11.3 分子伴侣(chaperones) 11.3.1 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

11.3.2 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.

11.4 Chromatin ◆染色质(Chromatin) ◆染色体(chromosome) ◆染色体与染色质比较 ●在化学本质上没有差异 ●在构型上不同 ●是遗传物质在细胞周期不同阶段的 不同表现形式。

11.4.1 DNA ◆DNA的三种构型 三种DNA构型中,沟的特征在遗传信息表达过程中起关键作用,调控蛋白都是通过其分子上氨基酸侧链与沟中碱基对两侧潜在的氢原子供体(=NH)或受体(O和N)形成氢键而识别DNA遗传信息的。另外, Z型DNA同细胞癌变有一定的关系。

三种不同构型的DNA

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.

不同生物端粒序列特征

DNA稳定遗传的三种功能位点

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。

Yeast artificial chromosome

11.4.2 Histone 组蛋白的性质和类型 ◆The histones are small proteins containing a high proportion of basic amino acids (arginine and lysine) that facilitate binding to the negatively charged DNA molecule. ◆There are five major types of histones--called H1, H2A, H2B, H3, and H4 . which are very similar among different species of eukaryotes ◆精细胞中是由精蛋白代替组蛋白，成熟的鱼类和鸟类的红细胞中H1则被H5所取代 ◆染色质中的组蛋白与DNA的含量之比为：1∶1。

组蛋白的功能 ◆核小体组蛋白(nucleosomal histone), 包括H2A、H2B、H3和H4, 作用是与DNA组装成核小体

虽然组蛋白分子的氨基酸序列是高度保守的，也会进行一些化学修饰，如酰基化、甲基化、磷酸化，以及ADP核糖化等。 组蛋白的化学修饰 虽然组蛋白分子的氨基酸序列是高度保守的，也会进行一些化学修饰，如酰基化、甲基化、磷酸化，以及ADP核糖化等。

组蛋白的化学修饰

组蛋白的化学修饰

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

11.4.3 Nonhistone 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。

非组蛋白参与染色体构建

Transcription Factor Motifs

Transcription Factor Motifs ◆锌指结构基序(模体）(zinc finger motif) The zinc ion of each finger is coordinated to two cysteines and two histidines. 有两种主要的锌指结构∶Cys2/His2指和Cys2/Cys2的锌指结构。

锌指结构基序

◆The helix-turn-helix motif ●由两个α螺旋组成的同型二聚体 ●两个α螺旋之间被一段转动的肽隔开 ●一个螺旋为识别螺旋(recognition helix),负责识别DNA大沟的特异碱基序列。 ●另一个螺旋没有碱基特异性,与DNA磷酸戊糖链骨架接触。

The helix-turn-helix motif

◆ The leucine zipper motif ●The leucine zipper contains four or five leucine residues spaced at intervals of seven amino acids, resulting in their hydrophobic side chains being exposed at one side of a helical region. ●This region serves as the dimerization domain for the two protein subunits, which are held together by hydrophobic interactions between the leucine side chains.

The leucine zipper motif

◆Helix-loop-helix (HLH) motif ●This motif is characterized by two -helical segments separated by an intervening loop. ●它与螺旋-转角-螺旋结构的差别在于∶两个螺旋的一侧还有一段疏水链，这样当螺旋-环-螺旋结构位于两个多肽之间时，这两个疏水的侧链就会将两个多肽链连在一起形成类似亮氨酸拉链的结构。

The helix-loop-helix (HLH) motif

Trans-acting factor and cis-acting element 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

金属硫蛋白基因的调控区

11.4.4 染色质的类型 ◆异固缩（heteropythosis) ◆常染色质(euchromatin) ◆异染色质(heterochromatin) ●结构性异染色质(active chromatin) ●兼性异染色质(inactive chromatin) ▲X失活（X-inactivation)

11.4.5 Nucleosome 核小体的结构特点 ◆由200个左右碱基对的DNA和四种组蛋白结合而成； Nucleosome is basic structural unit of chromatin 。 ◆由200个左右碱基对的DNA和四种组蛋白结合而成； ◆其中四种组蛋白(H2A、H2B、H3、H4 )各2分子组成八聚体的小圆盘，是核小体的核心结构； ◆146个碱基对的DNA绕在小圆盘外外面1 3/4圈。每一分子的H1与DNA结合, 起稳定核小体结构的作用； ◆两相邻核小体之间以连接DNA(linker DNA)相连, 长度为～80bp不等。

The organization of chromatin in nucleosomes ?

核小体

Structure of a chromatosome (A) The nucleosome core particle consists of 146 base pairs of DNA wrapped 1.65 turns around a histone octamer consisting of two molecules each of H2A, H2B, H3, and H4. A chromatosome contains two full turns of DNA (166 base pairs) locked in place by one molecule of H1. (B) Model of the nucleosome core particle. The DNA backbones are shown in brown and turquoise. The histones are shown in blue (H3), green (H4), yellow (H2A), and red (H2B).

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。

核小体与DNA的转录

核小体与基因转录

组蛋白乙酰化与基因转录

11.5.1 Chromosome packing ◆核小体(nucleosome) ◆螺线管(solenoid) ◆超螺线管(supersolenoid) ◆染色体(chromosome) 从核小体开始到染色体, DNA总共压缩: 压缩7倍 压缩6倍 压缩40倍 压缩5倍 DNA-→核小体→螺线管-→超螺线管-→染色单体

染色体的四级结构

30nm 纤维

11.5.2 中期染色体 形态和类型 中期染色体是以两条姐妹染色体(chromatid)构成,彼此以着丝粒相连。

11.5 染色体(chromosome) Chromatin fibers are thought to fold upon themselves further to form the compact metaphase chromosomes of mitotic cells, in which the DNA has been condensed nearly 10,000-fold

色体的电镜照片

染色体的主要结构 ◆着丝粒(centromere) ●主缢痕(Primary constriction) ◆动粒(kinetochore) ◆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

着丝粒与动粒

Centromere and kinetochore

主缢痕与主缢痕

11.5.3 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

11.5 Nucleolus The most prominent substructure within the nucleus is the nucleolus, which is the site of rRNA transcription and processing, and of ribosome assembly.

11.6.1核仁的结构与核仁周期 核仁的基本结构 ◆纤维中心(fibrillar centers FC) ◆致密纤维组分(dense fibrillar component, DFC) ◆颗粒区(granular component, GC)

Structure of the nucleolus

11.6.2 核仁的功能 核仁是细胞制造核糖体的装置。 ◆rRNA的合成 ◆rRNA前体的加工 ◆参与核糖体大小亚基的装配 ◆控制蛋白质合成的速度

核仁中进行的核糖体装配

11.7 Nuclear matrix ●Nuclear matrix is defined as the structural skeleton of the nucleus.(或称核骨架,nucleoskeleton) ● The concept of the nuclear matrix was proposed in 1975 on the basis of experiments in which nuclei were treated with DNAse to digest most of the DNA and extracted with high salt buffers to remove histones and other nuclear proteins. ● Such treatments left a residual network of fibers (the nuclear matrix), which was suggested to provide an internal structural framework for the nucleus, analogous to the role of the cytoskeleton as the structural framework of the cell. ● It has been proposed that the nuclear matrix serves to organize and anchor functional domains of the nucleus, including chromatin loops, DNA replication factories, splicing domains, and structures involved in mRNA transport.

核骨架

11.7.1 核基质的功能 ◆染色体骨架 (chromosome scaffold) ◆核纤层蛋白的骨架作用 ◆DNA复制时的染色质附着位点

染色体骨架

核纤层装配

参与DNA复制