▪ If X and Y interact, that brings the two domains together,
and activates transcription of a reporter gene
▪ The reporter gene then likely produces a product that can
be detected, like fluorescence or a catalyst for a
colorimetric reaction (e.g. if X-gal is used, the yeast cells will
turn blue)
- The yeast two hybrid system can be used to find unknown proteins that
interact with a known protein
o One would prepare a cDNA library linked to the coding region of
the transcription activating domain and express there hybrid genes,
along with a gene encoding the DNA binding domain
- Example: DNA-binding domain—Z hybrid gene, in yeast cells
o In practice, each yeast cell would make a different fusion protein
(AD–A, AD–B, AD–C, etc.).
o AD–D binds to BD–Z and activates transcription, but none of the
other fusion proteins can do this because they cannot interact with
BD–Z
o Once clones that activate transcription are found, the plasmid
bearing the AD–D hybrid gene is isolated and the D portion is
sequenced to find out what it codes for
Architectural Genome Organization: Chromatin/Nucleosomes
Chromatin
- The packaging problem:
o There are approx. 3.3 billion base pars over 23 chromosomes.
o Each base pair is approx. 0.34nm long (in B-DNA)
o The average chromosome is thus 5cm long, but a typical human cell has diameter 20 µm
o Human DNA’s total length is approx. 2m, so it must be condensed by a factor of over 100,000X
- Chromatin is composed of DNA and proteins (histones) that help chromatin fold so it can condense and
package itself into the cell's nucleus
- Euchromatin is relatively extended and open, and has the potential to be active
o Its dispersed throughput the nucleus
- Heterochromatin is highly condensed and often genetically inactive (no space for TFs to reach promoter
regions)
o Typically found at the telomeres and centromeres, and in permanently repressed gene sequences
▪ Constitutive: certain genomic regions are never expressed and often have just a structural
role (e.g. Telomeres)
▪ Facultative: specific regions or whole chromosomes that are expressed depending on intra
and extracellular conditions and cell identity (e.g. repressing one X chromosome in female
mammals)
o Heterochromatin can also silence genes as much as 3kb away.
Nucleosomes & Chromatin Packaging
- The first order of folding involves structures called nucleosomes, which have core histones around which
DNA winds
- They are the basic structural unit of chromatin.
- Each individual nucleosome core particle consist of a complex of 8 histone proteins, two of each histone
H2A, H2B, H3 and H4
o The histones are rich in lysine and arginine, whose positive charges stabilize the negative DNA
around the core
, o The H3 and H4 monomers bind to form a tetramer and are assembled on the DNA
o The H2A and H2B monomers form heterodimers which then associated to the
existing complex
o Approx. 146bps warp around a single histone core
o They are highly conserved throughout evolution, and essential for survival (likely
evolved early in eukaryotic evolution)
- The core histone proteins form an octamer histone core, around which DNA is
wrapped around the outside
o Having the DNA on the outside also minimizes the amount of bending (with risk of
breaking) that the DNA would have to do
- The overall structure resembles ‘beads on a string.’
- Transcription of reconstituted chromatin with an average of 1 core per 200bp of DNA
exhibits approx. 75% repression relative to naked DNA
o The remaining 25% is due to promoter sites that are
not covered by nucleosome cores
30nm Fiber
- The packaging of chromatin occurs at several successive levels
o the formation of the nucleosomes is the first stage,
and produces 10nm fiber.
o the 10nm fiber is then further folded into more
condensed 30nm fibers
- 30nm fiber is produced using H1 histones.
o the nucleosomes core particles form a regular arrangement where the nucleosomes are in contact
with each other
o the H1 histone binds to the linker DNA
▪ DNA enters and exists the histone core wrapping from the same point, where H1 binds
where the helices cross to tighten the fibers even more
- H1 is known as the linker histone.
- H1 can cause repression by binding to linker DNA
o Activators can prevent this effects if added at the same time as H1, but they cannot reverse the
effects of preformed nucleosome core
o If the activators get to the DNA first, they block the repressive action of H1
▪ If H1 reaches the DNA first, it stabilizes the nucleosomes and blocks activation
o Activators can team up with chromatin remodelling factors to push nucleosomes aside if they are
not fully stabilized by H1.
o TFs like Sp1 and GAL4 acts as both anti repressors and activators
- Nucleosome Repeat Length (NRL) determines the structure of 30nm fibre
o The length of DNA from the beginning of one nucleosome to the beginning of the
next varies from 165bp to 212bp
Radial Loops
- The 30nm fibre is further condensed in chromatin folding to form radial loops
- The 30nm fibre is arranged in 3D around a central scaffold
- The radial lops are then condensed further into chromosomes
Conservation of Histone Proteins
- Of all the histones, H1 is the one that varies the most among organisms (less conserved)
- H3 is the most conserved histone.
- High resolution electrophoresis of the histones will show that the histone genes are not single copy genes
like most protein coding genes in eukaryotes
o They are separated many times, but the copies are mainly identical
, o H1 genes show the greatest variation (e.g. 6 subspecies in the
mouse where histone genes are repeated 10-20 times in a
single genome)
- Birds, fish, amphibians and reptiles have another lysine rich histone
that could be an extreme variant of H1, but it is so different that it
generally has a distinct name (H5)
Post-Translational Modifications
- All histones are characterized for having a conserved fold, which provides the core of the quaternary
structure forming the nucleosomes.
- They also have an unstructured N-terminal region, which protrude outwards from the nucleosomes (approx.
25 amino acids long)
o Its role is to accept post translational modifications.
o The terminal can be modified by acetylase or deacetylase, and can interact with other nucleosomes
to compact DNA further
- These modifications can affect the density with which chromatin in packed
o Acetylase can acetylate chromatin, causing it to become more loose and flexible (i.e. euchromatin)
▪ Acetylation occurs on lysines
o Deacetylase can deacetylate the chromatin, which leads to methylation
▪ Methylation signals recruiting factors that promote the formation of heterochromatin.
▪ Methylation occurs on lysines or arginines.
- Other modifications include phosphorylation (on serines or threonine), ubiquitination (on lysines) and ADP
ribosylation (on glutamate)
- These modifications are at the heart of epigenetics, and allow for genome regulation in eukaryotes.
- Euchromatin histone tails are hyperacetylated (loosens the structure and induces transcriptional activation)
whilst heterochromatin histone tais are hypoacetylated(induces chromatin compaction and gene repression)
DNA Negative Supercoiling
- When the core is removed, it leaves negatively supercoiled dsDNA.
- Negative supercoiling makes the strand separation easier, which is required for replication and transcription
- The DNA wrapping around the histone core has several distortions.
- Chromatin with the histone removed leaves a supercoiled DNA duplex, which are only removed if the DNA is
nicked to relax the supercoiling
- EM studies reveal that the 30nm fiber forms loops between 35 and 85kb long, anchored to the central
matrix of the chromosome
- DNA winds about 1.65 times around the core, condensing the length by a factor 6-7
The Histone Fold
- All core histones contain the same fundamental histone fold, which consists of 3 α-helices linked by 2 loops.
- They also contain histone tails.
o The tails of H2B and H3 pass out of the core particle through a cleft formed from two adjacent DNA
minor grooves
o One of the H4 tails is exposed to the side of the core particle. Its rich is basic residues and can
interact strongly with ana acidic region of a H2A-H2B dimer in an adjacent nucleosome
o These types of interactions may play a role in nucleosome cross linking.
and activates transcription of a reporter gene
▪ The reporter gene then likely produces a product that can
be detected, like fluorescence or a catalyst for a
colorimetric reaction (e.g. if X-gal is used, the yeast cells will
turn blue)
- The yeast two hybrid system can be used to find unknown proteins that
interact with a known protein
o One would prepare a cDNA library linked to the coding region of
the transcription activating domain and express there hybrid genes,
along with a gene encoding the DNA binding domain
- Example: DNA-binding domain—Z hybrid gene, in yeast cells
o In practice, each yeast cell would make a different fusion protein
(AD–A, AD–B, AD–C, etc.).
o AD–D binds to BD–Z and activates transcription, but none of the
other fusion proteins can do this because they cannot interact with
BD–Z
o Once clones that activate transcription are found, the plasmid
bearing the AD–D hybrid gene is isolated and the D portion is
sequenced to find out what it codes for
Architectural Genome Organization: Chromatin/Nucleosomes
Chromatin
- The packaging problem:
o There are approx. 3.3 billion base pars over 23 chromosomes.
o Each base pair is approx. 0.34nm long (in B-DNA)
o The average chromosome is thus 5cm long, but a typical human cell has diameter 20 µm
o Human DNA’s total length is approx. 2m, so it must be condensed by a factor of over 100,000X
- Chromatin is composed of DNA and proteins (histones) that help chromatin fold so it can condense and
package itself into the cell's nucleus
- Euchromatin is relatively extended and open, and has the potential to be active
o Its dispersed throughput the nucleus
- Heterochromatin is highly condensed and often genetically inactive (no space for TFs to reach promoter
regions)
o Typically found at the telomeres and centromeres, and in permanently repressed gene sequences
▪ Constitutive: certain genomic regions are never expressed and often have just a structural
role (e.g. Telomeres)
▪ Facultative: specific regions or whole chromosomes that are expressed depending on intra
and extracellular conditions and cell identity (e.g. repressing one X chromosome in female
mammals)
o Heterochromatin can also silence genes as much as 3kb away.
Nucleosomes & Chromatin Packaging
- The first order of folding involves structures called nucleosomes, which have core histones around which
DNA winds
- They are the basic structural unit of chromatin.
- Each individual nucleosome core particle consist of a complex of 8 histone proteins, two of each histone
H2A, H2B, H3 and H4
o The histones are rich in lysine and arginine, whose positive charges stabilize the negative DNA
around the core
, o The H3 and H4 monomers bind to form a tetramer and are assembled on the DNA
o The H2A and H2B monomers form heterodimers which then associated to the
existing complex
o Approx. 146bps warp around a single histone core
o They are highly conserved throughout evolution, and essential for survival (likely
evolved early in eukaryotic evolution)
- The core histone proteins form an octamer histone core, around which DNA is
wrapped around the outside
o Having the DNA on the outside also minimizes the amount of bending (with risk of
breaking) that the DNA would have to do
- The overall structure resembles ‘beads on a string.’
- Transcription of reconstituted chromatin with an average of 1 core per 200bp of DNA
exhibits approx. 75% repression relative to naked DNA
o The remaining 25% is due to promoter sites that are
not covered by nucleosome cores
30nm Fiber
- The packaging of chromatin occurs at several successive levels
o the formation of the nucleosomes is the first stage,
and produces 10nm fiber.
o the 10nm fiber is then further folded into more
condensed 30nm fibers
- 30nm fiber is produced using H1 histones.
o the nucleosomes core particles form a regular arrangement where the nucleosomes are in contact
with each other
o the H1 histone binds to the linker DNA
▪ DNA enters and exists the histone core wrapping from the same point, where H1 binds
where the helices cross to tighten the fibers even more
- H1 is known as the linker histone.
- H1 can cause repression by binding to linker DNA
o Activators can prevent this effects if added at the same time as H1, but they cannot reverse the
effects of preformed nucleosome core
o If the activators get to the DNA first, they block the repressive action of H1
▪ If H1 reaches the DNA first, it stabilizes the nucleosomes and blocks activation
o Activators can team up with chromatin remodelling factors to push nucleosomes aside if they are
not fully stabilized by H1.
o TFs like Sp1 and GAL4 acts as both anti repressors and activators
- Nucleosome Repeat Length (NRL) determines the structure of 30nm fibre
o The length of DNA from the beginning of one nucleosome to the beginning of the
next varies from 165bp to 212bp
Radial Loops
- The 30nm fibre is further condensed in chromatin folding to form radial loops
- The 30nm fibre is arranged in 3D around a central scaffold
- The radial lops are then condensed further into chromosomes
Conservation of Histone Proteins
- Of all the histones, H1 is the one that varies the most among organisms (less conserved)
- H3 is the most conserved histone.
- High resolution electrophoresis of the histones will show that the histone genes are not single copy genes
like most protein coding genes in eukaryotes
o They are separated many times, but the copies are mainly identical
, o H1 genes show the greatest variation (e.g. 6 subspecies in the
mouse where histone genes are repeated 10-20 times in a
single genome)
- Birds, fish, amphibians and reptiles have another lysine rich histone
that could be an extreme variant of H1, but it is so different that it
generally has a distinct name (H5)
Post-Translational Modifications
- All histones are characterized for having a conserved fold, which provides the core of the quaternary
structure forming the nucleosomes.
- They also have an unstructured N-terminal region, which protrude outwards from the nucleosomes (approx.
25 amino acids long)
o Its role is to accept post translational modifications.
o The terminal can be modified by acetylase or deacetylase, and can interact with other nucleosomes
to compact DNA further
- These modifications can affect the density with which chromatin in packed
o Acetylase can acetylate chromatin, causing it to become more loose and flexible (i.e. euchromatin)
▪ Acetylation occurs on lysines
o Deacetylase can deacetylate the chromatin, which leads to methylation
▪ Methylation signals recruiting factors that promote the formation of heterochromatin.
▪ Methylation occurs on lysines or arginines.
- Other modifications include phosphorylation (on serines or threonine), ubiquitination (on lysines) and ADP
ribosylation (on glutamate)
- These modifications are at the heart of epigenetics, and allow for genome regulation in eukaryotes.
- Euchromatin histone tails are hyperacetylated (loosens the structure and induces transcriptional activation)
whilst heterochromatin histone tais are hypoacetylated(induces chromatin compaction and gene repression)
DNA Negative Supercoiling
- When the core is removed, it leaves negatively supercoiled dsDNA.
- Negative supercoiling makes the strand separation easier, which is required for replication and transcription
- The DNA wrapping around the histone core has several distortions.
- Chromatin with the histone removed leaves a supercoiled DNA duplex, which are only removed if the DNA is
nicked to relax the supercoiling
- EM studies reveal that the 30nm fiber forms loops between 35 and 85kb long, anchored to the central
matrix of the chromosome
- DNA winds about 1.65 times around the core, condensing the length by a factor 6-7
The Histone Fold
- All core histones contain the same fundamental histone fold, which consists of 3 α-helices linked by 2 loops.
- They also contain histone tails.
o The tails of H2B and H3 pass out of the core particle through a cleft formed from two adjacent DNA
minor grooves
o One of the H4 tails is exposed to the side of the core particle. Its rich is basic residues and can
interact strongly with ana acidic region of a H2A-H2B dimer in an adjacent nucleosome
o These types of interactions may play a role in nucleosome cross linking.
