Topic 8 Transcription Flashcards
1
Q
Differences in replication and transcription
A
Replication:
copies the entire genome once and only once per cell cycle
Both dna strands are the template for new DNA sysnthesis
Transcription:
Selectively copies only certain parts of the genome from one to multiple times
Only one of the dna strands is a template on which rna strand is built
2
Q
What is dna dependent dna sysnthesis and dna dependent rna sysnthesis
A
When dna is made using dna (replication)
When rna is made using dna (transcription using rna pol)
3
Q
Whag is the template strand for rna transcription called
What is the non template one called
Why
A
Non coding strand, antisense, 3-5
Coding, sense, 5-3
Because the sense strand is the exact same as the RNA except it’s has no U
4
Q
What is the purpose of the rna transcript dissociating from the dna a few ribonucleotide from the point of synthesis
A
To allow Multiple transcriptions of the same gene to happen at the same time
Translation can happen rapidly as soon as mRNA is made (can go straight to cytoplasm
5
Q
What is different in rna polymerase and dna polaymerase
A
RNA pol makes rna without using primers, dna pol needs primers
RNA pol has less proofreading mechanisms than dna pol
6
Q
Explain the parts that make up rna polymerase
A
Eukaryotes has 3 rna pol and prokaryotes has 1
Each pol has two alpha and 2 beta subunits
But the eukaryotic pol I,II,III have 7-11 extra subunits that are specific to each pol
7
Q
What are the role of each rna pol in eukaryotes
What is the expection
A
Pol I: transcribes the larger precursor ribosomal rna
Pol 2: protien coding gene
Pol3: tRNA and 5S ribosomal RNA
Even though we say that the pol II makes protien coding genes, it can also make no coding siRNA and microRNA
So they still have overlapping finction
8
Q
What are the steps to transcription
A
Initiation
Elongation
Termination
9
Q
Explain general transcription initiation
A
The promoter determines what region of the dna undergoes transcription
Three steps:
Form a closed complex structure by binding the rna pol to the promoter (this is the pre initiation complex)
The closed complex transformed into an open complex (this is the transcription bubble)
Then the initial transcribing complex makes the first 10 ribonucleotides
10
Q
Explain general transcription elongation and termination
A
Transcription happens 5-3
The rna synthesis continues by unwinding the dna in the front and reannealing it behind
The growing rna emerges from the template and weak proofreading happens
Termination: Transcription stops and rna is released from the complex
11
Q
What is the transcription start site
A
The +1 site
The very first nucleotide that is transcribed
12
Q
Prokaryote vs eukaryotes transcription
A
Prokaryotes:
Have only one RNA pol
Need only one initiation factor (the sigma factor)
Eukaryotes:
Have 3 RNA pol
Need several initiation factors (like general transcription factors) for promoter specific initiation
13
Q
What is needed for transcription in vitro (in the test tube)
A
The core promoter
This includes:
BRE: TFIIB recognition element (binds TFIIB)
TATA: the TATA box element (binds TBP)
Inr: initiatior (binds TFIID)
DPE: downstream promoter element (binds TFIID
14
Q
What is the core promoter
A
Minimal sequence needed for accurate transcription in vitro
15
Q
Whag is special about Inr
A
Inside the Inr sequence is the +1 site where everything downstream of that is transcribed as rna
Anything upstream is non coding
16
Q
What are the important transcription factors that bind to the core promoter in in vitro transcription
A
TBP: bind to TATA box and recruits ~10 other TAFs (TBP Accosiated factors)
TFIIB: bind the PIC after the TBP binds, sets the directionality for transcription, also brigdes between the TBP and pol II
TFIIF: bind to the promoter with pol II, the polII-TFIIF stabilized the DNA-TBP-TFIIB complex and recruits TFIIE and TFIIH
TFIIE: recruits and regulates TFIIH
TFIIH: uses atp to transition the pre initiation complex to open form by melting,
Phosphorylates the CTD (c term domain) of pol II which triggers the pol activity for transcription
Recruited other proteins to do 5’ capping and stabilize the rna
Also used in NER
17
Q
The formation of the PIC is
A
Sequential
18
Q
Explain how the PIC is formed in vitro
A
- The TBP binds to TATA box, TFIID is recruited, 11 TAFs are also recruited
- TFIIA AND TFIIB bind
- The pol II with TFIIF binds
Until this point the dna is in closed form
- TFIIE AND TFIIH bind to complete the PIC, TFIIH melts DNA and makes dna open form
- The CTD of rna pol II is phoporylsted by TFIIH, promoter escape happens and transcription elongation starts with the first few nucleotides being transcribed
19
Q
How does the TBP bind to TATA box in vitro trasncription
A
The TBP has a beta sheet which binds to the minor groove of the TATA BOX
The TBP binding changes the confirmation of the TATA minor groove to bend which widen the minor grooves to a flat structure
20
Q
How does TFIIB bind to the core promoter in vitro transcription
A
Bind to the major grooves lf the promoter region
This sets the unidirectionality of the transcription since it bind only to on side of the promoter (assymetric)
21
Q
What is PIC
A
Pre initiation complex
Protien complex containing pol and general transcription factors
22
Q
What is special about the rna pol II CTD
A
It has repeats for phosphorylation and it isn’t found on pol I AND III
23
Q
Why do we do. In vitro transcription
A
To understand the minimum players/requirement for transcription
24
Q
What is included in in vivo transcription upstream of the core promoter
A
Has the regulatory sequences needed to efficient transcription in vivo since dna is in chromatin form in vivo
Can be very far from the promoter
Includes:
Proximal promoter elements
Upstream activator sequences (UASs)
Enhancers
Boundary elements
Insulators
Silencers
25
Why are addition regulatory protien upstream of the core promoter needed for transcription in vivo
Because the dna template in vivo is in the chromatin form
So we need additional protiens to help relax the chromatin structure to allow for transcription factors to recognize the promoter region
26
What are the activators in vivo transcription
The activators recognize activation site upstream of the promoter
Recruit pol and stabilize the pol : promoter interaction
Bind to chromatin remodeling complexes: this includes chromatin remodeler and HAT so they can modify the nucleosome structure and open up the dna for transcription
27
What is the mediator complex in vivo transcription
Bridges/connects the CTD of the pol and the upstream activators
Regulate the activity of the TFIIH to regulate gene expression
28
What is different about mediator protiens between yeast and humans in vivo Includes
In human They do not bind one at a time in a sequential manner they instead form subcomplex structures called modules
Across yeast and humans some subunits are conserved and some aren’t. Across both they have similar shape and are larger than RNA pol
The Med17 subunit of the module is needed to polII transcription in vivo
The function of most individual subunits is unknown
29
What techniques can we use to understand the subunits in modules
Use immunoprecipitation
Make an antibody to a specific protien X, bind the antibody to the beads, when x comes down all thing is binds to comes down and we can see y and z come down with it using SDS page
This shows what protiens were specific to that module
30
What needs to happen for elongation to occur
The nucelosomes in the front of the polymerase need to be removed for transcription to occur
31
What removes the nucleosomes for transcription elongation to occur
FACT (FAcilitates chromatin transcription) dimer
Has the Spt16 and SSRP1 subunits which disassemble the histones in front of the RNA pol
But also important to reform the histones behind the pol after transcription is done
32
After elgonation has becgun what happens to the initiation factors
The initiation factors dissociate from pol II
Elongation factors are recruited to the CTD tail of pol II, this recruitment depends on the phosphorylation state of the tail
33
What are elongation factors
Give examples
Factors that stimulate elongation
The ELL protien family and TFIIS increase the rate of elongation by limiting the time that pol pauses
TFIIS can also proofread the new transcript
34
What are RNA processing enzymes
When are they recruited
5’ capping enzymes (puts cap on mRNA)
3’ polyadenylation and cleavage factors (to cleave the mRNA from the pol)
Both important for mRNA stability
Splicing factors
35
When are rna processing enzymes recruited
Recruitment of different processing enzymes depends on the phosphorylation state of the CTD tail of the pol II
if the CTD phosphorylated at diff sites, diff processing enzymes are recruited
the capping enzyme is recruited to the 5’ end of the mRNA to Add the 5’ cap and protect from 5-3 Exonucleases
36
When does rna intron splicing happen
As the rna is made, the introns are exposed
The splicing can happen before the rna sysnthesis is completed and while the 5’ cap is being added
37
Explain how the 5’ cap is formed
1. RNA triphosphatase removes the gamma phosphate from the 5’ end of the rna transcript (order is gamma beta alpha)
2. Guanalyltransferase adds GMP (guanosine monophosphate) to the beta phosphate of the rna transcript
3. Mehtyltransferase adds a methyl group to the guanine base from the GMP
This makes the 7 methyl guanylate cap at the 5’ end of the mRNA
38
What is the purpose of the 5’ cap
Stabilized the transcript
And signals that the transcript is correctly processed
39
Explain how the poly A tail is added
When transcription almost finished
The rna pol II transcribes the poly A signal AAUAAA
After this, the phosphorylated CTD tail recruits poly adenylation enzyme CPSF to the poly a signal on the mRNA
The recruited CPSF also recruits CstF to the poly A signal
The RNA pol stil transcribes a few nucleotides but falls off shortly
These cleavage factors cleave the rna downstream of the poly a signal to remove the mRNA from the pol II
Poly A polymerase (PAP) adds ~200 Adenines to the 3’ end of the mRNA
Then the poly A binding protien (PBP) coats the poly a tail
40
What is the purpose of the poly A tail
Stabilized the mRNA (protects from 3-5 Exonucleases)
Signals correct 3’ end processing
41
What are the models of transcription termination
Torpedo model
Allosteric model
42
Torpedo model
A 5’-3’ exonuclease (torpedo) called Xrn (humans) or Rat1 (eukaryotes)
After the poly A signal is added and the second mRNA is being made (since rna pol still transcribing some nucleotides) the torpedo degrades the second rna from pol II
This second rna is unprotected since no 5’ cap
The degredation from the exonuclease is faster than the sysnthesis speed of the pol so the pol II is chased out and dissociates
43
Allosteric model
When the second mRNA is being made, the pol II loses its processivity due to a conformational change
Pol II loses its affinity for the template and dissociates and stops transcription
44
What is the purpose of rna pol I
Why do we need it
Transcribes precursor/ ribosomal rna (non protien coding)
Meaning it only transcribes one gene that serves as the precursor RNA, dedicated to only one gene
We need this because that one gene has many isoforms that need to be transcribed in a very high level whcih is why pol I is dedicated to making all of those isofoms
45
How does pol I transcription happen
The rRNA precursor transcript has the UCE (upstream control element) and the core promoter
These are impotent for the regulation of pol I transcription
UBF protiens bind to the UCE, recruits SL1 to the promoter region
SL1 has TBP + 3 TAFS to bind to the TATA box in the promoter
Then pol I recruited for transcription of that transcript
46
What does pol III do
Transcribes tRNA, 5s rRNA and other small RNA
47
How does pol III transcription happen
The promoter is downstream of the start (+1) site, meaning the transcription starts upstream of the promoter and the promoter is transcribed
TBP recognize the TATA box, recruits TFIIIC to the promoter, leading to recruitment of TFIIIB at the start site (postion order is IIIB,TBP,IIIC)
Then pol III binds to the TFIIIB and displaces TFIIIC to start transcription
48
What is the primary point of gene regulation (first point we can regulate gene expression
Transcription initiation
49
What is transcription controlled by
Activators and repressors
50
What can influence the gene expression
Nucleosomes and their modifiers
Regulation of RNA splicing
51
What elements are upstream of the core promoter and what are the definitions
Promoter proximal elements
Upstream activator sequences (UASs): it’s an enhancer in yeast only found upstream of a gene but not a far distance
Enhancers: tight cluster of regulatory binding sites that affect long distances at either upstream or downstream of the gene
Boundary elements
Insulators: block promoter activation by binding to activators at the enhancer
Silencers
52
What are
regulatory binding sites
Regulatory sequences
Promoter
regulatory binding sites: binding stirs for different transcription factors
Regulatory sequences: the entire collection of regulatory binding sites for a gene
Promoter: region of DNA involved in the binding of the pre initiation complex
53
What am happens to gene regulation in more complex genes
As the genes get more complex , bacteria to human, the regulatory sequences and genes expression is more complex
54
What is a transcription activator in eukaryotes give an example
Explain the structure of it
GAL4 activates transcription of the gal1 gene (galactose) in S cerevisiae by binding to the UAS region
GAL4 is a dimer (made of two monomers that come together) and can recognize palindromic sequences
Has a modular structure: DNA binding domain and activation domain
55
Explain the GAL1 gene UAS
The UAS has four regulatory sequences the each bind a dimer of GAL4
It’s 275 BP upstream of the start site
56
What is special about activator binding to regulatory sequences
A different activator binds to reach regulatory sequences
but all of the regulatory sites dont need to be bound at the same time to regulate gene expression
57
Explain the domain swap experiment
It’s demonstrates the modularity of transcription factors like GAL4
WT gal4 activates transcription of the lacZ gene but Gal4 without the activation domain, the transcription of lacZ doesn’t happen
Then make a hybrid of gal4 and lexA (activation domain from gal4 bind it to the generic LEXa dna binding domain) found that the transcription of lacZ gets activated in the hybrid but not in the lexA dna binding domain
Showed that both the activation domain and the DSB binding domain is required for initiation and activation of transcription
58
Explain the yeast two hybrid assay
Detect protien protien interactions based on yeast transcription factors , and the target protiens function based on the function of the things it’s binding to
protien a (bait) has the DNA binding domain
By itself gives no transcription of the reporter gene because only has one of the essential domains
If mix protien a and a bunch of other protiens with activation domains them together and transcription occurs, this means protien a and b (has activation domain) interact and bind to each other to allow transcription
59
What are the ways to screen for transcription in the yeast two hybrid assay
Have his- Yeast cells that can’t make histidine properly and die without his media
the reporter gene expresses the his precursor so If transcription happens the yeast survive (are self suffienct), if not they die
Also can use GFP gene to see if green colour shows after transcription
60
What can be present in the dna binding domain of a transcription factor
Helix turn helix motif (in homeodomain protiens)
Zinc containing dna binding domains (zinc finger like TFIIIA or zinc cluster like Gal4)
Leucine zipper motif (like the GCN4 in yeast)
Helix loop helix
61
What can be present in the activation domain of a transcription factor
Hard to tell apart because they don’t have defined/distict motifs
So they are instead grouped by their amino acid composition: example GAL4 has acidic activation domain, or some are glu or pro rich
Have sticky surfaces since involved in protien protien interactions (but non specific so binds to any protien)
62
Explain the helix turn helix motif
Two aplha helices separated by a short turn
The recognition helix recognizes specific base pairs in the major groove
The other helix positions the recognition helix into the major groove
63
What are homeodomain protiens
Found in all eukaryotes, example of helix turn helix structure
Have 3 alpha helix , helix turn helix motif
Helix 1 terminal end interacts with the minor groove through arg amino acid to give additional contact
The helix 3 is serving as the recognition helix which interacts with the major groove though ser arg asn amino acids
All together helix 1 and 2 stabilize the 3rd helix into the major groove
64
Describe the zinc contains domain
Have the recognition helix connected to a beta sheet
In between is the zinc ion that coordinates with 2 his (from recognition helix) and 2 cys (from beta sheet)
This coordination makes the structure stable and function properly to act as a dna binding domains
65
Describe the leucine zipper motif
A dimer of two leucine zipper alpha helices
They flank and recognize both sides of the dna major groove
The top of the dimer is a coiled coil domain that has leucine residues to hold he two monomers together
66
Describe the helix loop helix (HLH) protien
Similar to helix turn helix but more loop than turn
Monomer of 1 short alpha helix and 1 long alpha helix comes together with another to form a dimer
Flank on both sides of the major groove for dna binding
67
How can we tell which transcription factor is interaction with what region on the dna
Through chromatin immunoprecipitation (ChIP)
68
Explain ChIP
Used to determine protien DNA interactions , called chromatin because any dna protien complex is called a chromatin structure
Want to find what dna a specific transcription factor (B) was interacting with through the promoter region (so separate from all another TF binding to their promoter)
They cross link/fix the Transcription factors to the dna promoter using formaldehyde
They shear/fragment the dna, do immunoprecipitation with it through anti-B-antibody beads (alpha-B-ab)
Then collect the dna protien b was interacting with and do two things:
Microarray: can tell the sequence of the dna
PCR: to amplify the dna sequence
All to see what DNA protien b was interacting with
69
What are the major functions of transcription activators
Protien recruitment:
Recruiting other TF
Recruiting nucleosome modifiers (to change chromatin structure)
Indirectly recruiting RNA pol
Reciruitng factors needed for initiation and elongation
They work cooperatively and synergistically (together and at the same time) to enhance transcription activation
70
How do activators recruit protiens
Bound to the enhancer region of the DNA upstream of start site
They recruit TFIID and the mediatior directly
Recruit RNA pol indirectly
The effects of its are additive meaning the more interactions with activators, the stronger the stimulation of transcription
71
How do activators recruit nucelosome modifiers
DNA is in heterochromatin (tight structure)
They recruit histone modifiers such as histone acetyl transferase that opens up the chromatin
Recruit nucleosome modifiers like the chromatin remodeling complex that use ATP to physically move nucleosomes and expose the dna (also like the SWI/SNF family of modification complexes)
72
What are enhancers and insulators
DNA sequences:
Enhancers are bound by activators to enhance transcription, far away upstream or downstream from the promoter region
Insulators are between the enhancer and promoter regions, protiens binding to the insulator blocks the signal of the enhancer and turn gene expression off
But if sequence is promoter enhancer insulator promoter , then the transcription can be selectively activated for the left side genes
Also if enhancer insulator promoter enhancer, the downstream enhancer can still activate the promoter upstream
73
Explain the looped out model for enhancer function
Because the enhancer region can fold and contact the pre initiation complex both downstream and upstream by folding
This is why the enhancer is distance and orientation independent
74
What is the LCR (locus control region and what does it do
A gene sequence which regulates the expression of a cluster of genes
In human it’s regulating the 5 globin genes in adult bone marrow which are temporally and spatially regulated in their expression
The expression order is eta gamma g gamma a delta beta
This sequential expression is done by the LCR region that is upstream of these genes
75
In the LCR in human how do we switch. On the globin genes in the correct order
We don’t know yet but the LCR in mouse gives an idea
76
Explain the LCR in mice
They have the GCR (global control region) which works like the LCR
The GCR controls HoxD gene expression which is invoked in body segmentation and the patterning of limbs
Don’t know exactly how it works but now we know that there is a regulatory element other than enhancer and insulator that regulates the gene expression (the GCR/LCR)
77
Explain the synergistic (combined) direct effects of activators
1. Two activators can bind to their sites and directly interact with each other
2. The two activators are spaced further apart and interact with a common protein to bridge the two activators
Together they have stronger transcription activation than alone
78
What is synergistic
Different activators can recruit a single protien by touching different parts of that protien
The effect of the activators working together is greater than the sum of individual alone
Synergy serves as a checkpoint to ensure proper signals are received
79
Explain the indirect effects of activators
Have the coiled nucleosome structure and Activetor B cant bind
1. Activator A recruits a nucleosome remodeler which reveals a binding site for activator B
2. Binding of Activator A unwinds the nucleosome , this reveals the binding site for activator B
Shows how they still work together synergistically
80
Explain the real life example of synergy with activators for S cerevisiae
S. Cerevisiae yeast has a and alpha haploid mating type, to express, the mat locus has to be digested by the endonuclease HO
This HO gene then needs to be expressed , but it is only expressed in the mother cells not daughter cells (daughter cells have HO expression suppressor)
This expression of HO is only in the G1-S transition of mitosis
To make the HO gene, first SWI5 (activator) binds to the activator region upstream and recruits chromatin remodeler and histone acetyl transferase to open the chromatin
The region between the first HO nucleotide and the activator region is now open for SBF (activator) to bind there
This SBF turns on the transcription of HO
Shows how the activators work synergistically/cooperatevely to activate expression
Swi5 is only active in mother cells, SBF only active in the correct cell cycle stage
81
Explain the real life example of synergy with activators for the human beta interferon gene
Beta interferon gets expressed upon infection
Upstream of the interferon genes is the enhancer region but its is bent and inaccesible
Architertural protiens HMGA1 bind to the enahncer region which recruit the histone modifiers to straighten out the enhancer region
This allows other activator: Jun/ATF, IRF, NF- kappa B, to bind to the enhancer region
form a complex called the enhanceososome to turn on Beta interferon expression by HMGA1 keeping the enhancer straight
82
What are the ways that transcription repressors can work
Only in prokaryotes: They block RNA pol binding sites by binding to the promoter
Interact with the RNA pol at the promoter to inhibit initiation of transcription
Competition: overlapping dna binding sites of the repressor and activator makes it so that when the repressor binds the activator is blocked
Inhibition: binding of repressor allows the repressor to interact/occlude with the activator to stop its function
Direct repression: Interfere with the activity of activators, mediators, or rna pols at the promoter to inhibit initiation of transcription
Indirect repression: Recruit histone modifiers like deactylase to further compact the chromatin structure and stop expression
83
Explain the combinatorial control of activators and repressors
Mutiple combinations of activators/ repressors on a single gene come together to allow regulation and activation of gene expression
A common regulator is found in the promoters of different genes (gene a and b) to regulate their expression and turn in multiple genes at the same time
They also each have their cell type specific regulators
84
Explain real life case of combinatorial control
S cerevisiae expressing its specific mating type
Has three types of cells: alpha haploid, a haploid, and a/alpha diploid
Each has a mat locus and each express their own cell type specific regulators
For a: a1 regulator
For alpha: alpha1 and alpha 2
For diploid: a1 and alpha2 (repressor)
In each of these 3 cell types they also have a common regulator called Mcm1
These regulators work in combination to express either the a, aplha, or haploid specific genes (aSG, alphaSG, hSG)
85
Explain each case for the mating type genes in S cervisae
These regulators work in combination to express either the a, aplha, or haploid specific genes (aSG, alphaSG, hSG) in each cell
a cell: hSG no regulators bound, hSG is expresss and cell stays in halpoid stage,
aSG has mcm1 bound to express aSG
Alphas g has nothing bound, gene off
Alpha cell: hSG no regulators bound, hSG is expresss and cell stays in halpoid stage,
aSG has mcm1 bound but also alpha 2 as a dimer (repressor) to stop aSG expression
The alphaSG promoter region has weaker binding to mcm1, so has alpha1 and mcm1 bound to allow tighter binding, turns on alphaSG
A/alpha diploid: hSG has a1 and alpha 2 bound, hSG is repressed by alpha 2 and cell stays in diploid stage
aSG has mcm1 and the alpha 2 dimer repressor so aSG off
There is no alpha1 in the diploid cell so alphaSG not expressed
So all three genes off for cells to stay in diploid stage
All of these combinatorial regulation of activators and repressors can help change cell type
86
How else can gene/transcription expression be regulated
What are they called
By signal transduction pathways
JAK/STAT pathway
MAPK pathway (mitogen activated protien kinase)
87
Explain the JAK STAT pathway
Have RTK, the cytokine ligand comes in
Receptor dimerizes, intracellular domain auto phosphorylates tyrosines, receptor activated
STAT with has SH2 domain which recognizes the phosphotyrosines, then STAT dimerizes
Once dimer, STAT goes into nucleus as a transcription activator to activate gene expression
STAT: signal transducer and activator of transcription
88
Explain the MAPK pathway
Have RTK, growth factor ligand, dimerizes, autopohsphoylates
This recruits adaptor with sh2 domain like SOS (which is a gef: guanine nucleotide exchange factor) and grb2
These bind gdp RAS (a small gtpase), whcih becomes active in GTP form (due to SOS)
The active RAS actives mapKKK to activate mapKK then mapK
When mapK active, it phosphorylates transcription activators like JUN to send them to nucleus and activate beta interferon expression
89
What is RAS
Why is the called MAPK mapkk mapkkk
Earliest discoevered oncogene
Named in order of discovery
90
How can gene silencing regulation happen
By histone modifiers and nucleosome remodelers forming heterochromatin
91
Explain the transcriptional silencing by heterochromatin formation (nucleosome remodeled and histone modifiers)
Most heterochromatin is at Telomeric regions because more packed and less transcription but below that is euchromatin
This happens because when telomere is being formed RAP1 dna binding protien binds then recruits sir2 histone deacetlyase (closes chromatin structure)
Also recruit sir3,4 which extends/spread the heterochromatin to a larger region to make larger telomeric closed regions
92
Where is heterochromatin mostly found
In telomeres and centromeric regions
Centromere has rRNA repeats which are very redundant and a lot of it isn’t needed, this is why more compact so less expression of that ribosomal rna
93
What are sir protiens
Yeast telomeric SILENCE INFORMATION REGUALTOR protiens
94
What can stop/regulate the spread of heterochromatin silenced region
Insulator elements can block the spread of histone modifications
Histone methyl transferases (close down the dna structure) repress the spreading of SIR2 mediated silencing (since now silencing using diff method)
Methylation of the histone H3 tail: can either increase or silence transcription (h3K4 methylated increase, h3k9 decrease
95
How can dna be modified to silence gene expression (silencing by dna modification)
DNA has cpG islands (CG repeats in the promoter region)
The cytosines get methylated on the 5’ end of this ring to become 5’ methyl cytosine
This causes leakage of gene suppression, meaning not fully suppressed
To fully suppress, protien MeCP2 need to bind to the 5 Me cytosine and recruit histone modifiers/nucleosome remodelers which fully close chromatin and silence
Sometimes want full silence not just a little
96
Explain how imprinting can determine gene expression in the offspring
Have maternal and paternal chromosome
The maternal chromosome: igF2, ICR (insulator), H19, enhancer
has enhancer downstream of h19 and igf2. H19 (non conding rna that suppresses cell division) is transcribed. The ICR insulator blocks the enhancer from also expressing igF2
Paternal: the H19 and the ICR insulator region is methylated. Since no insulator or h19, enhancer expresses igF2
Igf2 (insulin growth factor 2) allow cell division
This is the balance between igf2 expression and h19 expression between maternal and paternal balances cell division
97
Explain the disorder that happens when the imprinting is wrong
Explain symptoms
Beckwith-wiedermann syndrome
Caused by overexpresion of paternal genes (more igf2), or mutation in maternal genes (less H19) on chromosme 11
This result in over activity of igf2 so overgrowth of the cells
Overgrowth, enlarged tongue, midline abdominal wall defects, severe hypoglycaemia (low blood sugar after birth), increase risk of childhood cancers
98
Explain how the dna methylation can go from one generation to the next and what happens
Modification Passed on through cell division
the CpG sequence is palindromic meaning the opposite strand also has the CpG modification. After replication this needs to methylating needs to be added again to the daughter strand
Upon replication the hemimethylated CpG are methylated by a maintence methylase
The maintence methylase only recognizes the hemimethylates cytosine not the unmethylated one so that only the ones that are supposed to be methylated are methylated
99
Explain the speculations of why a1 doesn’t bind in the a cell hSG but does in the diploid cell hSG
In the diploid, a1 is expresses and acts as a corepressor to bind to alpha 2 and stop hSG transcription
In the a cell, alpha 2 isn’t expressed, so a1 doesn’t bind to the hSG as a corepressor
Or
a1 gets degraded very fast so in a cells there is no concentration of a1 to bind to hSG
in diploid cells the alpha 2 stabilized the a1 so it doesn’t degrade very fast and can now bind to hSG
