The Nucleus

The nucleus: organization, structure/function

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.

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

NUCLEUSNUCLEUS

http://upload.wikimedia.org/wikipedia/commons/3/38/Diagram_human_cell_nucleus.svg

nuclear pores

chromatin

nucleolus

nuclear envelope

nuclear pores

nucleus

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 thechromosomes 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

(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)

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.

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

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 3,000-4,000 pore complexes (10-12 pores per sq μm) 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)

CytoplasmicCytoplasmicfilamentfilament

CytoplasmCytoplasm

NucleusNucleus

CytoplasmicCytoplasmicringring

Inner ringInner ring

BasketBasket

Distal ringDistal ring

The Nuclear Pore ComplexThe Nuclear Pore Complex

RibosomeRibosome

~150Å

~2000Å

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

EM of nuclear pore complex

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

mRNAmRNA

mRNAmRNA

RibosomalRibosomalSubunitsSubunits

RibosomalRibosomalProteinsProteins

Nucleo-Cytoplasmic TransportNucleo-Cytoplasmic Transport

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

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

(importin)

NLS(cargo)

Pore opens

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)

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

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 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)

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

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

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


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


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


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


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


• 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


• 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

• 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

Chromatin Compaction

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

Fig. 9

Beads on a String—10 nm FiberBeads on a String—10 nm Fiber

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

10 nm filament; nucleosomes

histones (= 1g per g DNA) DNA

proteinpurification

H1

H3H2A

H2BH4

•Basic (arg, lys);•+ charges bind to - phosphates on DNA

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)

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

Nucleosome StructuresHistone octamer

2 H2A2 H2B2 H32 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 nucleosome1. Flexibility of DNAs: A-T riched minor groove inside and G-C

riched groove outside2. 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 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

30 nm Fiber30 nm Fiber

• 30 nm fiber is coil of 30 nm fiber is coil of nucleosomes with 6/turnnucleosomes with 6/turn

The 30 nm FiberThe 30 nm Fiber(Compacts DNA 7X more)(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


• 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)

74

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

Acid extraction removes histones from meta-phase chromosomes, leaving nonhistones...

Low power high power

Scaffold protein

DNA loops

Major non-histone proteins = topoisomerases!

• 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

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 Euchromatin—defined originally as

areas that stained lightly Heterochromatin—defined originally as

areas that stained darkly

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

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.

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

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

Covalent Modification of core histone tails

Acetylation of lysinesMethylation 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

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.

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.

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The Nucleus