分子生物学核小体.pptx

染色体与 DNAFigure CO:X chromosome in blue染色体2.1.1 染色体概述染色体(chromosome)是细胞核中载有遗传信息的物质，在显微镜下呈丝状或棒状，由核酸和蛋白质组成，在细胞发生有丝分裂时期容易被碱性染料着色，因此而得名。同一物种内每条染色体所带DNA的量是一定的，但不同染色体或不同种中间变化很大。原核细胞原核细胞染色体外裹着稀疏的蛋白质，其中一部分与DNA的折叠有关，另一些则参与DNA复制、重组及转录过程。真核细胞真核细胞的染色体中，DNA与蛋白质完全融合在一起，其蛋白质与相应DNA的质量比约为1:1.chromosome A discrete unit of the genome carrying many genes.Each consists of a very long molecule of duplex DNA and an approximately equal mass of proteins.It is visible as a morphological entity only during cell division.A thin section shows the bacterial nucleoid as a compact mass in the center of the cell原核细胞染色体(类核Nucleoid)The nucleoid spills out of a lysed E.coli cell in the form of loops of a fiberThe bacterial genome consists of a large number of loops of duplex DNAFIGURE 08:An unrestrained supercoil in the DNA path creates tension,but no tension is transmitted along DNA when a supercoil is restrained by protein bindingThe sister chromatids of a mitotic pair each consist of a fiber(30 nm in diameter)compactly folded into the chromosome真核细胞染色体类核；染色质；包装比例nucleoid The structure in a prokaryotic cell that contains the genome.The DNA is bound to proteins and is not enclosed by a membrane.chromatin The state of nuclear DNA and its associated proteins during the interphase(between mitoses)of the eukaryotic cell cycle.packing ratio The ratio of the length of DNA to the unit length of the fiber containing it.Specific Sequences Attach DNA to an Interphase Matrix（分裂间期基质）DNA is attached to the nuclear matrix at specific sequences called MARs or SARs.The MARs are A-T-rich but do not have any specific consensus sequence.chromosome scaffold（支（支架）架）A proteinaceous structure in the shape of a sister chromatid pair,generated when chromosomes are depleted of histones.Chromatin Is Divided into Euchromatin and HeterochromatinRegions of heterochromatin remain densely packed throughout interphase.chromocenter An aggregate of heterochromatin from different chromosomes.Regions of compact heterochromatin are clustered near the nucleolus and nuclear membrane常染色质和异染色质Euchromatin and HeterochromatinDNA Is Organized in Arrays of Nucleosomes（核小体）The core DNA is the length of 146 bp that is found on the core particles produced by prolonged digestion with MNase.Linker DNA is the region of 8 to 114 bp that is susceptible to early cleavage by nucleases.FIGURE 05:Micrococcal nuclease initially cleaves between nucleosomesThe Nucleosome Is the Subunit of All Chromatin A nucleosome contains 200 bp of DNA and two copies of each core histone(H2A,H2B,H3,and H4).DNA is wrapped around the outside surface of the protein octamer（八聚体）.The histone octamer has a structure of an H32-H42 tetramer associated with two H2A-H2B dimers.FIGURE 06:The nucleosome consists of approximately equal masses of DNA and histones(including H1)The Nucleosome Is the Subunit of All Chromatin The Nucleosome Is the Subunit of All Chromatin The nucleosome is a cylinder with DNA organized into 1 2/3 turns around the surfaceThe Nucleosome Is the Subunit of All ChromatinEach histone is extensively interdigitated with its partner.All core histones have the structural motif of the histone fold.N-and C-terminal histone tails extend out of the nucleosome.H1 is associated with linker DNA and may lie at the point where DNA enters or exits the nucleosome.Photos courtesy of E.N.Moudrianakis,Johns Hopkins University.FIGURE 10ab:The crystal structure of the histone core octamer is represented in a space-filling modelThe Nucleosome Is the Subunit of All ChromatinThe histone fold consists of two short a-helices flanking a longer a-helixHistone pairs(H3+H4 and H2A+H2B)interact to form histone dimersStructures from Protein Data Bank 1HIO.G.Arents,et al.,Proc.Natl.Acad.Sci.USA 88(1991):10145-10152.The Nucleosome Is the Subunit of All ChromatinThe histone fold domains of the histones are located in the core of the nucleosomeNucleosomes Are Covalently Modified Histones are modified by methylation,acetylation,phosphorylation,and other modifications.Combinations of specific histone modifications define the function of local regions of chromatin;this is known as the histone code.Histone tails have many sites of modificationNucleosomes Are Covalently ModifiedPhoto courtesy of Sean D.Taverna,Johns Hopkins University School of Medicine,and Haitao Li,Memorial Sloan-Kettering Cancer Center.Additional information at S.D.Taverna,et al.,Nat.Struct.Mol.Biol.14(2007):1025-1040.Numerous protein motifs recognize methylated lysinesNucleosomes Are Covalently ModifiedAcetylation during replication occurs on specific sites on histones before they are incorporated into nucleosomesNucleosomes Are Covalently ModifiedAcetylation associated with gene activation occurs by directly modifying specific sites on histones that are already incorporated into nucleosomesDNA Structure Varies on the Nucleosomal Surface DNA is wrapped 1.67 times around the histone octamer.DNA on the nucleosome shows regions of smooth curvature and regions of abrupt kinks.The structure of the DNA is altered so that it has an increased number of base pairs/turn in the middle,but a decreased number at the ends.Structures from Protein Data Bank:1P34.U.M.Muthurajan,et al.,EMBO J.23(2004):260-271.The Path of Nucleosomes in the Chromatin Fiber10 nm chromatin fibers consist of a string of nucleosomes.30 nm fibers have six nucleosomes/turn,which are organized into a two-start helix.Histone H1,histone tails,and increased ionic strength all promote the formation of the 30 nm fiber.The Path of Nucleosomes in the Chromatin FiberThe 10 nm fiber is a continuous string of nucleosomesThe 30 nm fiber is a two start helix consisting of two rows of nucleosomes coiled into a solenoidLevels of chromatin packagingReplication of Chromatin Requires Assembly of NucleosomesHistone octamers are not conserved during replication,but H2A-H2B dimers and H32-H42 tetramers are conserved.There are different pathways for the assembly of nucleosomes during replication and independently of replication.Replication of Chromatin Requires Assembly of NucleosomesNucleosome assembly in vivoNucleosomes Are Displaced and Reassembled During Transcription Most transcribed genes retain a nucleosomal structure,though the organization of the chromatin changes during transcription.Some heavily transcribed genes appear to be exceptional cases that are devoid of nucleosomes.RNA polymerase displaces histone octamers during transcription in vitro,but octamers reassociate with DNA as soon as the polymerase has passed.Nucleosomes Are Displaced and Reassembled During TranscriptionAn experiment to test the effect of transcription on nucleosomes shows that the histone octamer is displaced from DNA and rebinds at a new positionNucleosomes Are Displaced and Reassembled During TranscriptionNucleosomes are reorganized when transcription passes through a gene.Additional factors are required both for RNA polymerase to displace octamers during transcription and for the histones to reassemble into nucleosomes after transcription.染色体带型G（Giemsa)-banding generates a characteristic lateral series of bands in each member of the chromosome setThe human X chromosome can be divided into distinct regions by its banding pattern每条带约10000KbA magnified view of bands 87A and 87C shows their hybridization in situ with labeled RNA extracted from heat-shocked cellsChromosomes are pulled to the poles via microtubules that attach at the centromeres有丝分裂中期有丝分裂后期有丝分裂末期着丝点粘结蛋白微管A model of the overall structure of a regional centromereFIGURE 26:A large protein complex assembles at the CDE sequences and connects the chromosome to microtubulesA typical telomere has a simple repeating structure with a G-T-rich strand that extends beyond the C-A-rich strand端粒有简单的重复序列A typical telomere has a simple repeating structure with a G-T-rich strand that extends beyond the C-A-rich strandThe crystal structure of a short repeating sequence from the human telomere forms three stacked G quartets端粒封堵线性染色体的末端A loop forms at the end of chromosomal DNA染色体末端形成环状结构A loop forms at the end of chromosomal DNA端粒的功能1.保护染色体末端：2.端粒延长3.协助减数分裂中同源染色体配对The meiotic telomere cluster is visualized by telomere FISH端粒对生存是必需的1.在分裂细胞中存在端粒酶活性。2.端粒酶突变后，每次分裂，染色体变短。3.端粒消失后，细胞不能分裂。Mutation in telomerase causes telomeres to shorten in each cell divisionTelomere length is maintained at 350 bp in wild-type yeastReproduced from T.M.Nakamura,et al.,Science 277(1997):955-959 http:/www.sciencemag.org.Reprinted with permission from AAAS.Photo courtesy of Thomas R.Cech,Howard Hughes Medical Institute.真核细胞染色体的组成1.蛋白质蛋白质包括组蛋白和非组蛋白。组蛋白是染色体的结构蛋白，它与DNA组成核小体。组成：组成：DNA，组蛋白，非组蛋白，RNA富含赖氨酸和精氨酸组蛋白的特性组蛋白的特性1.进化上的极端保守性 特别是H3、H4，牛、猪、大鼠的H4完全相同2.无组织特异性 3.肽链上氨基酸分布的不对称性 N端碱性氨基酸 C端疏水氨基酸4.组蛋白的修饰作用 甲基化、乙酰化、磷酸化、多聚ADP糖基化等 5.富含赖氨酸的组蛋白H5 只存在于鸟类、两栖类等含有细胞核的红细胞中非组蛋白非组蛋白包括酶类和细胞分裂有关的蛋白（收缩蛋白、骨架蛋白、核孔复合物蛋白、肌动蛋白、肌球蛋白、微管蛋白、原肌蛋白）高速泳动蛋白（high mobility group protein,HMG蛋白）能与DNA结合，也能与H1作用，可能与超螺旋结构有关。DNA结合蛋白 可能是一些与DNA复制或转录有关的酶或调解物质。A24非组蛋白 C端与H2A相同，功能不详3.染色质和核小体染色质和核小体染色质DNA的Tm值比自由DNA高。染色质DNA的复制和转录活性比自由DNA低。DNA酶I对染色质DNA的消化慢于纯DNA。核小体是一种串珠状结构，由核心颗粒和连结线DNA两部分组成，可描述如下：每个核小体单位包括约200bp的DNA、一个组蛋白核心和一个H1；由H2A、H2B、H3、H4各两分子形成八聚体，构成核心颗粒；DNA分子以左手螺旋缠绕在核心颗粒表面，每圈80bp，共1.75圈，约146bp，两端被H1锁合；相邻核心颗粒之间为一段60bp的连接线DNA。