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The nucleus: organization,

structure/function

Nucleus

The Nucleus is a membraneenclosed organelle which house


most of the genetic information
and regulatory machinery
responsible for providing the cell
with its unique characteristics.

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

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

NUCLEUS
NUCLEUS

nuclear
envelope
nucleolus
nuclear
pores

chromatin

nucleus

nuclear
pores

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.

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

Nucleoplasm
The nucleoplasm: a highly viscous liquid,
similar to cytoplasm, which surrounds the
chromosomes and nucleolus

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

The nuclear envelope


The Components:

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

Lamin provides anchorage sites for chromatin

Functions of lamins
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

(Albertsetal)

(slidefromJessHurt,HMS)

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

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.

Gerace et al., 1984

Dissolution of the Nuclear Lamina

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

Breakdown of the Nuclear Membrane

Re-Formation of the Nuclear Envelope

Nuclear pore complex

Nuclear pore composed of a


nuclear pore complex measuring
70-90nm (inside diameter) with
9nm open channel
Typical mammalian celll has 3,0004,000 pore complexes (10-12 pores
per sq m) in the nuclear envelope

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

The nuclear pore complex


(NPC)

The Nuclear Pore Complex


Cytoplasmic
filament
Cytoplasm

Cytoplasmic
ring

~2000
Nucleus

Inner ring

~150
Ribosome

Basket
Distal ring

Nuclear pores are large protein complexes


Cytplasmic
fibrils

Cytosol

Cytoplasmic face

Nuclear envelope
Nucleus
Nuclear lamina
Annular subunit of central
channel or transporter
Nuclear basket or cage
Nuclear face

Multiple copies of ~100 different proteins (nuclear


pore proteins = NPPs) totaling >125 million daltons!

EM of nuclear pore complex

The nuclear pores on the membrane


Purpose of nuclear pores?
-allows for exchange of macromolecules
-NPCs are dynamic

Type of cargo transported?


-proteins, ribosomes, RNPs,
and RNAs

How is this achieved?


-Via Nups (proteins of the NPC)
-assembly/disassembly of cargos
via exchange of GDP for GTP by
Ran

Nucleo-Cytoplasmic Transport

Ribosomal
Subunits

mRNA

Ribosomal
Proteins

mRNA

Transport of large molecules is active requires GTP

Small molecules (< 60


kDa), or about 9 nm
diameter) enter or
exit nucleus by
passive diffusion
Nuclear pores also required for active export of RNPs
(including ribosome subunits, mRNA, tRNA etc.)

Larger molecules
must be actively
tranported:
(1)

binding to
transporter;
and

(2) transport thru


nuclear pore
using GTP

Import and export occur through same pores

Import into the nucleus


Protein import to
nucleus
Nuclear localization
signal (NLS)
Best-studied 1 or 2
sequences of +ve
charged

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

Simplified view of nuclear transport


NLS

(cargo)
(importin)

Pore opens

Energy for transport provided by G proteins


(GTP binding proteins; large family)
Molecular switches
Pi

GAP
GDP

GTP
GTPase
on

GTP

GEF

GTPase
off

GDP

GAP = GTPase Activating Protein


GEF = Guanine Nucleotide Exchange Factor

RAN

GTPase used in
nuclear transport

Pi

GAP
GDP

GTP
GTPase
on

GTP

GEF

GTPase
off

GDP

Nuclear import/export cycle is driven by GTP hydrolysis

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 and (form import receptor)
Involved in cytoplasm to nucleus transport
The subunit binds the nuclear localization signal
(NLS) of cargo protein to be transported to the
nucleus and the subunit interacts with the FG
nucleoporins
Exportins
Nucleus to cytoplasm transport
Nuclear transport factor 2 (NTF2)

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 RanGTP

Directional protein import is driven by GTP hydrolysis


NLS
Importin
Importin

Pi

NLS

Ran
GAP

Cytoplasm
Importin

Ran-GTP
Ran-GDP

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)

NTF2

Ran-GTP-importin exported from


nucleus thru pore
Ran-GAP stimulates GTP
hydrolysis in cytoplasm by Ran
Ran-GDP releases importin in
cytoplasm

Nucleus
NLS

Importin

Importin
Ran-GTP
GTP GDP

Ran-GTP

RanGEF

Ran-GDP

NLS

Ran-GDP transported into nucleus


(not shown)
Ran GEF stimulates nucleotide
exchange restoring Ran-GTP.

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

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

Export from the nucleus

DNA Organization in
Eukaryotic
Chromosomes

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

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

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

Eukaryotic chromosomal
organization

Histone proteins

Abundant
Histone protein sequence is highly conserved
among eukaryotesconserved 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

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

Eukaryotic chromosomal organization


Histone proteins
5 main types

H1attached to the nucleosome and involved in further


compaction of the DNA (conversion of 10 nm chromatin to
30 nm chromatin)
H2A
H2B
Two copies in each nucleosome
H3
histone octamer; DNA wraps
H4

around this structure1.75 times

This structure produces 10nm chromatin

Nucleosome structure
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

Chromatin Compaction

Fig. 9

Orders of chromatin
structure from
naked DNA to
chromatin to fully
condensed
chromosomes...

Beads on a String10 nm Fiber

DNA Molecules are highly condensed in chromosomes


Nucleosomes of interphase under electron microscope
Nucleosome: basic level of chromosome/chromatin organization
Chromatin: protein-DNA complex
Histone: DNA binding protein
A: diameter 30 nm; B: further unfolding, beads on a string conformation

10 nm filament; nucleosomes

protein
purification

histones

(= 1g per g DNA)

H1 Basic (arg, lys);


+ charges bind
H3 to - phosphates
H2A on DNA
DNA
H2B
H4

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)

nucleosomes

deoxyribonuclease I
(DNase I) digestion

Separate DNA
from protein
proteins
H1
2H3
2H2A
2H2B
2H4

DNA
146 nt fragments

the histone
octamer

Conclude: histones in a
nucleosome protect 146 nt
from DNase I attack.

Nucleosome Structures
Histone octamer
2 H2A
2 H2B
2 H3
2 H4

Structural Organization of the Core Histones

The Assembly of the Core Histones

Notice the long tails of the octamer

The bending of DNA in a nucleosome


1. Flexibility of DNAs: A-T riched minor groove inside and G-C
riched groove outside
2. DNA bound protein can also help

The function of
Histone tails

The function of Histone H1

Chromatin Remodeling

Cyclic Diagram for


nucleosome formation and
disruption

10 nm Fiber

A string of nucleosomes is seen under EM as a 10 nm


fiber

30 nm Fiber

30 nm fiber is coil of
nucleosomes with 6/turn

The 30 nm Fiber
(Compacts DNA 7X more)

Histone H1 : essential for the


solenoid
Secondary Structure

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

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)

Model for the organization of 30-nm chromatin fiber into looped domains that
are anchored to a nonhistone protein chromosome scaffold

74

Acid extraction removes histones from metaphase chromosomes, leaving nonhistones...


Low power

high power

DNA
loops
Scaffold
protein

Major non-histone proteins = topoisomerases!

Third order of
compaction
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

The many different orders of chromatin packing that give rise to the highly
condensed metaphase chromosome

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

Euchromatindefined originally as areas


that stained lightly
Heterochromatindefined originally as
areas that stained darkly

DNA compaction

Euchromatinchromosomes 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

DNA compaction

Heterochromatinchromosomes
or regions therein that are
condensed throughout the cell
cycle
Provided first clue that parts of
eukaryotic chromosomes do not
always encode proteins.

Chromatin
Modifications

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.

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

Histone tails are the


ones modified
Amino acids
available
for chemical
modification

Histone
tails
DNA
double
helix

Nucleosome
(end view)
(a) Histone tails protrude outward from a nucleosome

Acetylated histones
Unacetylated histones
(b) Acetylation of histone tails promotes loose chromatin
structure that permits transcription

Covalent Modification
of core histone tails
Acetylation of lysines
Methylation of lysines
Phosphorylation of
serines
Histone acetyl
transferase (HAT)
Histone deacetylase
(HDAC)

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.

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

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.

Acetylation of core histones


Example: Histone acetylation
H O
NCC
(CH2)4
NH3

Lysine

H O
NCC

N-ARTKQTARKSTGGKAPRKQLATKAARKSAP

(CH2)4
HNC0CH3

14

18

Acetylation

23

H3

Acetylated Lysine

Acetylation

Pierce, B. 2005. Genetics, a conceptual approach. 2nd Ed. WH Freeman.

Acetylation causes histones


to lose some of their positive
charge. This causes them to
bind less tightly to the
negatively charged DNA
backbone.

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.
Acetylation

Pierce, B. 2005. Genetics, a conceptual approach. 2nd Ed. WH Freeman.

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.