DNAse I x cleave
- Factors that influence ability of TF to bind = TF-TF interaction, TF-cofactor interaction, DNA modifications, DNA shape, genomic context
15
Q
AP1
A
- TFs often bind to promoter + enhancer as dimer
- Homodimer of C-Jun or heterodimer of C-Jun/c-Fos bind w/ different affinities
- AP1 recognition site has dyad symmetry
- AP1 TF bind AP1 have diff affinities (Fos:Jun hetemrodimer binds AP1 30x ↑ than Jun homodimer)
- Fos + Jun have 2 1/2 sites in NTD, CT leucine zipper
- Specificity of interactions btw Fos/Jun - residues of hydrophobic face (Leu zipper a+d) + residues surrounding hydrophobic face (e+g)
- Fos:Fos homodimers x form
16
Q
Detecting TF bound to DNA in vitro
A
- ChIP
- EMSA (radioactively labelled DNA incubated w/ nuclear protein, run on gel, compare to DNA w/o protein, complexes migrate slower than free DNA, Ab raised against protein complex, can Δ conditions
17
Q
Regulating processes via cooperativity
A
- E.g. reprogramming
- Most cells are differentiated
- Can add genes encoding TF Oct4, Klf4, could reprogram somatic cells back to pluripotent state
- If add different signals, can re-differentiate into different cell types e.g. fibroblast → neurone
- As reprogram, switch off somatic enhancers + switch on enhancers for pluripotency
18
Q
Modular TF experiment
A
- ‘Domain swap’ experiment
- GAL4 has NT DBD + TAD
- WT DBD + TAD binds enhancer us activator sequence → transcription
- Take away CTD + add different oligomerisation domain
- Replace activator w/ Lex operator
- Mix and match TAD + DBD
- Y2H (Gal4 DBD + bait, Gal4 TAD + prey)
19
Q
Consensus sequence vs responsiveness
A
- Based on steroid-thyroid hormone receptor family TF
- Respond to presence of hormones
- Bind to major groove on DNA
- Thyroid hormone bs = dyad symmetry, have GGT
- Also have DBD, TAD + hormone binding domain
- Experiment = oestrogen receptor + DBD of glucocorticoid receptor, aa substitution
20
Q
How do TF function
A
- us enhancer w/ ↑ bs + promoter w/ ↑ bs, work together
1. (like prokaryotes), A + B work to recruit RNAP + PIC to open DNA + get transcription, RLS = RNAP recruitment
2. Nucleosome depleted region → RNAP recruited → Pol stalls, waits for processing of transcript, RLS = release of promoter into early elongation + escape
21
Q
Enhancer
A
- Multiple transcription bs
- Enhancer activation coincides w/ DNAse I hypersensitivity
- Chromosomes around have specific PTMs
- Associated w/ divergent transcription
- 20x more enhancers than genes
- ‘Superenhancers’ - LCR that control major developmental switch
- Use NET-Seq
22
Q
How do enhancers interact with promoters
A
- Flies = enhancers show specificity for different types of promoter (housekeeping vs developmental)
- Housekeeping genes have proximal enhancers, developmentally regulated = distal enhancer
- Experiment (GALF4 fused to TF of interest)
- Coactivator = help enhancers talk to promoter, facilitate interaction btw TF bound to proximal/distal enhancer + PIC
- Co repressor = prevent enhancer talking to promoter
- Cohesin may stables the interactions
- TFIIH + mediator have CDks, act on RNAPII + help release from promoter
23
Q
Insulators + promoter interaction
A
- Not of insulator binding proteins e.g. CTCF (TF that interacts w/ DNA, needs to bind insulator)
- ICR = another insulator
- Methylation on females = different to paternal
- ICR separates promoter form enhancer
- When ICR = methylated, CTF x bind paternal but does bind maternal (no methylation), stops enhancer talking to promoter
