Chap 8 Nucleus and Chromosomes Nucleus of a Eukaryotic Cell Nuclear Envelope Nuclear Pore Complex...

Chap 8 Nucleus and Chromosomes

• Nucleus of a Eukaryotic Cell

• Nuclear Envelope

• Nuclear Pore Complex

• Chromatin

• Nucleolus and Ribosome Biogenesis

• Nuclear Matrix

8.1 The Nucleus of a Eukaryotic Cell

T nn r nu lhe i e c ear m m r ne b a e The outer nuclear membrane

Surrounded by two concentric membranes

8.1.1 Organization of the Nucleus

chromosomes

nuclear matrix

nucleoli

nucleoplasm

an interphase HeLa cell nucleus

showing some of the major components of the nucleus

8.1.2 Internal Architecture

contrast to the cytoplasm, the nucleus:

❖not individually enclosed by membranes

❖not visible using conventional light or

electron microscopy techniques

revised picture of nuclear structure

• Nuclear matrix: a proteinaceous scaffol

d-like network

• Nucleolus: the center for synthesis and

processing of rRNA molecules

8.2 The Nuclear Envelope

• The separation of a cell’s genetic material

from the surrounding cytoplasm may be

single most important feature that

distinguishes eukaryotes from prokaryotes.

Structure of the nuclear envelope

• outer nuclear membrane

• inner nuclear membrane

• nuclear lamina

• perinuclear space

• nuclear pore

nuclear lamina

Function of the nuclear envelope

a barrier between the nucleus and cytoplasm, as a distinct biochemical compartment

sole channels through the nuclear envelope

8.3 Nuclear Pore Complex

• Vertebrate: 50100 proteins

• Diameter: 120 nm, 125 MDa

• basketlike apparatus

• eightfold symmetry

Composition of the NPC

• Cytoplasmic ring

• Nuclear ring

• Spoke

• Central plug

Nuclear envelopes of Xenopus oocytes visualized by field emission in-lens SEM

Cut-away model of the NPC

Three-dimensional models of the NPC

A structure with eightfold symmetry

The NPC consists of an assembly of eight spokes arranged around a central channel

The spokes are connected to rings at the nuclear and cytoplasmic surfaces

Electron micrograph of NPC

NLSs direct nuclear proteins to the nucleus

The nuclear proteins are selective traffic across the nuclear envelope from the cytoplasm to the nucleus

The NLSs include histones, DNA polymerases, RNA polymerases, transcription factors, splicing factors transport through NPC

核孔运输特点◆被动运输◆主动运输●信号引导●双向性

Molecular traffic through NPC

核孔的被动运输

Small molecular traffic through the NPC by passive diffusion

Small molecules and some proteins with MW< 50

kD pass freely across the nuclear envelope in eith

er direction.

Most proteins and RNAs pass through the NPC b

y an active process in only one direction.

核孔的双向运输

mRNA 的输出

2.1 核蛋白运输机制 基本概念◆核蛋白 (nuclear protein)

◆核定位信号 (nuclear localization signals ,NLS)

◆核输出信号 (nuclear export signals, NES)

◆输入蛋白 (importin)

◆输出蛋白 (exportin)

核定位信号

Nuclear localization signals

核蛋白的输入◆核质蛋白 (nucleoplasmin) 实验◆核蛋白输入机理●输入蛋白●Ran 蛋白 :小 GTPase RanGAP:RanGTP 激活蛋白 :细胞质中

RCC1 ： Ran nucleotide-exchange factor1. 细胞核中

核质素的核定位信号及其作用

Receptors for the NLS transport proteins to the nucleus

Protein import through the NPC can be divided into two steps, distinguished by whether they require energy.

The first step does not require energy, proteins that contain NLSs bind to the NPC but do not pass through the pore.

The second step is an energy-dependent process that requires GTP hydrolysis.

Protein import through NPC

Role of the Ran protein in nuclear import

核蛋白输入机理

核内 RNA 与蛋白质输出◆mRNA 的输出

●异质核糖核蛋白 (heterogeneous

ribonucleoprotein, hnRNP)

●信使 RNP(messenger RNP, mRNP)

●mRNA 的输出

◆核内蛋白质输出

◆snRNA 的输出

Transport of RNA between nucleus and cytoplasm

• active, energy-dependent process

• ribonucleoprotein complexes rather

than naked RNAs

mRNA 的输出

snRNA 的输出

核内蛋白质的输出

3 分子伴侣 (chaperones)

3.1 分子伴侣的发现及种类◆The term “chaperone" was first used by Ron Laskey a

nd his colleagues to describe a protein (nucleoplasmin)

that is required for the assembly of nucleosomes from

histones and DNA.

◆Nucleoplasmin binds to histones and mediates their ass

embly into nucleosomes, but nucleoplasmin itself is not

incorporated into the final nucleosome structure.

◆Chaperones thus act as catalysts that facilitate assembl

y without being part of the assembled complex.

分子伴侣的概念及其特点◆1991 年 Ellis 等人提出 :

●由不相关类的蛋白质组成的一个家系

●它们介导其它蛋白质的正确装配

● 但自己不成为最后功能结构中的组分。

◆该概念有以下特点 :● 凡具有“介导”功能的蛋白 , 都称为分子伴侣 ,可以是完全不同的蛋白质 ;

● 作用机理尚不清楚 , 故用“介导”二字 ,伸缩性较大；

● 分子伴侣一定不是最终结构的组成部分 ,但不一定是一个分离的实体 ;

● 装配的涵意比较广 , 包括 : 帮助新生肽的折叠 ,越膜定位 , 亚基组装等。

◆It is important to note that :● Chaperones do not convey additional information

required for the folding of polypeptides into their correct three-dimensional conformations.

● The folded conformation of a protein is determined solely by its amino acid sequence.

● Rather, chaperones catalyze protein folding by assisting the self-assembly process.

●They appear to function by binding to and stabilizing unfolded or partially folded polypeptides that are intermediates along the pathway leading to the final correctly folded state.

分子伴侣的基本功能

◆分子内伴侣 (intromolecular chaperones)

◆分子伴侣的分布●从细菌到人 ,从动物到植物●细胞质、线粒体、叶绿体和微体

分子伴侣结构上的共同特点◆家族成员具有高度保守性◆家族成员结构上具有相似性◆大部分在体内为组成型表达 ,在刺激条件下会被进一步诱导。

Hsp60 的电镜三维镜象照片

3.2 functions of chaperones

◆帮助蛋白质折叠和装配◆蛋白质的转运和定位◆参与细胞器和细胞核结构的发生◆应激反应◆参与信号转导

Action of chaperones during translation

Actions of chaperones GroEL,GroES of E.coli in protein folding

Sequential actions of Hsp70

and Hsp60 chaperones

Action of chaperones in signaling

Action of chaperones during protein transport

Molecular Chaperones

◆应激反应●Molecular chaperones were initially identified as heat-sho

ck proteins, a group of proteins expressed in cells that have been subjected to elevated temperatures or other forms of environmental stress.

●The heat-shock proteins (abbreviated Hsp), which are highly conserved in both prokaryotic and eukaryotic cells, are thought to stabilize and facilitate the refolding of proteins that have been partially denatured as a result of exposure to elevated temperature.

●However, many members of the heat-shock protein family are expressed and have essential cellular functions under normal growth conditions.

●These proteins serve as molecular chaperones, which are needed for polypeptide folding and transport under normal conditions as well as in cells subjected to environmental stress.

8.4 Chromatin & Chromosome

• chromatin types

❖Heterochromatin

☺constitutive

☺facultative

❖euchromatin

The Functions of chromatin

• Storage of genetic information

• Precise segregation of replicated DNA

into two daughter cells

• Platform for transcription, replication,

recombination and DNA repair

Composition of Chromatin

• DNA: stable association with histones

• His tone: H1, H2A, H2B, H3, H4

• Nonhistone: not as stable as DNA-hist

one interactions

N terminal tails are

subject to covalent

modification-important

for transcription

Five major types of histones in calf thymus

Histone Mass Residue No Lys (%) Arg (%)

H1

H2A

H2B

H3

H4

22 500

13 960

13 774

15 273

11 236

215

129

125

135

102

29

11

16

10

11

1

9

6

13

14

The DNA in chromosomes is highly condensed

A scanning electron micrograph of a mitotic chromosome, showing the paired identical chromatids associated along their length and joined tightly at the centromere.

Nucleosomes: the lowest level of chromosome organization

• Nucleosome = a nucleosome core particle + linker DNA + a linker histone

• DNA length: 180-200 bp

• Nucleosome core particle = histone octamer

+ 146 bp DNA

Nucleosome: the basic units of chromatin structure

Kornberg R.(1974): beads on a string

30 nm fiber

beads on a string-nucleosome from interphase nucleus

The organization of chromatin in nucleosomes

?

核酸酶超敏感位点 (nuclease-

supersensitive site)

◆核小体与 DNA 的复制●八聚体的组蛋白进行部分解离 ;

●其中 (H3-H4)2四聚体在一起 ,并且在两条子代双链上随机分布 ;

●原核小体中的 H2A-H2B 则是以两个二聚体存在 ,并相互分离 ;

●随机与子代双链上原或新合成的 (H3-H4)

2四聚体结合组成核小体。

◆核小体与 DNA 的转录 即使正在转录的基因仍然有核小体结构 ,表明转录并不要求整个基因都处于无核小体状态。

Nucleosomes contains DNA wrapped around a protein core of eight histone molecules

• Nucleosome core particle is released from chro

matin by digestion of the linker DNA with a nu

clease. After dissociation of the isolated nucleo

some into its protein core and DNA, the length

of the DNA that was wound around the core ca

n be determined. Its length of 146 nucleotide p

airs is sufficient to wrap almost twice around t

he histone core.

The high-resolution structure of a nucleosome core particle

• The nucleosome core particle, as determined by

X-ray diffraction analysis, reveals how DNA is ti

ghtly wrapped around a disc-shaped histone core,

making 1.65 turns in a left-handed coil.

K. Luger et al. Nature 1997, 389: 251260

Histone depleted metaphase chromosomes

Chromosomes have several levels of DNA packing

• Packaging of nucleosomes into the 30-nm chromatin f

iber depends on histone H1, which is thought to pull t

he nucleosomes together into a regular repeating arra

y.

• Each DNA molecule is packaged into a mitotic chrom

osome that is 10,000 fold shorter its extended length.

Chromatin fibers may be packed according to a zigzag model

zigzag model

• The structure of the 30-nm chromatin fiber

may be a combination of these zigzag variati

ons. An interconversion between these three

variations may occur through an accordion-li

ke expansion and contraction of the fiber.

Problems

• How the long linear DNA molecules are

packaged into compact chromosomes?

DNA Packing

• Eukaryotic DNA is packaged into a set of

chromosomes

• Why compaction of DNA into chromosome is

essential

?

Simple Calculation

• Human: 3109 bp, 23 chromosomes

• 1.02 m/haploid, 2.04 m/cell

• The nucleus: 10 m in diameter

• The mitotic chromosome is ~ 1 m

• If no compaction, nucleus would be too s

mall to hold all DNA!!!

Compaction of chromatin is cell-stage dependentA. Interphase chromatin B. a mitotic chromosome,

which is duplicated already

Question: How this compaction is achieved?

Changes in nucleosome structure allow access to DNA

• Eukaryotic cells contain chromatin remodeling complexes, protein machines that use the energy of ATP hydrolysis to change the structure of nucleosomes temporarily so that DNA becomes less tightly bound to the histone core. The remodeled state may result from movement of the H2A-H2B dimers in the nucleosome core; the H3H4 tetramer is particularly stable and would be difficult to rearrange.

Chromatin remodeling complexes alter nucleosome structure

The remodeling of nucleosome structure has two important consequences

• First, it permits ready access to nucleosomal DNA by other proteins in the cell, particularly those involved in gene expression, DNA replication, and repair.

• Second, they can catalyze changes in the positions of nucleosomes along DNA; some can even transfer a histone core from one DNA molecule to another.

Metaphase Chromosomes

• Metaphase chromosomes are so highly condensed

that their morphology can be studied using light

microscope.

• Staining techniques yield characteristic patterns

of alternating light and dark chromosome bands.

• Genes can be localized to specific chromosome

bands by in situ hybridization.

Human metaphase chromosomes

Typical appearance of a metaphase chromosome

• Scanning electron micrograph of several human

metaphase chromosomes showing the paired ide

ntical chromatids associated along their length a

nd joined tightly at the centromere.

• The connections between chromatids consist of a

protein called cohesin that contains a number of

highly conserved subunits.

The sister chromotids of a

mitotic pair each consist of

a fiber (30 nm in

diameter) compactly folded

into the chromosome.

染色体的主要结构◆着丝粒 (centromere)

●主缢痕 (Primary constriction)

◆次缢痕 (secondary constriction)

◆动粒 (kinetochore)

◆核仁组织区 (nucleolar organizing

region, NOR)

◆随体 (satellite)

◆端粒 (telomere)

四种不同位置着丝粒的染色体

Centromere

• The constricted region of a chromosome

that is the position at which the pair of c

hromatids are held together.

• The centromeres serve both as the sites o

f association of sister chromatids and as t

he attachment sites for microtubules of t

he mitotic spindle.

着丝粒与动粒

Centromere and kinetochore

The Functions of centromeres

• Required for chromosome stability

• Sister chromatid pairing

• Mitotic and meiotic spindle attachment

• Chromosome movement

• Cell cycle checkpoint control

主缢痕与主缢

痕

Telomeres

• allow complete replication of the ends of

chromosomes

• protect them from erosion and fusion

with other DNA fragments

The telomere DNA sequences of a variety of eukaryotes

Organism Telomeric repeat sequence

Yeasts Saccharomyces cerevisiae Schizosaccharomyces pombeProtozoans Tetrahymena DictyosteliumPlant ArabidopsisMammal Human

G13T

G25TTAC

GGGGTT G18A

AGGGTTT

TTAGGG

Telomere signals on chromosomes after FISH with Cy3-labelled (CCCTAAA)3 probe

Telomere-mediated chromosome integrity in mammalian cells lacking telomerase or DNA repair factors

A dicentric (Dic) chromosome (pointed

arrow) in a human metaphase spread s

howing telomere signals (red) at the ter

mini and two centromeres (green) alon

g the chromosome arms.

端粒的形成

◆人工染色体 (artificial chromosome)

人工构建的含有稳定染色体的天然结构序列，即 ARS、 CEN 、 TEL 序列的微小染色体，可以象天然染色体一样在寄主细胞中稳定复制和遗传，称为人工染色体。

DNA 结构稳定遗传的功能序列◆ARS (autonomous replicating sequence)◆CEN (centromeric sequence) The centromere is a specialized region of the

chromosome that plays a critical role in ensuring the correct distribution of duplicated chromosomes to daughter cells during mitosis ◆ TEL(telomeric sequence)

The sequences at the ends of eukaryotic chromosomes, called telomeres, play critical roles in chromosome replication and maintenance.

5.3 Giant chromosone

◆多线染色体 (polytene chromosome)

●概念●时相：间期●存在的组织▲双翅目昆虫的幼虫组织内 , 如唾液腺、气管等。▲体积也相应增大●产生的原因：

Polytene chromosome

◆灯刷染色体 (lampbrush

chromosome) 灯刷染色体是卵母细胞进行减数第一

次分裂时 ,停留在双线期的染色体。

它是一个二价体 , 含 4 条染色单体。它 由轴和侧丝组成 ,形似灯刷。

灯刷染色体

8.5 Nucleolus and ribosome biogenesis

• The nucleolus is the most obvious structure seen i

n the nucleus of a eukaryotic cell when viewed in

the light microscope.

• It is the site of rRNA transcription and processing,

and of ribosome assembly.

Ultrastructure of nucleolus

❖fibrillar centers, FC

❖dense fibrillar component, DFC

❖granular component, GC

❖nucleolar associated chromatin

❖nucleolar matrix

DNA稳定遗传的三种功能位点

Electron micrograph of a thin section of a nucleolus in

a human fibroblast, showing its three distinct zones

Nucleolar fusion

Function of the nucleolus in ribosome and other ribonucleoprotein synthesis

• The nucleolus is a ribosome production factory, d

esigned to fulfill the need for large-scale producti

on of rRNA and assembly of the ribosomal subun

its.

• In addition to its important role in ribosome biog

enesis, the nucleolus is also the site where other R

NAs are produced and other RNA-protein compl

exes are assembled.

Nucleolar dynamics

• The nucleolus also plays an important role in cel

l-cycle regulation, senescence and stress response

s.

• It is demonstrated that the nucleolar proteome ch

anges significantly over time in response to chang

es in cellular growth conditions using a quantitati

ve proteomic approach for the temporal characte

rization of protein flux through cellular organelle

s.

The arrangement of rRNA genes

• The nucleolus is organized around th

e chromosomal regions that contain t

he genes for the 5.8S, 18S, and 28S r

RNA.

Ribosomal RNA genes

The rRNA transcription unit

Transcription of the rRNA genes

• 18S, 5.8S, 28S rRNA→RNA pol I,

a single unit

• 5S rRNA→RNA pol III

Processing of pre-rRNA

• The 45S pre-rRNA transcript contains external

transcribed spacers (ETS) at both ends and inte

rnal transcribed spacers (ITS) between the sequ

ences of 18S, 5.8S, and 28S rRNA. The pre-rRN

A is processed via a series of cleavages (

illustrated for human pre-rRNA) to yield the matu

re rRNA species.

Processing of rRNA

Ribosome Assembly

• Ribosomal proteins are imported to the nucleolus

from cytoplasm and begin to assemble on pre-rR

NA prior to its cleavage. As the pre-rRNA is proc

essed, additional ribosomal proteins and the 5S r

RNA assemble to form preribosomal particles. T

he final steps of maturation follow the export of p

reribosomal particles to the cytoplasm,

yielding the 40S and 60S ribosomal subunits.

Nonhistone proteins

Nonhistone chromosomal protei

ns include a large number of wi

dely diverse structural, enzymat

ic, and regulatory proteins.

非组蛋白的种类与性质◆序列特异性 DNA 结合蛋白 (sequence-sp

ecific DNA-binding protein) 。◆其他蛋白●以 DNA 作为底物的酶●作用于组蛋白的一些酶●调节基因表达的蛋白因子等◆非组蛋白的特性 : 呈酸性、带负电荷。

非组蛋白的功能除了一些酶以外，非组蛋白还具有以下功能∶◆参与染色体的构建 ;◆参与 DNA 复制 ;◆调控基因的表达。

Transcription Factor Motifs

Trans-acting factor and cis-acting element

◆反式作用因子 (trans-acting factor)

They can affect the expression of genes lo

cated on other chromosomes within the c

ell. ◆顺式作用元件 (cis-acting element)

They affect the expression of only linked

genes on the same DNA molecule.

Trans-acting factor and cis-acting element

8.6 Nuclear matrix

• The protein network in the nucleus is called the

nuclear matrix, a proteinaceous scaffold-like net

work that permeates the cell. It is composed of a

ctin and numerous other protein components tha

t have not been fully characterized, including co

mponents of the chromosomal scaffold that rear

ranges and condenses to form metaphase chrom

osomes during mitosis.

Terms or abbreviation

• Nuclear matrix or nuclear skeleton

• SARs: scaffold-associated regions

• MARs: matrix-associated regions

Histone depleted metaphase chromosomes

SARs(MARs)

• Scaffold(matrix) attachment region

• Regions of the chromosomes with sequences specific for topoiso

merase, HMG protein, and H1 binding

• Found only in untranscribed regions of chromosomes

• Spaced along the chromosomes, with the intervening regions co

ntaining one or more genes?

• Highly AT rich (65%) and several hundred bp long

DNA binds to the protein matrix

Summary

• The nucleus is the largest structure in the

eukaryotic cell. It consists of DNA, proteins,

and RNA, and plays a vital role in:

❖Protein synthesis

❖The passage of genetic information from one

generation to the next

Summary

• Nuclear envelope encloses the nucleus. It

consists of two layers of membrane.

• Nuclear pores are found at points of

contact between the inner and outer

membranes.

• Chromatin is the collective name for the

long strands of DNA and associated

proteins.

Summary

• Nucleoli are extremely dense structures i

n the nuclei and are highly active in rRN

A synthesis.

• The nuclear matrix consists of DNA, nucl

eoproteins, and structural proteins.