Chap 8 Nucleus and Chromosomes Nucleus of a

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Chap 8 Nucleus and Chromosomes • Nucleus of a Eukaryotic Cell • Nuclear Envelope

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 Surrounded by two concentric membranes The

8. 1 The Nucleus of a Eukaryotic Cell Surrounded by two concentric membranes The inner nuclear membrane The outer nuclear membrane

8. 1. 1 Organization of the Nucleus chromosomes nuclear matrix nucleoli nucleoplasm

8. 1. 1 Organization of the Nucleus chromosomes nuclear matrix nucleoli nucleoplasm

an interphase He. La cell nucleus showing some of the major components of the

an interphase He. La 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

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 scaffold-like network • Nucleolus:

revised picture of nuclear structure • Nuclear matrix: a proteinaceous scaffold-like network • Nucleolus: the center for synthesis and processing of r. RNA molecules

8. 2 The Nuclear Envelope • The separation of a cell’s genetic material from

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 •

Structure of the nuclear envelope • outer nuclear membrane • inner nuclear membrane • nuclear lamina • perinuclear space • nuclear pore

nuclear lamina

nuclear lamina

Function of the nuclear envelope a barrier between the nucleus and cytoplasm, as a

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: 50 100 proteins • Diameter: 120 nm,

8. 3 Nuclear Pore Complex • Vertebrate: 50 100 proteins • Diameter: 120 nm, 125 MDa • basketlike apparatus • eightfold symmetry

Composition of the NPC • Cytoplasmic ring • Nuclear ring • Spoke • Central

Composition of the NPC • Cytoplasmic ring • Nuclear ring • Spoke • Central plug

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

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

Cut-away model of the NPC

Cut-away model of the NPC

Three-dimensional models of the NPC A structure with eightfold symmetry The NPC consists of

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

Electron micrograph of NPC

NLSs direct nuclear proteins to the nucleus The nuclear proteins are selective traffic across

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

Molecular traffic through NPC

Molecular traffic through NPC

Small molecular traffic through the NPC by passive diffusion Small molecules and some proteins

Small molecular traffic through the NPC by passive diffusion Small molecules and some proteins with MW< 50 k. D pass freely across the nuclear envelope in either direction. Most proteins and RNAs pass through the NPC by an active process in only one direction.

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)

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)

Nuclear localization signals

Nuclear localization signals

Receptors for the NLS transport proteins to the nucleus Protein import through the NPC

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

Protein import through NPC

Role of the Ran protein in nuclear import

Role of the Ran protein in nuclear import

 核内RNA与蛋白质输出 ◆m. RNA的输出 ●异质核糖核蛋白 (heterogeneous ribonucleoprotein, hn. RNP) ●信使RNP(messenger RNP, m. RNP) ●m.

核内RNA与蛋白质输出 ◆m. RNA的输出 ●异质核糖核蛋白 (heterogeneous ribonucleoprotein, hn. RNP) ●信使RNP(messenger RNP, m. RNP) ●m. RNA的输出 ◆核内蛋白质输出 ◆sn. RNA的输出

Transport of RNA between nucleus and cytoplasm • active, energy-dependent process • ribonucleoprotein complexes

Transport of RNA between nucleus and cytoplasm • active, energy-dependent process • ribonucleoprotein complexes rather than naked RNAs

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

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 and 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 assembly into nucleosomes, but nucleoplasmin itself is not incorporated into the final nucleosome structure. ◆Chaperones thus act as catalysts that facilitate assembly without being part of the assembled complex.

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

◆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.

Action of chaperones during translation

Action of chaperones during translation

Actions of chaperones Gro. EL, Gro. ES of E. coli in protein folding

Actions of chaperones Gro. EL, Gro. ES of E. coli in protein folding

Sequential actions of Hsp 70 and Hsp 60 chaperones

Sequential actions of Hsp 70 and Hsp 60 chaperones

Action of chaperones in signaling

Action of chaperones in signaling

Action of chaperones during protein transport

Action of chaperones during protein transport

Molecular Chaperones

Molecular Chaperones

◆应激反应 ●Molecular chaperones were initially identified as heatshock proteins, a group of proteins expressed

◆应激反应 ●Molecular chaperones were initially identified as heatshock 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

8. 4 Chromatin & Chromosome • chromatin types ❖Heterochromatin ☺constitutive ☺facultative ❖euchromatin

The Functions of chromatin • Storage of genetic information • Precise segregation of replicated

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: H 1,

Composition of Chromatin • DNA: stable association with histones • His tone: H 1, H 2 A, H 2 B, H 3, H 4 • Nonhistone: not as stable as DNAhistone interactions

N terminal tails are subject to covalent modification-important for transcription

N terminal tails are subject to covalent modification-important for transcription

Five major types of histones in calf thymus Histone Mass Residue No Lys (%)

Five major types of histones in calf thymus Histone Mass Residue No Lys (%) Arg (%) H 1 22 500 215 29 1 H 2 A 13 960 129 11 9 H 2 B 13 774 125 16 6 H 3 15 273 135 10 13 H 4 11 236 102 11 14

The DNA in chromosomes is highly condensed A scanning electron micrograph of a mitotic

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

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 30 nm fiber beads on a string-nucleosome

Nucleosome: the basic units of chromatin structure 30 nm fiber beads on a string-nucleosome from interphase nucleus Kornberg R. (1974): beads on a string

The organization of chromatin in nucleosomes ?

The organization of chromatin in nucleosomes ?

核酸酶超敏感位点(nucleasesupersensitive site)

核酸酶超敏感位点(nucleasesupersensitive site)

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

Nucleosomes contains DNA wrapped around a protein core of eight histone molecules • Nucleosome core particle is released from chromatin by digestion of the linker DNA with a nuclease. After dissociation of the isolated nucleosome into its protein core and DNA, the length of the DNA that was wound around the core can be determined. Its length of 146 nucleotide pairs is sufficient to wrap almost twice around the histone core.

The high-resolution structure of a nucleosome core particle • The nucleosome core particle, as

The high-resolution structure of a nucleosome core particle • The nucleosome core particle, as determined by X -ray diffraction analysis, reveals how DNA is tightly wrapped around a disc-shaped histone core, making 1. 65 turns in a left-handed coil.

K. Luger et al. Nature 1997, 389: 251 260

K. Luger et al. Nature 1997, 389: 251 260

Histone depleted metaphase chromosomes

Histone depleted metaphase chromosomes

Chromosomes have several levels of DNA packing • Packaging of nucleosomes into the 30

Chromosomes have several levels of DNA packing • Packaging of nucleosomes into the 30 -nm chromatin fiber depends on histone H 1, which is thought to pull the nucleosomes together into a regular repeating array. • Each DNA molecule is packaged into a mitotic chromosome that is 10, 000 fold shorter its extended length.

Chromatin fibers may be packed according to a zigzag model

Chromatin fibers may be packed according to a zigzag model

zigzag model • The structure of the 30 -nm chromatin fiber may be a

zigzag model • The structure of the 30 -nm chromatin fiber may be a combination of these zigzag variations. An interconversion between these three variations may occur through an accordion-like expansion and contraction of the fiber.

Problems • How the long linear DNA molecules are packaged into compact chromosomes?

Problems • How the long linear DNA molecules are packaged into compact chromosomes?

DNA Packing • Eukaryotic DNA is packaged into a set of chromosomes • Why

DNA Packing • Eukaryotic DNA is packaged into a set of chromosomes • Why compaction of DNA into chromosome is essential?

Simple Calculation • Human: 3 109 bp, 23 chromosomes • 1. 02 m/haploid, 2.

Simple Calculation • Human: 3 109 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 small to hold all DNA!!!

Compaction of chromatin is cell-stage dependent A. Interphase chromatin B. a mitotic chromosome, which

Compaction of chromatin is cell-stage dependent A. 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

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 H 2 A-H 2 B dimers in the nucleosome core; the H 3 H 4 tetramer is particularly stable and would be difficult to rearrange.

Chromatin remodeling complexes alter nucleosome structure

Chromatin remodeling complexes alter nucleosome structure

The remodeling of nucleosome structure has two important consequences • First, it permits ready

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

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

Human metaphase chromosomes

Typical appearance of a metaphase chromosome • Scanning electron micrograph of several human metaphase

Typical appearance of a metaphase chromosome • Scanning electron micrograph of several human metaphase chromosomes showing the paired identical chromatids associated along their length and 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

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)

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)

Centromere • The constricted region of a chromosome that is the position at which

Centromere • The constricted region of a chromosome that is the position at which the pair of chromatids are held together. • The centromeres serve both as the sites of association of sister chromatids and as the attachment sites for microtubules of the mitotic spindle.

Centromere and kinetochore

Centromere and kinetochore

The Functions of centromeres • Required for chromosome stability • Sister chromatid pairing •

The Functions of centromeres • Required for chromosome stability • Sister chromatid pairing • Mitotic and meiotic spindle attachment • Chromosome movement • Cell cycle checkpoint control

Telomeres • allow complete replication of the ends of chromosomes • protect them from

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 Yeasts Saccharomyces cerevisiae Schizosaccharomyces

The telomere DNA sequences of a variety of eukaryotes Organism Yeasts Saccharomyces cerevisiae Schizosaccharomyces pombe Protozoans Tetrahymena Dictyostelium Plant Arabidopsis Mammal Human Telomeric repeat sequence G 1 3 T G 2 5 TTAC GGGGTT G 1 8 A AGGGTTT TTAGGG

Telomere signals on chromosomes after FISH with Cy 3 -labelled (CCCTAAA)3 probe Telomere-mediated chromosome

Telomere signals on chromosomes after FISH with Cy 3 -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 showing telomere signals (red) at the termini and two centromeres (green) along the chromosome arms.

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

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.

Polytene chromosome

Polytene chromosome

8. 5 Nucleolus and ribosome biogenesis • The nucleolus is the most obvious structure

8. 5 Nucleolus and ribosome biogenesis • The nucleolus is the most obvious structure seen in the nucleus of a eukaryotic cell when viewed in the light microscope. • It is the site of r. RNA transcription and processing, and of ribosome assembly.

Ultrastructure of nucleolus ❖fibrillar centers, FC ❖dense fibrillar component, DFC ❖granular component, GC ❖nucleolar

Ultrastructure of nucleolus ❖fibrillar centers, FC ❖dense fibrillar component, DFC ❖granular component, GC ❖nucleolar associated chromatin ❖nucleolar matrix

Electron micrograph of a thin section of a nucleolus in a human fibroblast, showing

Electron micrograph of a thin section of a nucleolus in a human fibroblast, showing its three distinct zones

Nucleolar fusion

Nucleolar fusion

Function of the nucleolus in ribosome and other ribonucleoprotein synthesis • The nucleolus is

Function of the nucleolus in ribosome and other ribonucleoprotein synthesis • The nucleolus is a ribosome production factory, designed to fulfill the need for large-scale production of r. RNA and assembly of the ribosomal subunits. • In addition to its important role in ribosome biogenesis, the nucleolus is also the site where other RNAs are produced and other RNA-protein complexes are assembled.

Nucleolar dynamics • The nucleolus also plays an important role in cellcycle regulation, senescence

Nucleolar dynamics • The nucleolus also plays an important role in cellcycle regulation, senescence and stress responses. • It is demonstrated that the nucleolar proteome changes significantly over time in response to changes in cellular growth conditions using a quantitative proteomic approach for the temporal characterization of protein flux through cellular organelles.

The arrangement of r. RNA genes • The nucleolus is organized around the chromosomal

The arrangement of r. RNA genes • The nucleolus is organized around the chromosomal regions that contain the genes for the 5. 8 S, 18 S, and 28 S r. RNA.

Ribosomal RNA genes

Ribosomal RNA genes

The r. RNA transcription unit

The r. RNA transcription unit

Transcription of the r. RNA genes • 18 S, 5. 8 S, 28 S

Transcription of the r. RNA genes • 18 S, 5. 8 S, 28 S r. RNA→RNA pol I, a single unit • 5 S r. RNA→RNA pol III

Processing of pre-r. RNA • The 45 S pre-r. RNA transcript contains external transcribed

Processing of pre-r. RNA • The 45 S pre-r. RNA transcript contains external transcribed spacers (ETS) at both ends and internal transcribed spacers (ITS) between the sequences of 18 S, 5. 8 S, and 28 S r. RNA. The prer. RNA is processed via a series of cleavages (illustrated for human pre-r. RNA) to yield the mature r. RNA species.

Processing of r. RNA

Processing of r. RNA

Ribosome Assembly • Ribosomal proteins are imported to the nucleolus from cytoplasm and begin

Ribosome Assembly • Ribosomal proteins are imported to the nucleolus from cytoplasm and begin to assemble on prer. RNA prior to its cleavage. As the pre-r. RNA is processed, additional ribosomal proteins and the 5 S r. RNA assemble to form preribosomal particles. The final steps of maturation follow the export of preribosomal particles to the cytoplasm, yielding the 40 S and 60 S ribosomal subunits.

Nonhistone proteins Nonhistone chromosomal proteins include a large number of widely diverse structural, enzymatic,

Nonhistone proteins Nonhistone chromosomal proteins include a large number of widely diverse structural, enzymatic, and regulatory proteins.

 Transcription Factor Motifs

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

Trans-acting factor and cis-acting element ◆反式作用因子(trans-acting factor) They can affect the expression of genes located on other chromosomes within the cell. ◆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

Trans-acting factor and cis-acting element

8. 6 Nuclear matrix • The protein network in the nucleus is called the

8. 6 Nuclear matrix • The protein network in the nucleus is called the nuclear matrix, a proteinaceous scaffold-like network that permeates the cell. It is composed of actin and numerous other protein components that have not been fully characterized, including components of the chromosomal scaffold that rearranges and condenses to form metaphase chromosomes during mitosis.

Terms or abbreviation • Nuclear matrix or nuclear skeleton • SARs: scaffold-associated regions •

Terms or abbreviation • Nuclear matrix or nuclear skeleton • SARs: scaffold-associated regions • MARs: matrix-associated regions

Histone depleted metaphase chromosomes

Histone depleted metaphase chromosomes

SARs(MARs) • Scaffold(matrix) attachment region • Regions of the chromosomes with sequences specific for

SARs(MARs) • Scaffold(matrix) attachment region • Regions of the chromosomes with sequences specific for topoisomerase, HMG protein, and H 1 binding • Found only in untranscribed regions of chromosomes • Spaced along the chromosomes, with the intervening regions containing one or more genes? • Highly AT rich (65%) and several hundred bp long

DNA binds to the protein matrix

DNA binds to the protein matrix

Summary • The nucleus is the largest structure in the eukaryotic cell. It consists

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.

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 in the nuclei and are highly active

Summary • Nucleoli are extremely dense structures in the nuclei and are highly active in r. RNA synthesis. • The nuclear matrix consists of DNA, nucleoproteins, and structural proteins.