The Nucleus
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Transcript of The Nucleus
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The nucleus: organization, structure/function
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Nucleus
The Nucleus is a membrane-enclosed organelle which house most of the genetic information and regulatory machinery responsible for providing the cell with its unique characteristics.
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Nucleus Control center for cellular activity for the
whole cell Chromosomes are localized and replicated Transcription takes place here Has double membrane known as nuclear
envelope, one characteristic of differentiation of eukaryotes from prokaryotes
Vary in size and shape depending on cell type
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Nucleus
The contents of the nucleus are enclosed by a complex nuclear envelope.
Nucleus has 3 components Chromatin Nucleolus : site for ribosomal RNA synthesis Nucleoplasm : Contain a variety of particles with other
molecules involved in maintenance and development of the cell. The particles are:
Heterogenous nuclear ribonucleoprotein particles Complexes of protein and pre-messager RNA Small nuclear ribonucleoprotein particles
All these constituents are found within what is refferred to as the nuclear matrix
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nuclear pores
chromatin
nucleolus
nuclear envelope
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nuclear pores
nucleus
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THE NUCLEUS: FUNCTIONS
It stores the cell's hereditary material, or DNA.
Site of DNA replication Site of DNA transcription to mRNA Ribosomal formation
Nucleolus: RNA & protein required for ribosomal synthesis
It coordinates the cell's activities, which include growth, intermediary metabolism, protein synthesis and cell division by regulating gene expression.
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Nucleolus Morphologically distinct region of the nucleus which
is only observed during interphase because it breaks down and decodenses during mitosis
The nucleolus is prominent within the nucleus.It is made up of protein and ribosomal DNA (rDNA)
It has no membrane It is the site of RNA transcription and
processing,and ribosome assembly Some cell types and organisms
(e.g. Paramecium) contain more than one nucleolus
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Nucleoplasm
• The nucleoplasm: a highly viscous liquid,similar to cytoplasm, which surrounds thechromosomes and nucleolus
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Nuclear envelope Forms barrier between nucleus and cytoplasm Segregates nucleoplasm from cytoplasm Has 2 membranes. Inner and outer
membranes of about 7-10nm in thickness. The membranes are separated by a gap
called perinuclear space of about 20-40nm thick
The outer membrane is continuous with the endoplasmic reticulum and contain ribosomes
At intervals the inner and outer membranes fuse to form nuclear pores
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The nuclear envelope• The Components:
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Nuclear cortex/Lamina An electron dense layer of fibrous material
on nucleoplasmic side of inner nuclear membrane. It is about 30-40nm thick and is also called nuclear lamina Function to funnel materials to nuclear pores Involved in pore formation Organize chromosomes by binding to interphase
chromatin to special sites on inner nuclear membrane
Organization of nuclear envelop and perinuclear chromatin
Composed of lamins a, b and c
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Lamin provides anchorage sites for chromatin
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(Alberts et al)
• Intermediate filament proteins• Form meshwork at inside of inner nuclear membrane (INM), some extend into nucleoplasm• Nuclear strength and architecture• DNA replication and mRNA transcription• Involved in apoptosis
Functions of lamins
(slide from Jess Hurt, HMS)
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Assembly and Disassembly of Nuclear Envelope •Nuclear envelope (NE) is a cell cycle
dependent structure that disperses at the onset of mitosis (late prophase) and reassembles around the reforming nucleus in the late telophase.
•Inhibition of protein synthesis by cycloheximide in late G2 phase has no apparent affect on nuclear assembly in telophase indicating that no new protein synthesis is required for reassembly of the nuclear envelope.
•This reassembly involves ~ 10,000 nuclear pores in a matter of minutes.
•The correlations of breakdown of the nuclear envelope, chromosome formation mitosis & NE reassembly after mitosis are essential for cell division and the ability of cells to divide in an orderly manner.
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Assembly and Disassembly of Nuclear Envelope contd…
• The proteins that compose the nuclear lamina (lamins A, B,C) are involved in the disassembly/reassembly of the nuclear envelope during cell cycle via phosphorylation (P)/dephosphorylation (deP).
• Yeast genetic studies have identified cdc2 as an essential gene for cell division in yeast. This is a cyclin dependant protein kinase called cyclin B-cdc2 (cdk1) kinase (cyclins are regulatory proteins that mediate the enzymatic activity of protein kinases) that plays a major role in the regulation of cell cycle.
• Lamin phosphorylation/ dephosphorylation during cell cycle by cdc2 (cdk1) kinase.
Gerace et al., 1984
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Dissolution of the Nuclear Lamina
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Lamins are filamentous proteins in the intermediate filament family
Lamin phosphorylation in prophase disassembles the nuclear lamina & allows for nuc. envel. breakdown
Laminins are extracellular proteins, unrelated
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Breakdown of the Nuclear Membrane
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Re-Formation of the Nuclear Envelope
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Nuclear pore complex
Nuclear pore composed of a nuclear pore complex measuring 70-90nm (inside diameter) with 9nm open channel
Typical mammalian celll has 3,000-4,000 pore complexes (10-12 pores per sq μm) in the nuclear envelope
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Nuclear pore complex Complex of 125 million daltons, 30X larger
than ribosome Made up of multiple copies of 100 different
proteins called nucleoporins Em show octagonal membrane embeded
structure with eight (10nm long) filaments joined to form a basket that extens to the nucleoplasm
The membrane embeded portion is attached directly to the nuclear lamina
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The nuclear pore complex (NPC)
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CytoplasmicCytoplasmicfilamentfilament
CytoplasmCytoplasm
NucleusNucleus
CytoplasmicCytoplasmicringring
Inner ringInner ring
BasketBasket
Distal ringDistal ring
The Nuclear Pore ComplexThe Nuclear Pore Complex
RibosomeRibosome
~150Å
~2000Å
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Nuclear pores are large protein complexes
Multiple copies of ~100 different proteins (nuclear pore proteins = NPPs) totaling >125 million
daltons!
Cytoplasmic face
Nuclear face
Cytosol
Nucleus
Annular subunit of centralchannel or transporter
Nuclear basket or cage
Cytplasmic fibrils
Nuclear lamina
Nuclear envelope
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EM of nuclear pore complex
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The nuclear pores on the membrane
• Type of cargo transported?
• How is this achieved?
• Purpose of nuclear pores?
-allows for exchange of macromolecules
-NPCs are dynamic
-proteins, ribosomes, RNPs, and RNAs
-Via Nups (proteins of the NPC)
-assembly/disassembly of cargos via exchange of GDP for GTP by Ran
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mRNAmRNA
mRNAmRNA
RibosomalRibosomalSubunitsSubunits
RibosomalRibosomalProteinsProteins
Nucleo-Cytoplasmic TransportNucleo-Cytoplasmic Transport
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Transport of large molecules is active - requires GTP
Small molecules (< 60 kDa), or about 9 nm diameter) enter or exit nucleus by passive diffusion
Larger molecules must be actively tranported:
(1) binding to transporter; and
(2) transport thru nuclear pore using GTP
Nuclear pores also required for active export of RNPs (including ribosome subunits, mRNA, tRNA
etc.)Import and export occur through same pores
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Import into the nucleus
• Protein import to nucleus– Nuclear localization
signal (NLS)• Best-studied – 1 or 2
sequences of +ve charged
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A nuclear localization signal (NLS) is necessary and sufficient for nuclear import of proteins
The “classical” signal for nuclear import includes multiple basic amino acids (K = lysine and R = arginine)…example P-P-K-K-K-R-K-V
NLS can be anywhere in protein sequence
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Simplified view of nuclear transport
(importin)
NLS(cargo)
Pore opens
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GAP
Molecular “switches”
GTPase
GTP
GTPase
GDP
Pi
GDP
GTP
“on” “off”GEF
Energy for transport provided by G proteins (GTP binding proteins; large
family)
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GAPGAP
GTPaseGTPase
GTPGTP
GTPaseGTPase
GDPGDP
Pi
GDP
GTP
“on” “off”
GEFGEF
GAP = GTPase Activating ProteinGAP = GTPase Activating Protein
GEF = Guanine Nucleotide Exchange FactorGEF = Guanine Nucleotide Exchange Factor
RANRANGTPase used inGTPase used in
nuclear transportnuclear transport
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Nuclear import/export cycle is driven by GTP hydrolysis
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Macromolecular transport through nuclear pore complex
• Requires FG-Nucleoporins• They line the channel of nuclear pore complex and
contain multiple repeats of short hydrophobic sequences rich in phenylalanine(F) and glycine(G) reisdues
• Transport receptors – karyopherins• Soluble• Importins: importin α and β (form import receptor)
– Involved in cytoplasm to nucleus transport– The α subunit binds the nuclear localization signal
(NLS) of cargo protein to be transported to the nucleus and the β subunit interacts with the FG nucleoporins
• Exportins– Nucleus to cytoplasm transport
• Nuclear transport factor 2 (NTF2)
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Import into nucleus• Free importin binds to to NLS of the cargo protein forming a
complex• Cargo moves through the NPC by interacting with FG
nucleoporins (act as steping stones; no energy required)• In the nucleoplasm, interaction of Ran-GTP with importin
causes a conformation change, decreasing affinity of importin for NLS, and release of protein cargo
• To initiate another cycle of import, the Ran-GTP –importin complex is transported back to the cytoplasm where a GTPase accelerating protein (GAP) stimulates Ran to hydrolyze the bound GTP to GDP and release of importin
• Ran-GDP binds to NTF2 and is returned back to the nucleoplasm where a guanine nucleotide exchange factor (GEF) causes release of GDP from Ran and rebuilding of Ran-GTP
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Directional protein import is driven by GTP hydrolysis
Cytoplasm
Nucleus
Importin (NLS receptor) binds cargo (with NLS) in cytoplasm
Importin-cargo transported into nucleus thru nuclear pore
Ran-GTP in nucleus binds importin, importin releases NLS (cargo)
Ran-GTP-importin exported from nucleus thru pore
Ran-GAP stimulates GTP hydrolysis in cytoplasm by Ran
Ran-GDP releases importin in cytoplasm
Ran-GDP transported into nucleus (not shown)
Ran GEF stimulates nucleotide exchange restoring Ran-GTP.
NLS
NLSRan-GDPRan-GTP
Importin
Ran-GTP
Importin
Ran-GTPImportin
NLS
Importin
NLS
Importin
Ran-GDP
+
RanGEF
GDPGTP
Ran
GAP
Pi
NTF2
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Export from the nucleus
Export eg RNA, tRNA rRNA Move as ribonucleoproteins (RNPs)
Except t-RNA – direct transport by exportin
Protein component contains nuclear export signal (NES)
A specific nuclear export receptor( Exportin) is required and recognize NES
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Export from the nucleus
In nucleoplasm, exportin binds to NES of cargo to be exported and also to Ran-GTP
Cargo diffuses through NPC via transient interactions with FG repeats in FG nucleoporins
A GTPAse Accelerating protein stimulates conversion of Ran-GTP to Ran-GDP in cytoplasm
NES containing cargo released in cytosol while exportin and Ran-GDP are transported back to the nucleoplasm
GEF then stimulates conversion of Ran GDP to Ran GTP in cytosol
Note: the two transport processes are similar except in export Ran GTP is part of the cargo which is not the case in import
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Export from the nucleus
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DNA Organization in Eukaryotic Chromosomes
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Eukaryotic chromosomal organization
• Many eukaryotes are diploid (2N)• The amount of DNA that eukaryotes have varies; the
amount of DNA is not necessarily related to the complexity (Amoeba proteus has a larger amount of DNA than Homo sapiens)
• Eukaryotic chromosomes are integrated with proteins that help it fold (protein + DNA = chromatin)
• Chromosomes become visible during cell division• DNA of a human cell is 2.3 m (7.5 ft) in length if placed
end to end while the nucleus is a few micrometers; packaging/folding of DNA is necessary
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Eukaryotic chromosomal organization
2 main groups of proteins involved in folding/packaging eukaryotic chromosomes Histones = positively charged
proteins filled with amino acids lysine and arginine that bond
Nonhistones = less positive
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Model for Chromatin Structure• Chromatin is linked together every 200
bps (nuclease digestion)
• Chromatin arranged like “beads on a string” (electron microscope)
• 8 histones in each nucleosome
• 146 bps per nucleosome core particle with 53 bps for linker DNA
• Left-handed superhelix
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Eukaryotic chromosomal organization
Histone proteins Abundant Histone protein sequence is highly conserved
among eukaryotes—conserved function Provide the first level of packaging for the
chromosome; compact the chromosome by a factor of approximately 7
DNA is wound around histone proteins to produce nucleosomes; stretch of unwound DNA between each nucleosome
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Eukaryotic chromosomal organization
• Nonhistone proteins– Other proteins that are associated with the
chromosomes– Many different types in a cell; highly variable in cell
types, organisms, and at different times in the same cell type
– Amount of nonhistone protein varies– May have role in compaction or be involved in other
functions requiring interaction with the DNA– Many are acidic and negatively charged; bind to the
histones; binding may be transient
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Eukaryotic chromosomal organization
• Histone proteins– 5 main types
• H1—attached to the nucleosome and involved in further compaction of the DNA (conversion of 10 nm chromatin to 30 nm chromatin)
• H2A• H2B• H3• H4
– This structure produces 10nm chromatin
Two copies in each nucleosome ‘histone octamer’; DNA wraps around this structure1.75 times
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• Chromosomes– Nucleosome
• Contains a nucleosome core particle
– 146 base pairs of supercoiled DNA– Around a core of eight histone
molecules
– Histone core• Two copies of H2A, H2B, H3 and
H4– Octamer
• H1 resides outside of core– Linker histone– Binds to linker DNA– Connecting one nucleosome core
particle to next
Nucleosome structure
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Chromatin Compaction
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Orders of chromatin structure from naked DNA to chromatin to fully condensed chromosomes...
Fig. 9
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Beads on a String—10 nm FiberBeads on a String—10 nm Fiber
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DNA Molecules are highly condensed in chromosomesNucleosomes of interphase under electron microscope
Nucleosome: basic level of chromosome/chromatin organization Chromatin: protein-DNA complex
Histone: DNA binding proteinA: diameter 30 nm; B: further unfolding, beads on a string conformation
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10 nm filament; nucleosomes
histones (= 1g per g DNA) DNA
proteinpurification
H1
H3H2A
H2BH4
•Basic (arg, lys);•+ charges bind to - phosphates on DNA
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Experiments using nucleases
• Experiment: Digest chromatin with rat liver nuclease at low concentration. (or micrococcal nuclease)
• Electrophoresis of the digested chromatin material.
A regular pattern of bands on the gel, approx. every 200 bp
→ Histones distributed evenly on DNA, and at point which they bind, protect DNA from nuclease digestion. (nuclease digests double stranded DNA)
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deoxyribonuclease I (DNase I) digestion
nucleosomes
•Conclude: histones in a nucleosome protect 146 nt from DNase I attack.
Separate DNA from protein
H1
2H32H2A
2H2B2H4
the histoneoctamer
proteins
146 nt fragments
DNA
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Nucleosome StructuresHistone octamer
2 H2A2 H2B2 H32 H4
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Structural Organization of the Core Histones
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The Assembly of the Core Histones
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Notice the long tails of the octamer
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The bending of DNA in a nucleosome1. Flexibility of DNAs: A-T riched minor groove inside and G-C
riched groove outside2. DNA bound protein can also help
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The function of Histone tails
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The function of Histone H1
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Chromatin Remodeling
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Cyclic Diagram for nucleosome formation and
disruption
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10 nm Fiber10 nm Fiber
• A string of nucleosomes is seen under EM as a 10 nm A string of nucleosomes is seen under EM as a 10 nm fiberfiber
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30 nm Fiber30 nm Fiber
• 30 nm fiber is coil of 30 nm fiber is coil of nucleosomes with 6/turnnucleosomes with 6/turn
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The 30 nm FiberThe 30 nm Fiber(Compacts DNA 7X more)(Compacts DNA 7X more)
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Histone H1 : essential for the solenoid
Secondary Structure
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Secondary Structure: Essential points
The Solenoid is stabilized by H1 molecules H1 has a globular body that binds to the
outward DNA And 2 terminal arms (N- and C-) contact
the adjacent nucleosomes (actually the correspondent H1 histones that binds to the nucleosomes)
1 tour of solenoid = 6 nucleosomes
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Eukaryotic chromosomal organization
• Compaction continues by forming looped domains from the 30 nm chromatin, which seems to compact the DNA to 300 nm chromatin
• Human chromosomes contain about 2000 looped domains
• 30 nm chromatin is looped and attached to a nonhistone protein scaffolding
• DNA in looped domains are attached to the nuclear matrix via DNA sequences called MARs (matrix attachment regions)
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74
Model for the organization of 30-nm chromatin fiber into looped domains that are anchored to a nonhistone protein chromosome scaffold
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Acid extraction removes histones from meta-phase chromosomes, leaving nonhistones...
Low power high power
Scaffold protein
DNA loops
Major non-histone proteins = topoisomerases!
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• 300 nm coiled chromatin fibers forms radial loops• Non histone proteins (~30% of chromosomal proteins)
are implicated in the process• Form a structural scaffolding to which loops of chromatin
are attached to nuclear matrix (or chromosome scaffold)• Scaffolding located in the long axis of the metaphase
chromosome• DNA is tightly bound to this internal scaffold at S/MARs
locus (scaffold/matrix attachment regions)• 2 scaffold proteins are found: topoisomerase II and
matrix attachment proteins
Third order of compaction
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The many different orders of chromatin packing that give rise to the highly condensed metaphase chromosome
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DNA compaction Level of DNA compaction changes
throughout the cell cycle; most compact during M and least compact during S phases
2 types of chromatin; related to the level of gene expression Euchromatin—defined originally as
areas that stained lightly Heterochromatin—defined originally as
areas that stained darkly
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DNA compaction
Euchromatin—chromosomes or regions therein that exhibit normal patterns of condensation and relaxation during the cell cycle Most areas of chromosomes in active
cells Usually areas where gene expression
is occurring
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DNA compaction
Heterochromatin—chromosomes or regions therein that are condensed throughout the cell cycle
Provided first clue that parts of eukaryotic chromosomes do not always encode proteins.
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Chromatin Modifications
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Chromatin Modifications• Chromatin modifications affect the availability of
genes for transcription:– The physical state of DNA in or near a gene is
important in helping control whether the gene is available for transcription.
– Genes of heterochromatin (highly condensed) are usually not expressed because transcription proteins cannot reach the DNA.
• DNA methylation seems to diminish transcription of that DNA.
• Histone acetylation seems to loosen nucleosome structure and thereby enhance transcription.
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Histone Modifications
• In histone acetylation, acetyl groups are attached to positively charged lysines in histone tails
• This loosens chromatin structure, thereby promoting the initiation of transcription
• The addition of methyl groups (methylation) can condense chromatin; the addition of phosphate groups (phosphorylation) next to a methylated amino acid can loosen chromatin
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Amino acidsavailablefor chemicalmodification
Histone tails
DNA double helix
Nucleosome(end view)
(a) Histone tails protrude outward from a nucleosome
Unacetylated histones Acetylated histones
(b) Acetylation of histone tails promotes loose chromatinstructure that permits transcription
Histone tails are the ones modified
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Covalent Modification of core histone tails
Acetylation of lysinesMethylation of lysines
Phosphorylation of serines
Histone acetyl transferase (HAT)
Histone deacetylase (HDAC)
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DNA Methylation
DNA methylation is the attachment of methyl groups (-CH3) to DNA bases after DNA is synthesized.
Inactive DNA, such as that of inactivated mammalian X chromosomes, is generally highly methylated compared to DNA that is actively transcribed.
Comparison of the same genes in different types of tissues shows that the genes are usually more heavily methylated in cells where they are not expressed.
In addition, demethylating certain inactive genes (removing their extra methyl groups) turns them on.
At least in some species, DNA methylation seems to be essential for the long-term inactivation of genes that occurs during cellular differentiation in the embryo.
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chromatin can be covalently modified by
methylation• DNA methylation of cytosines
CH3 CH3
CH3 CH3
CH3
• Only certain cytosines can be methylated.• Sequence context matters.
– In animals, CG– In plants, CG and CNG
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Histone Acetylation
Histone acetylation is the attachment of acetyl groups (-COCH3) to certain amino acids of histone proteins; deacetylation is the removal of acetyl groups. When the histones of nucleosome are
acetylated, they change shape so that they grip the DNA less tightly.
As a result, transcription proteins have easier access to genes in the acetylated region.
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Acetylation of core histones
• Example: Histone acetylation
N-ARTKQTARKSTGGKAPRKQLATKAARKSAP9 14 2318
H3 Acetylation
H-N-C0-CH3
––N–C–C––
(CH2)4
OH
––N–C–C––
(CH2)4
NH3+
OH
Lysine Acetylated Lysine
Acetylation
Acetylation causes histones to lose some of their positive charge. This causes them to bind less tightly to the negatively charged DNA backbone.
Pierce, B. 2005. Genetics, a conceptual approach. 2nd Ed. WH Freeman.
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Consequences of chromatin modificaton
• Histone modification can reduce the positive charge on the proteins, thus altering their attraction for negatively charged DNA and loosening chromatin packing.
• Modification of both histones and cytosines can provide recognition sites for binding of other regulatory proteins, which in turn can alter chromatin packing and gene expression.
Acetylation
Pierce, B. 2005. Genetics, a conceptual approach. 2nd Ed. WH Freeman.