CHROMATIN STRUCTURE
CHROMATIN STRUCTURE
procaryotic chromatin is packaged somewhat but not nearly as much as eucaryotic chromatin
Eukaryotic chromatin = DNA (1 part) + proteins (2 parts)
chromatin’s proteins are divided into two types: histones and nonhistones (such as transcription factors
The human genome is about 2 yards long but the average size of a nucleus is 10um (microns) in diameter.
What happens to chromatin structure during transcription and replication?
Enzymes need access to DNA, which is never naked, so changes are necessary in chromatin structure for these processes to take place.
Levels of Organization — the Packing Ratio
Packing Ratio
Linear DNA
Nucleosome
Nucleosomes around 30 nm fiber
Attachment of 30 nm fiber to matrix/core
1000X (interphase nucleus, which is when transcription and replication occur)
Mitotic chromosome
10000X (mitotic chromosome is even more condensed so that it can be packaged during cell division)
Nucleosome — 67 nm DNA around 11 nm histone core (beads on a string), the fundamental unit of organization, the 6X comes from 67/11
EXPERIMENTS FROM THE TEXTBOOK
To Analyze the Nucleosome (FIGURE 19.17)
break open the cells
isolate the nuclei
put nucleus in hypotonic media (low salt)
nucleus is ruptured and chromatin spills out
beads = histone core
fiber = linker DNA
Spacing of Nucleosomes
lyse nuclei in hypotonic media
mild digestion by micrococcal nuclease, which cuts both strands of the DNA helix for a short time and with little enzyme for incomplete digestion (not a restriction enzyme because it only has little sequence specificity)
centrifuge mixture on sucrose gradient in excess of 50000 RPM (low density/low sucrose at top of gradient and high sucrose/high density at bottom).
The particles will move according to shape and size (histones and DNA are still complexed together)
fractionate according to gradient
extract DNA so that DNA goes into solution and get rid of the protein
run agarose gel with each lane corresponding to a fraction in order to separate DNA according to size
micrococcal nuclease (MN) seems to cut in 200 bp intervals and the DNA of each lane/fraction is of a specific length that is a multiple of 200 bps
linker region DNA is more accessible to MN cutting and the linker region is every 200 bps so nucleosomes are spaced every 200 bps
200 bp = mononucleosome
400 bp = dinucleosome
600 bp = tirnucleosome etc…
How is long is the linker and how long is the DNA around the histone core?
digest mononucleosome with MN for longer
200
165 bp
146 bp — tightly bound to histones and this number is constant and universal
What’s in a nucleosome?
Nucleosome = core particle + linker DNA
2 molecules of each core histone è
H2A, H2B, H3, H4
1 molecule of H1
200 bp of DNA
mononucleosome = core particle + linker
core particle = core histones + 146 bp
200 bp DNA = 67 nm (6X packing)
FIGURE 19.4
DNA wraps almost twice around when its 67 nm of DNA is wound around 11 nm core.
FIGURE 19.5
DNA’s two turns, each
diameter, take up almost all the 6nm height of the nucleosome
FIGURE 19.6
Two regions that are
apart on linear DNA are close to together on the nucleosome.
DNA helix with 10.5 bp per turn is wrapped around nucleosome.
Is the DNA at every point in this wrapping equivalent in structure?
To determine if DNA at all points in wrapping is equivalent
use DNAase I, which cuts only one strand
isolate and denature DNA
FIGURE 19.14
on a flat glass surface, DNAase I cuts every 10.5 bps
cutting periodicity = structural periodicity = 10.5 bp/turn
once a turn the DNA is more accessible and so is cut there
FIGURE 19.13
same experiment with core particle instead of glass surface and DNAase I
cuts every 10.7 bp in some regions and every 10 bp at other regions
high resolution mapping indicates that there is variation in cutting periodicity along the DNA that is wrapped around the core particle
location of helix on core changes the structure slightly
some regions are more stressed than others
TAKE HOME MESSAGE:
THIS HAS IMPLICATIONS FOR DNA BINDING PROTEINS.
What’s the role of H1?
binds linker DNA
required for 30 nm fiber, somehow involved in organizing nucleosomes
FIGURE 19.19
extraction under high salt conditions yields 30 nm fiber in presence, but not in absence of H1
FIGURE 19.20
guess at 30 nm structure è
helix of nucleosomes with a diameter of 30 nm (what the polymerases deal with)
It is thought that H1 binds at enter and exit of helix at each nucleosome and interacts with other H1s at the helix center, holding all this together at the 40X level of packing
30 nm fiber thought to be organized into loops, with some regions attached to the matrix
Is there a consensus sequence for attachment to the matrix?
What’s the sequence?
What are the proteins that make up the matrix?
FIGURE 18.8
isolate chromatin
extract (remove) histones
treat
with complete digestion by a combination of restriction enzymes. (No histones on the loops so restriction enzymes can cut) but the regions bound and protected by the matrix will not be cut
extract DNA away from protein and sequence these matrix associated regions (MARs)
no consensus sequence
very A-T rich
occur in many cases near 5’ regulatory region of transcription units ( not all regulatory regions are near attachment sites)
DNA may be organized into transcription unit(s) loops
MARs = SARs (scaffold attachment regions)
start with mitotic chromosomes, extract out histones
use restriction enzyme (RE) DNAase to find scaffold attachment regions on chromosome scaffold
thought that same proteins are involved in SAR and MAR attachments and that the matrix may be a loose scaffold
The Histone Octamer
FIGURE 19.21
crystallization tells us how the histones fit together
H2A-H2B dimmer on either side
All four histone proteins have histone fold structure
FIGURE 19.23
Histone fold = helix + loop + helix + loop + helix ( + loop + helix for two of the core histones)
A LOT OF DNA BINDING PROTEINS HAVE SIMILAR HELIX-LOOP-HELIX
FIGURE 19.25
N terminal tails stick out from the core and are subject to post translational modifications
Modifications of amino acid in tail can lead to changes in affinity of histone core with DNA by making the core less positive and bind the negatively charged DNA less tightly
Examples: acetylation (of lysine), methylation, phosphorylation of serine of histone during mitosis to increase condensing)
IMPORTANT FOR TRANSCRIPTION
How does transcription machinery deal with chromatin structures?
How does RNA polymerase deal with nucleosomes?
How do DNA-binding proteins (tx factors) interact with DNA?
Look in 10 different cells to see if nucleosomes on our specific gene are arranged in the same or random places.
Does the promoter always lie in the linker region?
FIGURE 19.29 and 19.30
How can we tell if nucleosomes are in specific positions?
we’re interested in the specific red sequence (let’s say it’s a promoter)
take nuclei from many cells and treat with MN to get mononucleosomes
digest with restriction enzyme that will cut just adjacent to our sequence and look to see if RE site is in same place relative to linker region
run agarose gel and get all fragments between 0-200 bp.
Use radioactive P-32 in a probe we hybridize to our red sequence, melting the probe and incubating with the gel in order to locate our piece of DNA
if this yields (and it does!) a unique, distinct band it’s because RE site is fixed and MN site is fixed from the structure
if we observed instead a continuous smear on the gel it would mean that the MN site is at random distances from the RE site (this is not what we find!)
In many cases, position of histone octamers is NOT random.
THIS HAS IMPORTANT IMPLICATIONS FOR TRANSCRIPTION.
This is called nucleosome positioning or nucleosome phasing.
Two Types of Nucleosome Positioning
FIGURE 19.31
Translational positioning
linear placement of DNA relative to histone
changing location of histone alters which turns are in the linker region
FIGURE 19.32
Rotational positioning
exposure of DNA helix on nucleosome surface
changing location of turns by rotating affects the accessibility of the double helixbecause of the exposure or lack of exposure due to protection by the nucleosome surface
Changes in positioning are associated with transcription.
Experiment:
FIGURE 19.38
URA3 gene in yeast has an inducible promoter and we can turn gene expression on and off and look at the two positioning cases
Nucleosome positioning can change depending on the &transcriptional state& of the gene
when the
URA3 gene isturned off, nucleosomes have particular positions but when gene expression is turned on the nucleosome positions are randomized, return to ladder positions when turned off again
ladder è
smear è
Hypersensitive Sites
isolate chromatin from specific cell type (specific because of gene expression particular to cell type)
let’s use RBCs and b
-globin gene ON (-300 to —50 region)
digest with DNAase I incompletely
certain sites are 100X more sensitive to cleavage
use gel and hybridize probe
FIGURE 19.41
often have hypersensitive sites in promoter region
cleavage sites needn’t be continuous
probably not bound to nucleosomes as need to be bound by transcription factors, which will later protect them
continue experiment with plasmidcontaining b
-globin gene in a test tube and only see hypersensitivity if RBC protein extract is added before or at the same time as histones.
Histones, if added first,
can block the binding of proteins from RBC extract.
DNA-binding proteins can influence nucleosome position.
DNAase Sensitivity
FIGURE 19.42
occurs all along transcription unit, not just promoter
if we look at sensitivity of b
-globin isolated from RBCs we get digestion è
sensitivity
but not with b
-globin from muscle è
no sensitivity
gene OFF 10% degraded
gene ON 50% degraded
transcribed genes are more open, making transcription unit more susceptibleanonymised是什么意思_anonymised在线翻译_anonymised什么意思_anonymised的意思_anonymised的翻译_英语单词大全_911查询
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&2017 　京ICP备号-6　京公网安备30　Cell Nucleus - Commanding the Cell
The cell nucleus acts like the brain of the cell. It helps control eating, movement, and reproduction. If it happens in a cell, chances are the nucleus knows about it. The nucleus is not always in the center of the cell. It will be a big dark spot somewhere in the middle of all of the . You probably won't find it near the edge of a cell because that might be a dangerous place for the nucleus to be. If you don't remember, the cytoplasm is the fluid that fills cells.
Life Before a Nucleus
Not all cells have a nucleus. Biology breaks cell types into
(those with a defined nucleus) and
(those with no defined nucleus). You may have heard of chromatin and DNA. You don't need a nucleus to have DNA. If you don't have a defined nucleus, your DNA is probably floating around the cell in a region called the nucleoid. A defined nucleus that holds the genetic code is an advanced feature in a cell.
Important Materials in the Envelope
The things that make a eukaryotic cell are a defined nucleus and other organelles. The nuclear envelope surrounds the nucleus and all of its contents. The nuclear envelope is a membrane similar to the
around the whole cell. There are pores and spaces for RNA and proteins to pass through while the nuclear envelope keeps all of the chromatin and nucleolus inside.
When the cell is in a resting state there is something called chromatin in the nucleus.
is made of DNA, RNA, and nuclear proteins. DNA and RNA are the nucleic acids inside of the cell. When the cell is going to , the chromatin becomes very compact. It condenses. When the chromatin comes together, you can see the chromosomes. You will also find the nucleolus inside of the nucleus. When you look through a microscope, it looks like a nucleus inside of the nucleus. It is made of RNA and protein. It does not have much DNA at all.
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Useful Reference Materials
Wikipedia:
http://en.wikipedia.org/wiki/Cell_nucleus
Encyclopaedia Britannica:
/EBchecked/topic/101396/cell/37399/Structural-organization-of-the-nucleus