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  The inner nuclear membrane The inner nuclear membrane  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 scaffold-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: 50  100 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 either direction. Most proteins and RNAs pass through the NPC by 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 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 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 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.

 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- shock 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, H4H1, H2A, H2B, H3, H4 Nonhistone: not as stable as DNA- histone interactions

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

Five major types of histones in calf thymus Histone Mass Residue NoLys (%)Arg (%) H1 H2A H2B H3 H

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 organizationthe lowest level of chromosome organization Nucleosome = a nucleosome core particle + linker DNA + a linker histone DNA length: bp Nucleosome core particle = histone octamer 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 moleculeseight 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 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.making 1.65 turns in a left-handed coil

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

Histone depleted metaphase chromosomes

Chromosomes have several levels of DNA packingDNA packing Packaging of nucleosomes into the 30-nm chromatin fiber depends on histone H1, 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

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?

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

Simple Calculation Human: 3  10 9 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 chromatinB. 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

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 identical chromatids associated along their length and joined tightly at the centromere.Scanning electron micrograph 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 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.

着丝粒与动粒着丝粒与动粒

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

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 OrganismTelomeric repeat sequence Yeasts Saccharomyces cerevisiae Schizosaccharomyces pombe Protozoans Tetrahymena Dictyostelium Plant Arabidopsis Mammal Human G 1  3 T G 2  5 TTAC GGGGTT G 1  8 A 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 showing telomere signals (red) at the termini and two centromeres (green) along 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。

灯刷染色体灯刷染色体

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 rRNA transcription and processing, and of ribosome assembly.

Ultrastructure of nucleolusnucleolus ❖ 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, designed to fulfill the need for large-scale production of rRNA 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 cell-cycle 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 rRNA genes The nucleolus is organized around the chromosomal regions that contain the genes for the 5.8S, 18S, and 28S rRNA.

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 internal transcribed spacers (ITS) between the sequences of 18S, 5.8S, and 28S rRNA. The pre- rRNA is processed via a series of cleavages (illustrated for human pre-rRNA) to yield the mature rRNA species.illustrated for human pre-rRNA

Processing of rRNA

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

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

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

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 MARs: matrix-associated regions

Histone depleted metaphase chromosomes

SARs(MARs) Scaffold(matrix) attachment region Regions of the chromosomes with sequences specific for topoisomerase, HMG protein, and H1 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

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 in the nuclei and are highly active in rRNA synthesis. The nuclear matrix consists of DNA, nucleoproteins, and structural proteins.