10-真核基因表达的调控.ppt

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,第九章 真核基因表达的调控,本章的主要内容：,1.,真核基因表达调控的主要特点。,2.,Dhfr基因加压筛选的机制。,3.,免疫球蛋白（抗体）基因表达过程中的染色体重排机制。,4.,mRNA前体的可变剪接机制。,5.,RNA编辑的机制及其生物学意义。,6.,细胞周期的主要调控因素有哪些？,前 言,真核生物与原核生物基因表达调控的特点比较：,1.,真核生物的基因数目比原核生物的多，而且多数基因组基因含有内含子以及功能不清的重复序列等；,原核的染色质是裸露的DNA，而真核的染色质则是由DNA与组蛋白紧密结合形成为核小体；,在原核细胞中，染色质的结构对基因的表达没有明显的调控作用，而在真核中，这种作用是明显的。,2.,在原核基因转录的调控中，既有激活物的调控（正调控），也有阻遏物的调控（负调控），二者同等重要。在真核中虽然也有正调控成分和负调控成分，但迄今已知的主要是正调控。而且一个真核基因通常有多个调控序列，必须有多个激活物同时特异地结合上，才能启动基因的转录；,3.,原核基因的转录和翻译通常偶联在一起，而真核基因的转录是在细胞核中进行，翻译在胞质中进行；,4.,生成的初级转录物需在核中进行转录后的加工和运输，所以，真核基因的表达有多种转录后的调控机制；,5.,真核生物大都为多细胞生物，在个体发育过程中逐步分化形成各种组织和细胞类型。分化是不同基因表达的结果。不同类型的细胞，功能不同，基因表达的情况也不一样。某些基因仅特异地在某种细胞中表达，称为细胞特异性或组织特异性表达，因而具有调控这种特异性表达的机制。,6.,真核生物对外界环境条件变化的反应和 原核生物十分不同。同一群原核生物细胞处在相同的环境条件中，对环境条件的变化会作出基本一致的反应；而真核生物常常只有少部分细胞基因的表达直接受到环境条件变化的影响和调控，其他大部分间接或不受影响。,第一节 染色体水平的调控,一、染色体丢失,某些生物，在个体发育的早期，体细胞的染色体部分丢失，而性细胞的染色体数目保持不变。,例子：,马蛔虫,（Parascaris equoorum）：2n=2;,动物极,植物极,小表瘿蚊,（Mayetiole destructor）：2n=40。,极细胞区：2n=40,2n=8,二、染色体扩增,染色体扩增的本质是细胞内特定基因拷贝数的专一性大量,扩增。,基因组序列的选择性扩增。,以,dhfr,基因的扩增为例，说明基因组序列的选择性扩增机制。,二氢喋啶 FH2 FH4 CoF衍生物 嘌呤或嘧啶,（1）,合成酶；,（2）,还原酶。,（1）,（2）,Figure 17.28 The,dhfr,gene can be amplified to give unstable copies that are extra-chromosomal(double minutes)or stable(chromosomal).Extra-chromosomal copies arise at early times.,Figure 17.29 Amplified copies of the,dhfr,gene produce a homogeneously staining region,(,HSR,),in the chromosome.,CHO野生型,CHO,r,Figure 17.30 Amplified extrachromosomal dhfr genes take the form of,doubleminute chromosomes,as seen in the form of the small white dots.Photograph kindly provided by Robert Schimke,.,2.,dhfr,基因扩增的机理和特点,（1）机理：,非同源重组,（2）特点：,A.,在用MTX加压筛选的初期，细胞大部分或全部是不稳定的；,B.,dhfr,基因的单细胞拷贝数的变化范围为40400，随着加压程度的提高，拷贝数逐渐增加，对MTX的耐受力也相应提高；,C.,dhfr,基因的长度为31kb，但被扩增的DNA长度可达5001 000kb；,D.,当MTX压力解除后，,dhfr,基因将逐渐减少；,E.,当与,dhfr,基因相连的外源DNA导入细胞后，有整合到内源,dhfr,基因位点的趋势。,3.,dhfr,加压系统的应用,EPO（促红细胞生成素）的高效表达。,pCMV/EPO,CHO cell line/dhfr-,Stable High Expression,MTX,Transformation,EPO,Extraction,三、染色体重排,（一）酵母交配型的转变,The mating type of a cell is determined by the genetic information present at the,MAT locus,.,MATa,allele at this locus are type a;likewise,the,MAT,allele are type.Cells of opposite types can mate;cells of the same type cannot.,Recognition of cells of opposite mating type is accomplished by the secretion of,pheromones,.cells secrete-factor(13 amino acids);,a,cells secrete a-factor(12 amino acids).,A cell of one mating type carries a surface receptor for the pheromone of the opposite type.,When an a cell and an cell encounter one another,their pheromones act on each other to arrest the cells in the G1 phase of the cell cycle followed by cell and nuclear fusion to produce an,a/,diploid cellFigure 17.1 Mating type controls several activities.,1.不同交配型的特点,Figure 17.2 The yeast life cycle proceeds through mating of,MATa,and,MAT,a,haploids to give heterozygous diploids that sporulate to generate haploid spores.,2.不同交配型的接合过程,Figure 17.3 Either a or,a,factor/receptor interaction triggers the activation of a G protein,whose,bg,subunits transduce the signal to the next stage in the pathway.,3.交配型的信号传递,Figure 17.4 The same mating type response is triggered by interaction of either pheromone with its receptor.The signal is transmitted through a series of kinases to a transcription factorthere may be branches to some of the final functions.,Figure 17.5 Changes of mating type occur when silent cassettes replace active cassettes of opposite genotype;when transpositions occur between cassettes of the same type,the mating type remains unaltered.,4.,交配型的转换,80%90%,5%,5%,5.不同交配型转换的机制,W,W,X,X,X,Y,Y,Ya/,a,Z1,Z1,Z1,Z2,Z2,MATa/,a,HMRa,HML,a,/,a,Figure 17.6 Silent cassettes have the same sequences as the corresponding active cassettes,except for the absence of the extreme flanking sequences in,HMRa,.Only the Y region changes between a and,a,types.,Figure 17.7 In diploids the,a,1 and,a,2 proteins cooperate to repress haploid-specific functions.In a haploids,mating functions are constitutive.In,a,haploids,the,a,2 protein represses a mating functions,while,a,1 induces,a,mating functions.,Figure 17.10 Cassette substitution is initiated by a double-strand break in the recipient(,MAT,)locus,and may involve pairing on either side of the Y region with the donor(,HMR,or,HML,)locus.,（二）抗体的多样性,1.免疫球蛋白的产生、种类、结构,The,immune response,of vertebrates provides a protective system that distinguishes foreign proteins from the proteins of the organism itself.Foreign material(or part of the foreign material)is recognized as comprising an,antigen,.,The immune system provides a striking and extensive case in which the content of the genome changes,when recombination creates active genes in lymphocytes.,B cells,，,T cells,mature in the thymus.Each class of lymphocyte uses the rearrangement of DNA as a mechanism for producing the proteins that enable it to participate in the immune response.For practical purposes,we usually reckon that a mammal has the ability to produce,10,6,-10,8,different antibodies.,B淋巴细胞的分化过程：,骨髓干细胞,前B淋巴细胞,未成熟B淋巴细胞,成熟B淋巴细胞,外周B淋巴细胞,抗体,骨髓,外周血,Figure 24.1 Humoral immunity is conferred by the binding of free antibodies to antigens to form antigen-antibody complexes that are removed from the bloodstream by macrophages or that are attacked directly by the complement proteins.,Figure 24.17 Immunoglobulin type and function is determined by the heavy chain.J is a joining protein in IgM;all other Ig types exist as tetramers.,Figure 24.4 Heavy and light chains combine to generate an immuno-globulin with several discrete domains.,Each antibody is an immunoglobulin tetramer consisting of,two identical,light chains,(L)and two identical,heavy chains,(H),.If any light chain can associate with any heavy chain,to produce 10,6,10,8,potential antibodies requires 10,3,10,4,different light chains and 10,3,10,4,different heavy chains.,不同Ig家族的V、D、J、C基因片段数,V D J C,人 小鼠 人 小鼠 人 小鼠 人 小鼠,L,6 4,L,1000 30 12 4 4 9 8,家族,Figure 24.5 The,lambda C,gene segment is preceded by a J segment,so that V-J recombination generates a functional lambda light-chain gene.,2.轻链和重链基因的结构与重组,Figure 24.6 The,kappa C,gene segment is preceded by multiple J segments in the germ line.V-J joining may recognize any one of the J segments,which is then spliced to the C gene segment during RNA processing.,Figure 24.7,Heavy genes,are assembled by sequential joining reactions.First a D segment is joined to a J segment;then a V gene segment is joined to the D segment.,Figure 24.8 The,lambda,family consists of V gene segments linked to a small number of J-C gene segments.,Figure 24.9 The human and mouse,kappa,families consist of V gene segments linked to 5 J segments connected to a single C gene segment.,3.抗体的多样性,Figure 24.10 A single gene cluster in man contains all the information for,heavy-chain,gene assembly.,4.重组机制,RAG(recombination active gene)基因在抗体基因的重组过程中发挥了重要作用。,构成抗体多样性的因素：,轻、重链基因多样性的物质基础：1）,V,基因的多样性；,2）,D,基因片段的多样性；,3）,J,基因片段的多样性；,4）,C,基因的多样性。,轻链,V,-,J-C,重组方式的多样性；重链,V,-,D-J-C,重组方式的多样性；,轻链,V-J,和重链,V-D,连接处碱基的插入或缺失；,轻链与重链的组合方式。,5.抗体的表达与分泌,Figure 24.15 A V gene promoter is inactive until recombination brings it into the proximity of an enhancer in the C gene segment.The enhancer is active only in B lymphocytes.,第二节 染色质水平的调控,一、染色质的组成与结构,Chromatin:,A mixture is composed of DNA、histones and nonhistones.,Euchromatin(lower density),Heterochromatin（higher density）：,无表达活性。,Nucleosome:,contains 200bp of DNA,organized by an octamer of small,basic proteins-,histones,into a bead-like structure.They form an interior core;the DNA lies on the surface of the particle.,The nucleosome provides the,first level of organization,giving a,packing ratio,of 6.Its components and structure are well characterized.,染色体,DNA,线状，无分支。不同生物的,DNA,长度相差很大。,生物,bp,DNA,长度,（,mm）,染色体数,(对),大肠杆菌,4.0,10,6,1.4,1,酵母,（,S.cerevisiae,）,1.4 10,7,4.6,16,果蝇,（,D.,malanogaster,）,1.7 10,8,56,4,人,3.9,10,9,990,23,Packing ratio,(,堆积比):,the ratio of the length of DNA to,the unit length of the fiber containing it.,2.组蛋白,（Histones）:,H1,H2A,H2B,H3 and H4,在进化过程中，不同生物之间，同种生物的不同发育时期均高度保守，尤其H2A,H2B,H3 and H4。,小牛胸腺组蛋白,组蛋白 氨基酸数 分子量,（kDa）,精,（%）,赖,（%）,H1 215 23.0 1 29,H2A 129 14.0 9 11,H2B 125 13.8 6 16,H3 135 15.3 13 10,H4 102 11.3 14 11,Unit evolutionary period,(单位进化周期)：两个进化系趋异后氨基酸序列改变1%的时间间隔。H3:3亿年；H4:6亿年。,3.,非组蛋白,（Nonhistones）,The,nonhistones,include all the proteins of chromatin except the histones.They are more variable between tissues and species,and they comprise a smaller proportion of the mass than the histones.They also comprise a much larger number of proteins,so that any individual protein is present in amounts much smaller than any histone.,The functions of nonhistone proteins include control of gene expression and higher-order structure.,RNA polymerase,may be considered to be a prominent nonhistone.,The,HMG,(high-mobility group)proteins comprise a discrete and well-defined subclass of nonhistones(at least some of which are transcription factors).,4.核小体,Figure 19.1 Chromatin spilling out of lysed nuclei consists of a compactly organized series of particles.The bar is 100 nm.,Figure 19.2 Individual nucleosomes are released by digestion of chromatin with micrococcal nuclease.The bar is 100 nm.,Figure 19.7 Micrococcal nuclease digests chromatin in nuclei into a multimeric series of DNA bands that can be separated by gel electrophoresis.,Figure 19.3 The nucleosome consists of approximately equal,masses of DNA and histones(including H1).The predicted mass,of the nucleosome is 262 kD.,1）组成,Figure 19.4 The nucleosome may be a cylinder with DNA organized into two turns around the surface.,2）结构,Figure 19.6 Sequences on the DNA that lie on different turns around the nucleosome may be close together.,Figure 19.10 Microccocal nuclease initially cleaves between nucleosomes.Mononucleosomes typically have 200 bp DNA.End-trimming reduces the length of DNA first to 165 bp,and then generates core particles with 146 bp.,Figure 19.8 Each multimer of nucleosomes contains the appropriate number of unit lengths of DNA.,Figure 19.21 In a symmetrical model for the nucleosome,the H3,2,-H4,2,tetramer provides a kernel for the shape.One H2A-H2B dimer can be seen in the top view;the other is underneath.,3）组装,Figure 19.27 In vitro,DNA can either interact directly with an intact(crosslinked)histone octamer or can assemble with the H3,2,-H4,2,tetramer,after which two H2A-H2B dimers are added.,Figure 19.22 The crystal structure of the histone core octamer is represented in a space-filling model with the,H3,2,H4,2,tetramer shown,in white,and the,H2A-H2B,dimers shown,in blue,.,H2A-H2B,H3,2,-H4,2,Figure 19.19 The 10 nm fiber（left）to the 30nm fiber which has a coiled structure.,5.30nm 纤丝的形成,Figure 19.20 The 30 nm fiber may have a helical coil of 6 nucleosomes per turn,organized radially.,6.辐射状环结构的形成,Figure 18.7 Histone-depleted chromosomes consist of a protein scaffold to which loops of DNA are anchored.,Figure 18.9 The sister chromatids of a mitotic pair each consist of a fiber(30 nm in diameter)compactly folded into the chromosome,.,（一）异染色质化,（二）活泼转录区DNA对核酸酶的敏感性提高,超敏感位点:,仅在发生基因表达的细胞中才能发现有这种对核酸酶超敏感的位点，可见它与基因的转录有关。已发现，这些位点通常位于被转录基因的5端1000bp的侧翼内，但也有位于离5端更远一些、3端和甚至基因内部的。许多超敏感位点相当于已知的调控蛋白所结合的位点。,（三）正在转录的染色质处DNA的甲基化程度降低,（四）正在转录区的组蛋白和有关蛋白质发生改变,活泼转录的染色质趋于缺乏组蛋白H1，而且其它的核心组蛋白则被乙酰基化或与,泛素（ubiquitin）,相结合而被修饰。,染色质的体外重组实验,二、染色质的结构对基因表达的调控,染色质体外重组实验：,用盐或稀酸处理染色质，可将DNA、组蛋白和非组蛋白分开，经离子交换层析、乙醇沉淀等技术处理可分别获得上述三种组分。,来自不同细胞的染色质组分混合后，能够重新组建成染色质。,将无转录活性细胞的DNA和组蛋白与有转录活性细胞的非组蛋白混合，则产生转录活性。,第三节 转录和转录后加工的调控,一、顺式作用元件与反式作用因子,（一）顺式作用元件（Cis-acting sequences）,启动子核心成分：如,TATA box；,上游启动子成分：如,CAAT box，GC box、,八聚体（,octamer,）,以及,ATF,结合位点等；,远上（下）游序列：如增强子，酵母的,UAS（upstream activator sequences），,静息子等；,特殊细胞中的启动子成分：淋巴细胞中的,Oct,和,B,等。,哺乳动物RNA polymerase 启动子上游转录因子结合的序列元件,组件 保守序列 DNA长度 结合因子 大小,（kDa）,丰度,/细胞,分布,TATA box TATAAA -10bp TBP 27?普遍,CAAT box GGCCAATCT -22bp CTF/NF1 60 300000 普遍,GC box GGGCGG -20bp SP 1 165 60 000 普遍,Octamer ATTTGCAT -20bp Oct-1 76?普遍,Octamer ATTTGCAT 23bp Oct-2 52?淋巴细胞,B GGGACTTTCG -10bp NF,B 44?淋巴细胞,B GGGACTTTCG -10bp H2.TH1?普遍,ATF GTGACGT -20bp ATF?普遍,结合于反应性元件的特殊转录因子,调节剂 反应元件 保守序列 长度 反应因子 大小（kDa）,热休克 HSE,CNNGAANNTCCNNG,27bp HSTF 93,糖皮质激素 GRE,TGGTACAAATGTTCT,20bp 受体 94,佛波酯 TRE,TGACTCA,22bp AP1 39,血清 SRE,CCATATTAGG,20bp SRF 52,（二）反式作用因子(Trans-acting element),通用反式作用因子：一般细胞中普遍存在，如,TBP，SP1，CTF/NF-1，Oct-1,等;,特殊组织和细胞中的反式作用因子：,Oct-2;,与反应性元件结合的反式作用因子:见下表。,反式作用因子的调控作用途径：,1）蛋白质与DNA相互作用；,2）蛋白质之间的相互作用；,3）蛋白质与配基结合；,4）蛋白质自身的修饰。,Figure 21.2 The activity of a regulatory transcription factor may be controlled by synthesis of protein,covalent modification of protein,ligand binding,or binding of inhibitors that sequester the protein or affect its ability to bind to DNA.,二、调控蛋白与DNA的结合方式,调控蛋白通常是结合在特异的DNA序列上而发挥作用的。而且调控蛋白通常有独立的结合DNA的结构域。,（一）螺旋转角螺旋（helix-turn-helix）,（二）锌指（,Zinc Finger,）,（三）同源异型结构域（,homeodomains,HD）,Figure 10.13 The structure of a monomer of Lac repressor identifies several independent domains.,（一）螺旋转角螺旋（helix-turn-helix,）,The,helix-turn-helix,motif was originally identified as the DNA-binding domain of phage repressors.One-helix lies in the wide groove of DNA;the other lies at an angle across DNA.,Figure 10.15 Inducer changes the structure of the core so that the headpieces of a repressor dimer are no longer in an orientation that permits binding to DNA.,Figure 21.12 Helix 3 of the homeodomain binds in the major groove of DNA,with helices 1 and 2 lying outside the double helix.Helix 3 contacts both the phosphate backbone and specific bases.The N-terminal arm lies in the minor groove,and makes additional contacts.,（二）锌指（Zinc Finger,）,The,zinc finger,motif comprises a DNA-binding domain.It was originally recognized in factor TFIIIA,which is required for RNA polymerase III to transcribe 5S rRNA genes.It has since been identified in several other transcription factors(and presumed transcription factors).A distinct form of the motif is found also in the steroid receptors,The,steroid receptors,are defined as a group by a functional,relationship:each receptor is activated by binding a particular,steroid.The glucocorticoid receptor is the most fully analyzed.,Together with other receptors,such as the thyroid hormone,receptor or the retinoic acid receptor,the steroid receptors are members of a superfamily of transcription factors with the,same general,modus operandi.,Figure 21.3 Transcription factor SP1 has a series of three zinc fingers,each with a characteristic pattern of cysteine and histidine residues that constitute the zinc-binding site.,Figure 21.4 Zinc fingers may form,a,-helices that insert into the major groove,associated with,b,-sheets on the other side.,Figure 21.5 The first finger of a steroid receptor controls specificity of DNA-binding(positions shown in red);the second finger controls specificity of dimerization(positions shown in blue).The expanded view of the first finger shows that discrimination between GRE and ERE target sequences rests on two amino acids at the base.,三、调节蛋白与蛋白质结合的方式,在真核细胞中，许多转录因子以二聚体结合在DNA上。,这些调控蛋白除了与DNA结合的结构外，还有与蛋白质结合,的结构域。这些结