Phil Mitchells lectures. Flashcards
What is the definition of transcription?
‘Recognition of DNA sequences by DNABP which allows targeting of RNAP to specific genes.’
What is an example of a ‘high rate’ gene?
Pol I transcriptional unit.
How can you visualise transcription?
Miller spreads.
What can you see on Miller Spreads?
The density of polymerases.
On a miller spread where are the RNA molecules that have been expressed more?
On the right.
If genes have the same copy number they are expressed at the same level. True or false?
False.
What is the definition of a transcription factor?
Proteins that recognise certain DNA sequences.
Is the promoter region large in Euks and Proks?
Euks.
In what two ways can TF regulate gene expression?
- Stimulate RNAP.
2. Alter the structure of chromatin.
When the chromatin is looser is it transcribed more or less?
More.
What are Barr Bodies?
The condensed form of the X chromosome.
What are receptors in the cytoplasm called?
Nucleoreceptors.
Name one type of pathway that is only activated for limited periods?
Cell growth and divison.
What type of receptor is the Her2 receptor?
Surface.
What drug can block the Her2 receptor?
Herceptin.
What level are most genes regulated at?
Transcriptional. (Euk and Prok.)
Do post transcriptional events matter more in proks or elks?
Euks.
Most human genes contain introns with one main pattern of DNA splicing. True or false?
False, most have multiple patterns of splicing.
What do multiple splicing patterns of eukaryotic genes result in?
More than one protein per gene.
What percentage of inherited genetic disorders in humans are a result of pre-mRNA splicing?
30%.
In humans what is the most common genetic disorder caused by pre-MRNA splicing?
Spinal Muscular Atrophy- causes lack of lung function an death at 2-3 years.
Gene expression can be regulated post-transcriptionally in what two ways?
- Level of translation.
2. mRNA stability.
Why are some transcripts localised to specific regions of the cell?
Ensures targeted expression.
Ribonucleases provide activities that are central to ________, _______ ________ and _________.
- RNA processing.
- Quality control.
- Degradation.
What is the expression called when the gene can be switched on?
Inducible expression.
What is a model of a inducible genetic control system in budding yeast?
GAL switch.
What is the major degradation machinery in eukaryotic cells?
Exosome.
What are the four major steps in the general pathway of eukaryotic gene expression?
- Chromatin remodelling.
- Processing of nuc. MRNP.
- Translation.
- mRNA turnover.
When does GAL induce a signal?
When galactose is in a medium.
How are most genes expressed?
Constitutively.
Eukaryotic transcriptional control helps the cell respond to the environment, like in prokaryotes. Why else is it important (3 reasons)?
- Celluar differentiation.
- Development.
- Cell signalling.
What can lose to control of regulated gene expression in multicellular organisms result in?
Developmental defects and cancer.
Apart from RNAP directed area being much larger than the directed are in proks, what else is it?
Much more dispersed.
Many genes can be influenced by a larger number of factors and sites in proks than in euks. True or false?
False, this is true for euks.
Can a gene be regulated by different factors acting on different promoter regions at different times?
Yes.
Transcription can be subject to control by multiple sets of regulatory factors as a result of more than one signal. What can this result in?
The generation of RNA transcripts with distinct 5’ ends from differently regulated promoters.
What does the sex lethal gene control?
Sex determination in fruit flies.
In what gender of fruit fly does the PE (young) promoter express the sex lethal gene?
Female.
In what gender of fruit fly does the PL (adult) promoter express the sex lethal gene?
Male and female.
What sort of transcripts do the PL and PE promoters generate in fruit flies?
Distinct but overlapping.
Is the PL or the PE promoter further upstream?
PL.
What is a Cis- regulatory element?
Regions of non coding DNA that regulate the transcription of nearby genes.
What do highly inducible genes contain?
A TATA box.
Where is the TATA box found?
30 nucleotides upstream of the transcription start site.
Highly inducible genes are highly expressed. True or false?
False.
What is the ‘real’ meaning of the promoter?
The region in which RNAP binds to upstream of gene.
Only some genes have an initiator. True or false?
True.
What are initiator sequences?
Highly degenerate sequences around the transcription start site.
Why are CpG islands formed?
As CG tends to be under represented and all the genes cluster together.
What is a CpG island?
Clusters of CG bp in the promoters of some genes.
What part of the CpG island is methylated?
m5C.
Where are CpG islands methylated?
Within the genome.
When are CpG islands unmethylated?
In transcribed genes.
Genes from CpG islands can be transcribed from multiple sites. True or false?
True.
What is the purpose of deletion analysis?
To find regulatory elements within a promoter region.
Describe deletion analysis.
Deletion analysis is used to find regulatory elements within a promoter region. Short sections of the promoter regions are removed from either end (or both) and the sequence is inserted into a vector and cloned upstream of a suitable reporter gene. Can then assy the extracts for the level of BG or luciferase protein.
Does linker scanning mutagenesis or deletion analysis allow you to see regulatory elements within in a promoter region?
Deletion analysis.
Does linker scanning mutagenesis or deletion analysis allow you to determine short regions/ sequences and their requirement in transcriptional control?
Linker scanning mutagenesis.
Describe linker scanning mutagenesis.
Short regions within the complete promoter region are assayed for their requirement in transcriptional control. Short, overlapping sequences within the promoter region are mutated to generate a series of constructs, each containing randomised nucleotides within a specific region of the same length DNA.
What gene was linker scanning mutagenesis first carried out on?
Thymidine kinase gene.
What three sequences were found in the thymine kinase gene via linker scanning mutagenesis?
TATA box, and two promoter proximal elements (PE1 and PE2.)
What are enhancer elements?
DNA sequences found long distances away from the transcription start site which can increase transcription levels.
Although most enhancers are site specific, what enhancer works on most mammalian genes?
SV40.
What is the size of the SV40 enhancer?
366bp (small).
What direction does the SV40 enhancer work in?
Both.
Enhancer elements are found close to where SV40 binds. True or false?
False.
Where are enhancers often found?
Expression plasmids.
What is SV40 enhancer composed of?
Multiple elements that collectively have maximum activity..
Describe an RNA detection assay.
Hybridise each RNA to one labelled DNA molecule. Add S1 nuclease which degrades ss RNA but not RNA bound to DNA. Analyse amount of detected fragment to determine RNA level.
What is found in the promoter region of a typical mammalian gene?
TATA box elements and a number of promoter proximal elements.
Where are promoter proximal elements often found?
200 nucleotides upstream from the transcription start site.
What regulatory element can be found in introns?
Enhancer elements.
How far away from the transcription start site can enhancer regions be found (in both directions)?
10s of kilobases in both directions.
Yeast genes are smaller than mammalian genes. Where are the TATA box elements located?
90 nucleotides upstream of the transcription start site.
Yeast has less regulatory elements. What are the two options it can have?
- Upstream activating sequence (UAS).
2. Upstream regulatory sequence ( URS).
What do yeast genes tend not to have?
CPG islands.
What do most yeast genes not have?
Introns.
Specific transcription factors are found upstream of every gene. True or False?
False.
How do specific transcription factors alter gene expression?
- Indirectly interact with polymerases.
2. Interact with the chromatin.
Why do specific transcription factors tend to function in groups?
Allows a wider diversity of complexes.
What type of structure do transcription factors have?
Modular domain.
What are general transcription factors required for?
Assembly of RNA polymerase molecules at the beginning of genes.
Explain the process in which general transcription factors act.
The TATA box binding protein (TBP) in TFIID binds to DNA. This recruits TFIIB. RNAP can then bind with TFIIF. TFIIE and TFIIH then recruited.
In what groove does the TATA box bind?
Minor.
What does TFIIH do?
Unwinds double stranded DNA for transcription.
What is needed in addition to general transcription factors for activity at the RNA pol2 promoters?
Mediator complex.
What is the mediator complex made form?
Over 30 different proteins.
The mediator complex is very conserved. True or false?
True.
What are specific transcription factors?
Regulatory proteins that stimulate or repress the basel level of transcription by RNA polymerase II.
What does basel mean in regards to transcription?
Non stimulated, non repressed level of transcription that is independent of additional factors.
What makes up basel transcription?
The mediator complex and general transcription factors.
In prokaryotes transcription factors interact with RNA polymerase directly. What happens in eukaryotes?
Polymerase is regulated indirectly through interactions with the mediator complex or by changing the structure of chromatin.
What percentage of human genes encode for transcription factors?
10%.
Is the mediator complex always bound to pol2?
Yes.
How many specific transcription factors are found in yeast?
Over 150.
How many specific transcription factors are found in humans?
Over 3000.
What does transcriptional regulation involve?
Combinational control by multiple STFs.
What two domains do STFs have?
DNA binding domain and an activation/repression domain.
What do STfs bind to?
Coactivators and corepressors.
What separates the two domains in a STF?
Flexible linker region.
What does the regulatory activation/ the regulatory repression domain bind to?
Other proteins that are involved in transcriptional regulation.
What does the Gal4 activator do?
Induces expression of genes responsive to galactose.
What is a UAS?
Upstream activating sequence. It is found in the promoter region in response to galactose.
What are the DNA binding domain and the activation domain of Gal4 fused separately to experimentally?
‘Bait’ and ‘Prey’ proteins.
When can the Gal4 protein promote transcription of the reporter gene containing a UAS for gal?
When the bait and prey proteins interact.
Deletions of proteins such as Gal4 in the N and C terminal showed the modular structure of STFs. A small deletion in the DNA binding domain showed both domains lost their binding ability. What terminal was this found at?
N.
What three reporter genes were use in the yeast two-hybrid analysis (bait and prey experiment.)?
His3, ADE2, LacZ.
Is the bait or the prey protein the protein of interest in the yeast two-hybrid analysis?
Bait.
What can be used to make a series prey proteins?
A library of plasmids.
The yeast two-hybrid analysis assay is used to look at interactions between yeast proteins. True or false?
False, it can look at interactions between any proteins.
What will yeast mutants that have mutated ADE be?
Red.
What do enhancer regions consists of?
Multiple regulatory elements/ control elements that bind to transcription factors in a cooperative manner.
DNA binding proteins can bind to the minor groove in the DNA within the enhancer region causing it to bend. What is an example of such a DNA binding proteins?
HMG1.
How is an enhancer complex containing multiple subunits assembled?
Through a large number of protein-protein and protein-DNA interactions.
What is the best characterised enhancer?
B- Interferon complex.
What sort of dimers can transcription factors function as?
Homo or hetero dimers.
What type of dimers is the enhancer complex B-Interferon made from?
Heterodimers.
When will the heterodimers found in the enhancer complex B-interferon bind to adjacent regulatory elements?
When HMG1 is present.
How can multiple weak interactions between STFs be increased?
Through interactions within the DNA.
Using genetics, where can TF’s be isolated from?
Yeast, flies and worms.
What is needed for galactose utilisation?
Gal2 permase, and the enzymes Gal1, Gal7 and Gal10.
What regulates the response to galactose?
- Gal80 regulator protein.
2. Galactose sensor G3.
How have TF from mammalian cells been isolated?
Biochemical approaches.
What transcription factor regulates the GAL switch in yeast?
Gal4.
Gal80 allows the genes for galactose utilisation to be transcribed. When is its nuclear pool depleted?
When Galactose binds to Gal3, meaning Gal80 can bind to Gal3 in the cytoplasm.
What can be used to isolate transcription factors that interact with a regulatory element with a known sequence?
Gel-shift assays.
In gel-shift assays what is incubated together?
Short double-stranded DNA molecule with the regulatory element is incubated with a fractionated nuclear extract.
Why can gel-shift assays recognise fractions with the cognate-specific transcription factor?
Because DNA complexed to proteins moves slower than normal DNA.
Multiple cycles of a gel-shift assay allow purification of DNA binding proteins. Are these always transcription factors?
No.
Explain the in vitro assay for transcription factors.
Purified DNA binding protein is incubated with a sample containing the cognate promoter region and a sample that does not. Can they see if the protein stimulates or inhibits transcription. IN VITRO.
Explain the in vivo assay for transcription factors.
Two plasmids are put into host, one with the suspected TF and one with the reporter gene under control by the cognate regulatory element. Measure amount of rMRNA produced. If the DNABP is actually a tf expect the amount of rMRNA to be increased.
In vivo assays of transcription factors allows you to see if the isolated DNABP from gel-shift assays is actually a TF. What else can it help you do?
Mutational analysis of the STF and the specific promoter region to see which parts are the necessary parts.
Loss of function of repressor proteins can cause increase gene expression. What is an example of a protein this affects?
Wilms Tumour Protein (WT-1).
What can mutations in the Wilms Tumour Protein (WT-1) cause?
Nephroblastoma.
Is more known about activation domains or DNA binding domains?
DNA binding domains as they are less varied.
Are there a group of transcription factors with highly acidic or basic activation domains?
Acidic- Gal4 is included in this.
When are acidic activation domains poorly structured?
When they are not associated with any other proteins.
When are acidic activation domains well structured?
When they are associated with co-activators.
Can the activator/ co-activator interaction be regulated?
Yes.
What is CREB?
cAMP response element binding protein.
What is CREB an example of?
A transcriptional activator.
What is CREB’s co-activator called?
CBP binding protein.
When can the transcriptional activator CREB interact with the co-activator CBP?
When it is phosphorylated.
For CREB to interact with CBP it has to be phosphorylated. What does this phosphorylation?
PKA- protein kinase A.
What regulates PKA, the kinase needed to phosphorylate CREB?
cAMP.
What does the transcriptional activator CREB bind to?
cAMP response elements- CREs.
CREB, the transcriptional activator, can bind to CREs in the genome. Where are CREs found?
Upstream of genes transcriptionally activated by the cAMP-dependant signalling pathway.
Acidic activation domains are poorly structured when not bound to other proteins. How are domains of nuclear-receptors different to this?
They have structure domains.
When are the activation domains of nuclear receptors active?
When they are bound to their ligands.
What triggers the interaction between the transcriptional activator and the co activator in nuclear receptors?
A hormone which causes a conformational change.
What does estrogen bound to its receptor generate?
Hydrophobic pocket that binds an amphipathic helix.
Estrogen bound it its receptor generates a pocket. What can this do?
Bind a amphipathic helix from the coactivator.
What can bind to the hydrophobic pocket of the estrogen receptor causing it to be sterically blocked?
Tamoxifen.
How do transcription activators and repressors bind to regulatory elements in the DNA?
In a sequence specific manner.
What groove in the DNA do activators/repressors bind to?
Major.
Transcription activators/ repressors bind to regulator sequences in the DNA through a sequence specific manner. How does this actually happen?
An alpha recogintion helix from the protein inserts into the DNAs major groove.
How are contacts made between the transcription activator/repressor and the DNA? What type of contacts are made?
Between amino acid residues in the recognition helix and the edges of bases within the DNA. The contacts are normally both hydrophobic and ionic.
Where is the homeodomain fold only found?
In eukaryotes. It is very similar to the helix-turn-helix found in phage transcriptional repressors such as cro.
How many amino acids make up the a-helical homeodomain?
60
What encodes for the a-helical homeodomain?
Homeobox.
What percentage are nucleotides are conserved in the homeodomains of different eukaryotes?
75%.
What do homeodomain proteins play a key role in?
Embryogenesis, development and gene expression patterns.
How are the genes encoding many homeodomain proteins found?
In Hoax gene clusters.
What does the order of Hoax gene clusters correlate to?
Spatial expression within the embryos.
What is the homeodomain fold?
A transcription factor.
Whats the biggest family of transcription factors in mammalian cells?
Zinc finger proteins.
What are the two major forms of zinc finger proteins?
C2H2 zinc finger and the C4 zinc finger.
Zinc finger proteins domains are folded around a central zinc ion through 4 contacts with 4 amino acid residues. What two combinations of amino acids can this be?
4 cysteine (C4) or 2 cysteines/ 2 histidines (C2H2).
How are the residues involved in the zinc finger model found in the primary sequence and why?
Two closely separated pairs- because the introverting looped peptide sequence when the structure is drawn out.
Transcription factors contain zinc fingers in fours. True or false?
False, they can contain multiple though.
How do proteins with zinc fingers bind DNA?
As a monomer.
What type group of zinc finger proteins contain nuclear receptors?
C4.
What are nuclear receptors transcription factors for?
Steroid hormones.
How many zinc finger domains are found in C4 zinc finger proteins?
Two. DNA is subsequently bound as a hetero or homo dimer.
What are basic leucine zipper proteins?
Coiled coiled dimers linked through parallel amphipathic helices with leucine in every 7th position.
What type of dimer is a leucine zipper protein?
Homo or hetero dimer.
What does the coil coil structure of the basic leucine zipper protein refer to?
Extended alpha helix regions of the tow subunits coiled around each other.
What charge status are the a helices involved in the coil coil in the basic leucine zipper?
Amphipathic, with the hydrophobic side making contact with the other subunit.
What contributes to the strong interactions between the two helices of the leucine zipper proteins?
The fact that every seventh residue is a leucine residue, contributing massively towards the hydrophobic interactions. These represent the ‘zip’.
Many transcription factors are leucine zipper proteins. How do these interact with the DNA?
The alpha helices of the zipper are extended at their N terminal and grip the DNA at adjacent major grooves.
What contacts are present between the leucine zipper protein and the DNA?
Base specific contacts and electrostatic interactions between the basic residues and the phosphodiester backbone.
What does the general term ‘basic zipper’ refer to? (bZIP)
The larger group of proteins that can have residues other than leucine along the dimerisation surface.
There is a domain similar to the bZIP domain where the extended alpha helices are interrupted with loop regions. What is this domain called?
Basic-helix-loop-helix. (bHLH)
What generates alternative heterodimers?
The combination of different transcription factors.
What is increased due to the formation of alternative heterodimers?
The diversity of DNA sequences that can be recognised/ expands the way the factors can be regulated.
Apart from the formation of alternative heterodimers, what else is another combination control mechanism of transcriptional control?
Binding of unrelated DNA binding proteins.
What may two proteins that bind weakly to adjacent sites within the DNA be able to form in isolation?
A stable transcription complex through intermolecular protein-protein interactions.
Two proteins that would normally bind to adjacent sites in the DNA may be able to bind to each other in isolation. What is this called?
Cooperative binding.
What is cooperative binding dependant on?
Relative position of the two recognition elements within the DNA.
Cooperative binding can happen between distinct DNA binding proteins. What is an example of this?
NFAT and API bind cooperatively to the IL2 promotor-proximal region.
What does cooperative binding between NFAT and API require?
Specific spacing of the recognition site within the DNA.
Some transcription factor dimers recognise the same DNA sequence. What does this result in?
Different activation domains together in different combinations at the same site.
Some transcription factor dimes recognise different DNA sequences. What does this result in?
An increased number of potential targets.
What type of DNA binding protein can bind to repressors?
bZIP.
The structure of eukaryotic chromosomes varies during the cell cycle and the chromosomes of interphase nuclei are less condensed meaning they are yet to be packaged into chromatin. True or false?
False. The chromosomes are still highly packaged into chromatin.
What is packaged into chromatin?
Chromosomes, RNA and protein.
Chromatin contains 50% of histone protein by mass. True or false?
False. It contains 50% of histone and non histone protein by mass.
What percentage of an average cell nucleus is taken up by DNA’s double helix?
5%.
What percentage of a sperm nucleus is taken up by the DNA double helix?
60%.
DNA packaged within the chromatin of a somatic cell during interphase must remain accessible to proteins involved in what?
- Transcription.
- Replication.
- DNA repair.
Is DNA always accessible?
No.
When chromatin is extracted from the nucleus under low salt concentrations and in the absence of mg2+ what is it said to resemble?
‘Beads on a string.’
Chromatin extracted from the nucleus under low salt concentrations and in the absence of mg2+ is said to resemble ‘beads on a string’. What are these beads/ whats the string?
The beads are the nucleosomes and the string is linker DNA.
What do nucleosomes consist of?
DNA would around a core of histone proteins.
How big are nucleosomes in diameter?
10nm.
When chromatin is extracted from the nucleus under physiological conditions (salt and mg2+ present) it does not resemble ‘beads on a string’. What does it resemble instead?
More compact 30nm fibres.
Can you move between the differently compacted chromatin fibres?
Yes.
What is the most compact chromatin fibre you can isolate?
100nm.
What are the core histone proteins?
H2A, H2B, H3, H4.
How many of the each of the core histone proteins is the histone made up off?
2 of each, 8 in total.
What dimers are formed between the core histone proteins?
H2A/H2B H3/H4 (there are two of each.)
How do the dimers made from the core histone proteins interleave?
In a ‘handshake like interaction.’
Are histone found in prokaryotes or eukaryotes?
Eukaryotes.
What can the core of a nucleosome be described as?
Octameric.
What way does the DNA wind around the histones?
Left handed.
What are the specifications of the DNA turn around a histone?
147bp makes a 1.7 left handed turn.
The linker region between the nucleosomes is a constant length. True or false?
False. The length can vary.
What can the length of the linker DNA vary between?
10 and 100 base pairs. IN MULTIPLES OF TENS.
Why are DNA linker regions always in multiples of tens?
As that is one helical turn.
What histone protein is bound to the DNA as it enters and exits the nucleosome?
H1.
What can histone proteins be described as?
Highly basic.
Are histone proteins globular?
Fairly, but they also have non globular tails.
What are three examples of some histone variants found in some nucleosomes?
H2AX, H3.3, and CENP-A.
What are the flexible domains of histones also called?
Histone tails.
What core histones have a globular tail only at their N terminus?
H3 and H4.
What core histone proteins have a globular tail at both termini?
H2A and H2B.
H2AX is a histone varient. What is its role?
Functions in DNA repair and is widely distributed throughout the genome.
CENP-A is a histone varient. What is its role?
Involved in attachment of the chromosomes to the microtubules during mitosis.
H3.3 is a histone varient. What is its role?
Found in actively transcribed genes.
What is most DNA that is packaged in sperm packaged with?
Arginine rich proteins called protamines.
What can post translational modifications of core histones lead to?
Changes in the chromatin.
What are the two forms of chromatin?
Heterochromatin and euchromatin.
Chromatin is now distinguished through the use of epigenetic markers, how was it originally determined?
Cytologically.
What type of chromatin is more densely packed ?
Heterochromatin.
What are the two types of heterochromatin?
Constitutive and facultative.
Where is constitutive chromatin found?
Repetitive DNA sequences
Satellite DNA
Centromeric DNA regions
Telomeric DNA regions.
What type of heterochromatin is stably inherited through cell division?
Constitutive heterochromatin.
What is the main difference between constitutive heterochromatin and facultative heterochromatin?
Facultative heterochromatin can become decondensed.
What type of chromatin is transcriptionally active?
Euchromatin.
What size fibres can euchromatin be found in?
10nm and 30nm.
In what cell type is heterochromatin densely packed?
Interphase cells.
What is heterochromatin used to refer to?
Transcriptionally inactive DNA.
What are two markers of actively transcribed DNA?
AcH3K9 and MeH3K4.
What are two markers of heterochromatin?
MeH3K9 and MeH3K27.
Explain the charge patch hypothesis.
When lysine residues are acetylated they loose their charge meaning there are less interactions between two nucleosomes / between the DNA and a nucleosome meaning the DNA is more loosely packed and can be transcribed.
What facilities the structural changing between the condensed state and the decondensed sate of chromatin?
Acetylation and methylation of core histones.
What specific interaction does AcHK16 inhibit?
The electrostatic interaction between the H2 and H4 core histone proteins in adjacent nucleosomes.
What is the histone code?
The cumulative collection of core histone modifications.
Histone modifications can affect chromatin structure in trans and cis interactions. What are cis and trans interactions?
Trans are interactions with factors. Cis are interactions with nucleosomes.
The markers for heterochromatin are MeH3K9 and MeH3K4. Why are these different to acetylated lysines?
Methylation does not effect the charge status.
MeH3K9 and MeH3K27 are both markers for heterochomaitn. What mark is required for heterochromatin formation?
MeH3K9.
What are H3K9 methylated nucleosomes recognised by?
Proteins containing a chromodomain.
What is the most abundant protein found in heterochromatin?
HP1.
What do many proteins containing a chromodomain also contain?
A structurally related chromoshadow domain.
What is the purpose of the chromoshadow domain?
Allows for protein protein interactions.
What does chromatin immunoprecipitation allow?
The analysis of protein/DNA interactions in vivo.
How are chromatin immunprecipitation interactions stabilised?
Chemical cross linking.
Why is the ChIP technique possible?
Because of the large availability of antibodies to specific histone modifications.
What are the steps for ChIP?
- Proteins are chemically cross linked to DNA.
- Cells lysed.
- DNA fragmented.
- DNA fragments bound tp a specific protein are purified with available antibodies.
- DNA analysed by PCR/ microarray.
What is the difference between using PCR or a microarray in the last step of ChIP?
PCR determines wether the protein is bound to a specific gene whereas microarray studies the localisation of the protein in a genome wide scale.
What can be used as a chemical cross linker inChIP?
Thermaldyhyde.
What are antibodies specific to in ChIP?
Acetylated or methylated histones.
What do you need to read the histone code?
Proteins that specifically recognise modified nucleosomes.
What part of the histone does the chromodomain bind to?
The histone tail.
How does the chromodomain bind to on the histone tail?
Methylated lysines.
What core histone protein binds H1 as a missing strand?
H3.
What is the purpose of the chromoshadow domain?
The chromosome domain on that protein can recognise a chromosome domain on another protein. Allows recruitment of more factors.
How does heterochromatin spread?
From an initiation point until it reaches the boundary.
What is heterochromatin spreading depend on?
- H3K9 trimentylation.
- HP-1.
- Histone methlytransferase Suv3-9/Clr4.
What methyltransferase involved in heterochromatin spreading also contains a chromodomain?
Suv3-9.
How is the HMT activity of Suv3-9 stimulated?
Interaction of its chromodomain to an adjacent H3K9me3 nucleosomes.
What does binding of the Suv3-9 chromodomain to H3K9me3 promote?
Methylation of the neighbouring nucleosomes.
H3 binds H1 as a missing strand. What structure does this make?
Beta fold.
When are Hox genes maintained in a transcriptionally state/ active state?
During late stages of embryogenesis and through adult life.
What protein complex regulates Hox genes in late embryogenesis?
Polycomb.
What protein complex regulates Hox genes in adult life?
Trithroax.
What are PRC1 and PRC2.
The two major complexes found in the Polycomb complex.
What major complex in the polycomb complex is recruited by transcriptional repressors early during embryogenesis?
PRC2.
What is found in the PRC2 complex of the polycomb complex?
H3K27 specific histone methyltransferase subunit, also called the enhancer of zest.
What is the enhancer of zest?
The H3K27 specific histone methyltransferase subunit found in PRC2 of the polycomb complex.
What does the polycomb complex associate with?
Transcriptional repressors during early embryogenesis.
What does the enhancer of zest do?
Methylate specific H3K27 nucleosomes around the repressor.
What is the purpose of the PRC1 subunit in the Polycomb complex?
Condenses the structure of the chromatin.
How does the PRC1 subunit condense the chromatin?
It contains the Pc subunit which can bind to MeH3K27 containing nucleosomes and condense their structure.
What is Pc in the PRC1 subunit?
A dimeric chromodomain subunit.
What is the purpose of the polycomb repressive complex?
Allows the repression of certain hox genes in certain regions of the organism throughout its lifetime.
What does the trithorax complex contain?
A H3K4 specific HMT.
How does the thithorax complex act as a repressor?
It contains a H3K4 specific HMT which bind relatively stably to Me3H3K4 keeping them methlayed.
What is Methylated H3K4 a marker for?
Transcriptionally active chromatin. ODD CASE.
What are the two mating types of bakers yeast?
Mata and Matalpha.
What happens when a yeast mother cell divides?
It switches mating type.
What chromosome in yeast are the mating type loci found?
3.
What are the three mating type loci found on chromosome 3 in yeast?
HMLalpha, HMRa and MatA.
What mating type locus is central in yeast/ where gene conversion occurs?
MatA.
Recombination of the mating type locus in cell division in yeast is dependant on what?
HO endonucelase.
Repression of the HMLalpha and HMRa is dependant on what?
Adjacent silencer regions.
In yeast where does heterochromatin silence gene expression?
rDNA locus, telomeres, centromeres and mating type loci.
What binds to yeast DNA in silencer regions?
Rap1.
What protein does Rap1 (bound to silencer regions in yeast) recruit?
Sir proteins.
What is Sir2?
A histone deactlyase (H3K9, H4K16).
What does histone hypoacetylation allow?
Chromosome decondensation.
If a gene is inserted close to a silencer region what happens?
Its expression is blocked.
Once Sir2 has removed the acetyl group what can bind?
Sir2/Sir3/Sir4.
Once the Sir2/Sir3/Sir4 complex has bound to a deactylated nucleosome what happens?
The adjacent nucleosome is deacetylated. This only stops once a boundary element is reached.
What does packaging of the 10nm fibre into the 30nm require?
Linker histone H1 and the tails of the core histones.
Two classes of models have been proposed for the structural organisation of the 30nm fibre. What are these?
The solenoid model and the zig-zag ribbon model.
What is the solenoid model?
Chain of nucleosomes on the 30nm fibre are wound into a single coil.
What is the zig-zag ribbon model?
Linker DNA of the 30nm fibre stretches across a two-stranded left-handed double helix of nucleosomes.
In vivo when can the 10nm fibre be seen as a 30nm fibre?
Physiological salt conditions.
The solenoid model is more accepted than the zig-zag ribbon model. True or false?
False. They are equally accepted.
What can the zig zag model for the 30nm structure be described as?
Spherical and slightly flat, like stacked coins.
Is the 30nm strand still transcriptionally active?
Yes, anything higher is too condensed though.
The core histones are subjected to extensive post-translational modification. What are examples of these?
- Acetylation of lysine residues.
- Mono/ di/ tri methylation of lysine residues.
- Phosphorylation of serines.
- Phosphorylation of threonines.
- Ubiquination of lysine.
- Symmetrically/ asymmetrically dimethylated arginines.
Modifications to the core histone proteins can be mutually exclusive or mutually dependant. Name an example of a mutually exclusive modification?
Methylation of lysine which blocks acetylation.
Modifications to the core histone proteins can be mutually exclusive or mutually dependant. Name an example of a mutually dependant modification?
Ubiquitination of H2B is required for methylation of H3K4.
Why can the tails of histones make interactions with DNA and other proteins?
They are non rigid.
What do HATs do?
Add acetyl groups to the epsilon amino group of lysine residues.
What do HDAC do?
Readily reverse the actions of HATs.
HMTs methylate lysine side chains. What does this do in turn?
Prevents acetylation.
Is acetylation or methylation more easily reversed?
Acetylation.
What reverses methylation?
Lysine-Specific demethylases.
How can the ‘chromatin landscape’ of genes be studied?
Nuclease sensitivity assays.
