第八章基因表达调控课件.ppt

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1、,Chapter 9 Gene expression controls,1 Basic conception and principle,一、基因表达的概念二、基因表达特性三、生物学意义四、基本原理,一、基因表达的概念,protein,transcription,translation,DNA,RNA,基因表达就是基因转录及翻译过程。,二、基因表达特性,基因表达时空性时间特异性（阶段特异性）空间特异性（细胞特异性、组织特异性）基因表达方式组成性表达（constitutive gene expression）管家基因（Housekeeping gene）：相对缺少时间和空间特异性而持续表达的 一

2、类基因。诱导和阻遏表达（induction and repression）：受环境因素影响而呈现出基因表达升高（诱导）或降低（阻遏）的现象。,Programming Gene Expression.Complex biological processes often involve coordinated control of the expression of many genes.The maturation of a tadpole into a frog is largely controlled by thyroid hormone.This hormone regulates ge

3、ne expression by binding to a protein,the thyroid hormone receptor,shown at the right.In response to hormone binding,this protein binds to specific DNA sites in the genome and modulates the expression of nearby genes.,Diagram of the human globin family of genes on chromosome,Highly expressed protein

4、-encoding genes of the pancreas and liver(as percentage of total mRNA pool),转录,翻译,转录水平调控,转录产物：加工、转运、降解,翻译水平,基因表达的多级调控,D N A,R N A,PROTEIN,基因激活,翻译后：加工、转运、降解,三、基因表达调控的生物学意义,适应环境、维持生长和增殖 维持个体发育与分化,四、基因转录激活调节基本要素,（一）基因表达的调控序列（元件,element）：一段特异DNA序列 1、原核生物基因表达调控单元操纵元（operon）调控区：启动序列（启动子,promoter,P）操纵序列(op

5、erator,O)编码区：编码序列（结构基因）,P O A B C,调控序列（区）编码序列（区）,启动子(promoter,P),启动子概念：能被RNA聚合酶识别、结合并启动基因转录的一段DNA序列。启动子组成特点：有两段保守序列(共有序列,consensus sequence）,决定启动子转录活性大小。-10区：TATAAT(Pribnow box),与RNA聚合酶核心酶结合-35区：TTGACA,与因子结合,操纵序列(operator,O)：是阻遏蛋白结合位点，与启动子序列毗邻，常交叉、重叠。介导负性调节 激活蛋白结合序列-介导正性调节,间隔,-35区,间隔,RNA起始,TTGACA,N1

6、7,TTAACT,N7,A,TTTACA,N16,TATGAT,N7,A,TTTACA,N17,TATGTT,N6,A,TTGATA,N16,TATAAT,N7,A,CTGACG,N18,TACTGT,N6,A,-10区,+1,trp,tRNATyr,lac,Rec A,ara B A D,TTGACA,TATAAT,共有序列,2、真核生物基因表达调控序列顺式作用元件（cis-acting element）:影响自身基因表达活性的序列，位于基因的两侧或中间：启动子（promoter）增强子(enhancer)沉默子(silencer),B,A,RNA聚合酶,B,A,DNA,DNA,转录起始点,

7、mRNA,mRNA,图 14-2 顺式作用元件概念,转录起始点,RNA聚合酶,(二)基因表达的调节蛋白,1、原核生物：特异因子：决定RNA聚合酶对一个或一套识别启动序列的特异性识别和结合能力，如因子：当细胞从基本的转录机制转入各种特定基因表达时，需要不同的因子。阻遏蛋白（repressor）结合操纵序列，阻遏基因表达 激活蛋白（activator）与启动子附近DNA序列结合，促进RNA聚合酶与启动子结合2、真核生物：转录因子(transcription factor)，可分为：反式作用因子（trans-acting factor）,大多数转录因子由某一基因表达后，与特异的顺式作用元件作用（DN

8、A-蛋白质）激活另一基因的转录。顺式作用因子（cis-acting factor）：极少数可识别和结合自身基因的调节序列，调节自身基因的开启或关闭。,蛋白质,mRNA,PA,DNA,PB,转录起始点,mRNA,顺式调节,蛋白质,图 14-3 反式与顺式作用蛋白,反式调节,B,A,（三）调节机制：通过非共价键分子间相互作用,蛋白质 DNA相互作用（protein-DNA interaction）：反式作用因子与顺式作用元件特异识别和结合。蛋白质蛋白质相互作用(protein-protein interaction):蛋白质之间相互作用可直接或间接结合DNA。,（四）RNA聚合酶活性：启动子序列影

9、响其与RNA聚合酶的亲合力。调节蛋白影响RNA聚合酶的活性。,第二节 原核基因转录调节,乳糖操纵元(lac operon)色氨酸操纵元(trp operon)基因重组(genetic recombination)SOS反应(SOS response),（一）结构组成：调控区：Plac、O、CAP结合位点和 编码区：三个结构基因（Z、Y、A）操纵元外有调节基因（lacI）编码阻遏蛋白,一、乳糖操纵元（lactose operon）,图162乳糖操纵子的结构,（二）调节方式：阻遏蛋白的负性调节 CAP的正性调节 协调调节,IPTG(乳糖类似物),阻遏蛋白的负性调节,CAP的正性调节,CAP,腺苷酸

10、环化酶催化ATP生成cAMP;cAMP磷二酯酶水解cAMP产生5-AMP,图163乳糖操纵子对基因表达的调节,协调调节,当阻遏蛋白封闭转录时，CAP对该系统不能发挥作用；没有CAP时，即使阻遏蛋白从operator上解离仍无明显的转录活性；若有葡萄糖和乳糖时，细菌优先利用葡萄糖-分解代谢阻遏（catabolic repression）,2,CAP,cAMP,CAP位点,低乳糖,阻遏蛋白,操纵序列,高乳糖,RNA聚合酶,mRNA 5,图 14-5 CAP,阻遏蛋白，cAMP和诱导剂对lac操纵子的调节 1.当葡萄糖浓度低，cAMP浓度高时 2.当葡萄糖浓度高，cAMP浓度低时,启动序列,启动序列

11、,启动序列,启动序列,1,二、色氨酸操纵子（trp operon）,（一）色氨酸操纵元组成：调控区：启动子（Ptrp）、操纵序列（Otrp）、前导序列（trpL）、衰减子序列（trp a）；及远离的调节基因（trpR）结构基因：trpE、trpD、trpC、trpB、trpA（二）调节方式：阻遏蛋白的负调控；辅阻遏物(色氨酸)+辅阻遏蛋白=阻遏蛋白 衰减子（trp a）机制,阻遏蛋白的负调控,色氨酸操纵子的结构,色氨酸操纵子对转录的调控机制,图166 噬菌体的开关事件操纵基因的结构,衰减子（attenuator）：提前终止转录，调节基因表达的DNA序列。位于转录起始点前，存在前导序列（162n

12、t）中，当trp浓度很高时，在与转录偶联的翻译过程中，在mRNA分子上形成一个不依赖因子的终止结构。,转录衰减机制,1,2,3,4,转录衰减机制,在高浓度色氨酸环境中，能形成色氨酰-tRNA，核糖体在翻译过程中能通过片段1，同时影响片段2和3之间的发夹结构形成，但片段3和4之间能形成发夹结构，这个结构就是P因子不依赖的转录终止结构，因此RNA聚合酶的作用停止。当色氨酸缺乏时，色氨酰-tRNA也相应缺乏，此时核糖体就停留在两个相邻的色氨酸密码的位置上，片段1和2之间不能形成发夹结构，而片段2和3之间可形成发夹结构，结果使色氨酸操纵子得以转录。,调节效应：,trp Attenuation:10-f

13、old repressiontrp repressor:70-fold repressionTotal regulatory effect:700-fold repression,第三节 真核基因转录调节,真核基因组结构特点真核基因表达调控特点真核基因转录激活调节,一、真核基因组结构特点,结构庞大：人 30亿（3109）bp,基因占6%单顺反子（monocistron）重复序列多 基因不连续性(断裂基因):外显子（exon）、内含子（intron）,二、真核基因表达调控特点,三种RNA聚合酶（RNA-Pol、）活性染色体结构变化 调节蛋白结合位点附近对核酸酶敏感 RNA酶前方DNA呈负超螺旋，

14、后方呈正超螺旋 5侧翼区CpG序列低甲基化 组蛋白被修饰，核小体结构不稳定 正性调节占主导 转录与翻译分离 转录后修饰、加工,三、真核基因转录激活调节,（一）顺式作用元件 启动子：含有一组转录控制组件（module）转录起始点 TATA 盒:位于-25-35bp处，是TFIID结合位点，控制转录的准确性和频率 CAAT盒 GC 盒 增强子（enhancer）：增强启动子转录活性的DNA序列。含有若干功能组件增强体（enhanson）是TF结合的核心序列 位于启动子内、或交错、或相邻、或其它部位 远距离起作用，专一性不强，无方向性 沉默子（silencer）负性调控元件，当结合特异调控蛋白因子时

15、，对基因转录起阻遏作用。,General pattern of cis-acting control elements that regulate gene expression in yeast and multicellular organisms(invertebrates,vertebrates,and plants).(a)Genes of multicellular organisms contain both promoter-proximal elements and enhancers as well as a TATA box or other promoter eleme

16、nt.The latter positions RNA polymerase II to initiate transcription at the start site and influences the rate of transcription.Enhancers may be either upstream or downstream and as far away as 50 kb from the transcription start site.In some cases,promoter-proximal elements occur downstream from the

17、start site as well.(b)Most yeast genes contain only one regulatory region,called an upstream activating sequence(UAS),and a TATA box,which is 90 base pairs upstream from the start site.,Nucleotide sequence of the human-globin gene,Nucleotide sequence of the human-globin gene,（二）反式作用因子（转录调节因子，转录因子）,1

18、、转录因子分类 基本转录因子：是RNA聚合酶结合启动子所必需的一组蛋白因子，决定三种RNA转录的类别。如RNA pol.：TFII D,TFIIA,TFIIB,TFIIE,TFIIF,TFIIH等 特异转录因子：个别基因转录所必需，决定该基因的时空特异性表达。转录激活因子：通常为增强子结合蛋白（enhancer-binding protein,EBP）转录抑制因子：多数为沉默子结合蛋白,2.转录调节因子结构 DNA结合域(DNA binding domain):锌指结构(zinc finger)碱性亮氨酸拉链（basic leucine zipper,bZIP）碱性螺旋-环-螺旋（basic

19、helix-loop-helix,bHLH）转录激活域(activation domain)：酸性激活域(acidic activation domain)谷氨酰胺富含域(glutamine-rich domain)脯氨酸富含域(proline-rich domain)二聚化结构域(dimerization domain)：ZIP、HLH,图167锌指结构,图168亮氨酸拉链,Major classes of transcription factors.Twelve distinct families of transcription factors have been classified

20、by their DNA-binding motifs.Four examples of these transcription factors implicated in the control of cell proliferation and differentiation are depicted here.Conserved regions unique to each family include regions of basic amino acids functioning as DNA-binding sites(solid rectangles)and putative h

21、elical regions associated with DNA binding or dimerization(cylinders)are shown.Transcriptional activation domains(solid triangles)are generally not conserved even within a family,generating considerable diversity of functional transactivation among interacting family members.Each family has at prese

22、nt more than half a dozen members.The classes shown possess a POU domain(Pituitary-1,pit-1),a basic helix-loop-helix domain(HLH,MyoD),a nuclear receptor type of zinc-finger domain(glucocorticoid receptors),or the basic region/leucine zipper domain(bZIP;CCAAT/enhancer binding proteins,C/EBP).,One typ

23、e of zinc finger protein.This protein belongs to the Cys-Cys-His-His family of zinc finger proteins,named after the amino acids that grasp the zinc.This zinc finger is from a frog protein of unknown function.(A)Schematic drawing of the amino acid sequence of the zinc finger.(B)The three-dimensional

24、structure of the zinc finger is constructed from an antiparallel sheet(amino acids 1 to 10)followed by an a helix(amino acids 12 to 24).The four amino acids that bind the zinc(Cys 3,Cys 6,His 19,and His 23)hold one end of the a helix firmly to one end of the b sheet.,DNA binding by a zinc finger pro

25、tein.(A)The structure of a fragment of a mouse gene regulatory protein bound to a specific DNA site.This protein recognizes DNA using three zinc fingers of the Cys-Cys-His-His type arranged as direct repeats.(B)The three fingers have similar amino acid sequences and contact the DNA in similar ways.I

26、n both(A)and(B)the zinc atom in each finger is represented by a small sphere.,Interaction between DNA and proteins containing zinc fingers.(a)GL1 is a monomeric protein that contains five C2H2 zinc fingers.-Helices are shown as cylinders,Zn+2 ions as spheres.Finger 1 does not interact with DNA,where

27、as the other four fingers do.(b)The glucocorticoid receptor is a homodimeric C4 zinc-finger protein.-Helices are shown as purple ribbons,-strands as green arrows,Zn+2 ions as spheres.Two helices(darker shade),one in each monomer,interact with the DNA.Like all C4 zinc-finger homodimers,this transcrip

28、tion factor has two fold rotational symmetry;the center of symmetry is shown by the yellow ellipse.In contrast,heterodimeric nuclear receptors do not exhibit rotational symmetry.,The Helix-Turn-Helix Motif Also Mediates Dimerization and DNA Binding,The DNA-binding helix-turn-helix motif.The motif is

29、 shown in(A),where each white circle denotes the central carbon of an amino acid.The carboxyl-terminal a helix(red)is called the recognition helix because it participates in sequence-specific recognition of DNA.As shown in(B),this helix fits into the major groove of DNA,where it contacts the edges o

30、f the base pairs,Some helix-turn-helix DNA-binding proteins.All of the proteins bind DNA as dimers in which the two copies of the recognition helix(red cylinder)are separated by exactly one turn of the DNA helix(3.4 nm).The second helix of the helix-turn-helix motif is colored blue.The lambda repres

31、sor and cro proteins control bacteriophage lambda gene expression,and the tryptophan repressor and the catabolite activator protein(CAP)control the expression of sets of E.coli genes.,.(a)In leucine-zipper proteins,basic residues in the extended helical regions of the monomers interact with the DNA

32、backbone at adjacent major grooves.The coiled-coil dimerization domain is stabilized by hydrophobic interactions between the monomers.(b)In bHLH proteins,the DNA-binding helices at the bottom(N-termini of the monomers)are separated by nonhelical loops from a leucine-zipper-like region containing a c

33、oiled-coil dimerization domain.,Interaction of homodimeric leucine-zipper and basic helix-loop-helix(bHLH)proteins with DNA,Leucine zipper homodimers bind to symmetric DNA sequences,as shown in the left-hand and center drawings.These two proteins recognize different DNA sequences,as indicated by the

34、 red and blue regions in the DNA.The two different monomers can combine to form a heterodimer,which now recognizes a hybrid DNA sequence,composed from one red and one blue region.,Transcription-Factor Interactions Increase Gene-Control Options alter their DNA-binding specificity.,A homeodomain bound

35、 to its specific DNA sequence.The homeodomain is folded into three a helices,which are packed tightly together by hydrophobic interactions(A).The part containing helix 2 and 3 closely resembles the helix-turn-helix motif,with the recognition helix(red)making important contacts with the major groove(

36、B).The Asn of helix 3,for example,contacts an adenine.Nucleotide pairs are also contacted in the minor groove by a flexible arm attached to helix 1.The homeodomain shown here is from a yeast gene regulatory protein,but it is nearly identical to two homeodomains from Drosophila,which interact with DN

37、A in a similar fashion.,The binding of a gene regulatory protein to the major groove of DNA.Only a single type of contact is shown.Typically,the protein-DNA interface would consist of 10 to 20 such contacts,involving different amino acids,each contributing to the binding energy of the protein-DNA in

38、teraction.,（三）mRNA转录激活及其调节 在启动子上转录因子和RNA聚合酶顺序识别、结合和组装成转录起始复合物：TFD-启动子复合物（TATA+TFD中的TBP+TAF）+TFA、B、F、H+RNA-pol(与TFD、B聚合)+EBP(与TAF结合),前起始复合物,稳定的起始复合物,TFA,TBP,TATA,TFB,TAF,TAF,TAF,EBP,TFIIF,H,Pol,DNA,图 14-10 增强子结合因子（EBP）、TBP相关因子（TAF）与转录起始复合物的形成,H,真核基因转录调节要点：,基本方式：顺式作用元件与反式作用因子相互作用调节（DNA-protein,protein-protein）；转录激活：在启动子上转录因子和RNA聚合酶顺序识别、结合和组装成转录起始复合物；顺式作用元件和反式作用因子各自不同的组合可产生多种调节类型；转录因子 种类、浓度和性质不同，决定了真核基因表达的时空特异性。,思考题,概念：管家基因、组成性表达、诱导与阻遏、操纵元、启动子、操纵序列、顺式作用元件、反式作用因子、增强子、沉默子、基本转录因子、TATA盒、阻遏蛋白、CAP、衰减子简述基因转录激活的基本要素。简述Lac operon 调节过程。简述真核基因转录调节因子种类和结构。,