大学生物遗传学：第十一章原核基因表达调控.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,*,第一节 操纵子,operon,Table of content,几个重要概念,一、调控可分为正调控与负调控,二、结构基因簇中各成员是协同调控的,三、Lac操纵子基因的表达受阻抑物控制,四、乳糖操纵子是可诱导的,五、阻抑物由小分子诱导物所控制,六、用顺式作用的组成型突变来鉴定操纵基因,七、用反式作用的突变来鉴定调控基因,八、多亚基蛋白质具有特殊的遗传性,九、阻抑物结合于操纵基因上,Table of content,十、阻抑物结合诱导物后从操纵基因上脱离,十一、阻抑物单体具有多个结构域,十二、阻抑物由两个二聚体组成四聚体,十三、阻抑物与DNA的结合是通过别构作用来调节的,十四,、,突变体的表表型与结构域的构型有关,十五、阻抑物与三个操纵基因结合并与RNA聚合酶相互作用,十六、操纵基因与低亲和力位点竞争性地结合阻抑物,十七、阻遏可发生在多个基因座上,十八、启动子与操纵基因相对位置具有多样性,十九 小 结,阻抑物（repressor）,能阻止基因表达的蛋白质，它可与DNA操纵基因结合来阻止转录或结合RNA来阻止蛋白质的翻译。,操纵子（operon）,细菌基因表达和调控的单位，包括结构基因和能被调控基因产物识别的 DNA控制元件。,lactose operon,The simplest form of the regulatory model,：,A regulator gene codes for a protein that acts at a target site on DNA,一、调控可分为正调控与负调控,负调控（negative control）,阻抑物与操纵基因结合来阻止有关基因的表达。,正调控（positive control）,调控因子通过与启动子元件结合来激活基因的表达。,In negative control,a trans-acting repressor binds to the,cis,-acting operator,to turn off transcription.,In positive control,trans,-acting factors must bind to,cis,-acting sites,in order for RNA polymerase to initiate transcription at the promoter.,正调控和负调控的共同点：,调控蛋白作为反式作用因子,，通过与顺式作用元件相互作用激活或抑制基因表达。,虽调控蛋白与DNA结合长度较长，但仅靠识别DNA上的短序列(常小于10 bp)起作用。,二、结构基因簇中各成员是协同调控的,对同一条代谢途径中的某些蛋白质，通常编码它们的基因在DNA上也紧密排列在一起，作为一个转录单位转录出多顺反子mRNA。,The,lac,operon occupies 6000 bp of DNA.The long,lacZ,gene is followed by the,lacY,and,lacA,genes.,三、,Lac,操纵子基因的表达受阻抑物控制,位于,lac,基因簇起点的启动子与操纵基因具有重叠区域；,阻抑物与操纵基因结合就可控制,lacZYA,基因簇的转录。,阻抑物是由,lacI,基因编码、具有4个相同亚基的四聚体。,Repressor and RNA polymerase,bind at sites that overlap around the,transcription startpoint of the,lac,operon,.,-5+21,lacI,基因编码的阻抑物(repressor),四、乳糖操纵子是可诱导的,可以对操纵子进行诱导的小分子，其结构通常与操纵子所表达的酶的底物的结构相同或类似。,-半乳糖苷（含乳糖）是,lacZYA,基因编码的酶可识别的底物。,-半乳糖苷不存在时，乳糖操纵子的表达水平极低。加入-半乳糖苷可诱导乳糖操纵子中三个结构基因的转录。,Addition of,inducer,results in rapid induction of,lac,mRNA,and is followed after a short lag by synthesis of the enzymes;removal of inducer is followed by rapid cessation of synthesis.,Results of experiment,when cells of,E.coli,are grown in the,absence of a-galactoside,there is no need for-galactosidase,and they contain very few molecules of the,enzymesay,90 around the center of symmetry.,七、应急应答产生(p)ppGpp,应急应答（stringent response）：,当细菌发现它们处于很差的生长环境，没有足够的氨基酸来维持蛋白质合成时，它们就会停止大部分活动，这种现象称为,应急应答,。,应急应答,产生两种非正常的核酸堆积物,有时又称预警子(alarmone)，即ppGpp和pppGpp，统称(P)PPGPP。,(P)PPGPP的作用是协同调控大量的分子活动。,(P)PPGPP水平和细菌生长速率一般呈相反关系。,应急应答使得rRNA和tRNA的合成量大幅减少（10-20%），一些mRNA的合成也减少（1/3）。蛋白质的降解速率增加，许多代谢调节作用开始发生。,八、(p)ppGpp由核糖体生成,应急因子,RelA,是一种(p)ppGpp合成酶，约与5%的核糖体结合在一起。,当A位被空载tRNA占据时，RelA被激活。,一个空载tRNA进入A位就生成一个(p)ppGpp,Stringent factor catalyzes the synthesis of pppGpp and ppGpp;,ribosomal proteins can dephosphorylate pppGpp to ppGpp.,In normal protein synthesis,the presence of aminoacyl-tRNA in the A site is a signal for peptidyl transferase to transfer the polypeptide chain,followed by movement catalyzed by,EF-G,;,But under stringent conditions,the presence of,uncharged tRNA,causes RelA protein to synthesize(p)ppGpp and to expel the tRNA.,九、ppGpp有许多作用,ppGpp阻止rRNA的转录。在编码rRNA的操纵子中，启动子上转录起始被特异地抑制。,ppGpp能缩短许多或大部分模板的转录延伸期，原因可能是ppGpp引起的不断增高的RNA聚合酶的暂停反应。,The figure summarizes the systems that are used to,control rRNA transcription in response to growth rate.,Nucleotide levels control initiation of rRNA transcription.,This means that the production of ribosomes,and thus,the level of protein synthesis,in turn respond to the levels of ATP and GTP,which reflect the nutritional condition of the cell.,十、翻译是可调控的,一种翻译调节方式：,与mRNA上靶区结合的蛋白质通过阻遏核糖体识别起始区来实现阻抑物的功能,，这和与DNA结合的阻抑物防止RNA聚合酶识别启动子的机制是相对应的。,A regulator protein may block translation by binding to a site on mRNA that overlaps the ribosome-binding site at the initiation codon.,Proteins that bind to sequences within the initiation regions of mRNAs may function as translational repressors.,另一种翻译调控方式：,一个顺反子的翻译需要前一个顺反子翻译所引起的二级结构变化来调控。,Secondary structure can control initiation.Only one initiation site is available in the,RNA phage,but translation of the first cistron changes the conformation of the RNA so that other initiation site(s)become available.,The cistrons always are expressed in a set order.,十一、r-蛋白合成的,自主调控,r-蛋白（核糖体蛋白，,ribosomal proteins,）操纵子的翻译是由此操纵子的表达产物来控制的，该产物可与多顺反子mRNA上某个位点结合。,Genes for ribosomal proteins,protein synthesis factors,and RNA polymerase subunits are interspersed in a small number of operons that are autonomously regulated.,Translation of the r-protein operons is autogenously controlled and responds to the level of rRNA.,A mechanism to link r-protein synthesis to rRNA synthesis.,About,autogenous regulation,:,Autogenous regulation:,the gene coding for the regulatory product is itself one of the targets for regulation,.,negative autogenous regulation:,the regulatory protein inhibits expression of a,contiguous set of genes,within the operon.,十二、T4噬菌体p32合成的自主调控过程,p32与其自身的mRNA结合来阻遏翻译的起始。,T4噬菌体基因,p32,表达的,蛋白质p32,在遗传重组、DNA修复和复制中起着主要作用，它的功能是由它与单链DNA的结合能力来决定的。,基因的无义突变使无活性蛋白质过量表达，因此当,p32蛋白,功能被阻遏后，就会有更多的该,p32蛋白,产生，这个效应发生在翻译水平上。,Excess gene 32 protein(p32),binds to its own mRNA,to prevent ribosomes from initiating translation.,Gene 32 protein binds to various substrates with different affinities,in the order single-stranded DNA,its own mRNA,and other mRNAs.Binding to its own mRNA prevents the level of p32 from rising 10,-6,M.,十三、自主调节常用来控制大分子聚合物的合成,微管前体，即游离的微管蛋白，可抑制微管蛋白mRNA的翻译。,Tubulin is assembled into microtubules when it is synthesized.,Accumulation of excess free tubulin,induces instability in the tubulin mRNA,by acting at a site at the start of the reading frame in mRNA or at the corresponding position in the nascent protein.,Note:,在那些将合成为大分子聚合物的蛋白质中，自主调节是一种普遍的调控方法。,2,）蛋白质,自主调控,是一种内在的自我限制系统，在自主调节的情况中，关键的参数是蛋白质本身的浓度。,十四、可变性二级结构调控衰减作用,调控RNA中必需发夹结构的形成可以使转录终止发生衰减。,最直接的衰减机制涉及到促使发夹结构,稳定或不稳定,的蛋白质。,Attenuation occurs when a terminator hairpin in RNA is not prevented from forming.,十五、大肠杆菌色氨酸操纵子,(,trp,operon),受衰减作用控制,在启动子和色氨酸基因簇的第一条基因之间存在一个衰减子(attenuator)(又称,内在终止子,）,The,trp,operon consists of five contiguous structural genes preceded by a control region that includes a promoter,operator,leader peptide coding region,and attenuator.,衰减子的终止作用与色氨酸水平有关：,1）当存在足够色氨时，便会有效地发生终止作用；,2）当色氨氨酸缺乏时，RNA聚合酶就会继续转录结构基因（,转录增加十倍）,。,本质：,衰减作用受mRNA二级结构改变的控制并取决于核糖体结合mRNA的位置。,An attenuator controls the progression of RNA polymerase into the,trp,genes.,RNA polymerase initiates at the promoter and then proceeds to position,90,where it pauses before proceeding to the attenuator at position,140,.,十六、衰减作用可以为翻译所控制,色氨酸操纵子的前导区有一个由,14个密码子,组成的可读框，其中包含编码色氨酸的两个密码子。,衰减子处的RNA结构取决于这个读框是否被翻译。,The trp leader region can exist in alternative base-paired conformations.The center shows the four regions that can base pair.,Region 1 is complementary to region 2,which is complementary to region 3,which is complementary to region 4.,On the left is the conformation produced when region 1 pairs with region 2,and region 3 pairs with region 4.On the right is the conformation when region 2 pairs with region 3,leaving regions 1 and 4 unpaired.,当存在色氨酸时，前导序列被翻译，使得衰减子能够形成引起终止的发夹结构。,当不存在色氨酸时，核糖体停在色氨酸密码子处，形成的二级结构会阻止发夹结构的形成，于是转录得以继续。,The alternatives for RNA polymerase at the attenuator depend on the location of the ribosome,which determines whether regions 3 and 4 can pair to form the terminator hairpin.,1,4,3,2,1,2,3,4,In the presence of tryptophan tRNA,ribosomes translate the leader peptide and are released.This allows hairpin formation,so that RNA polymerase terminates.,In the absence of tryptophan tRNA,the ribosome is blocked,the termination hairpin cannot form,and RNA polymerase continues.,十七、反义RNA（,antisense RNA）,可以抑制基因表达,RNA引入真核生物细胞时，可以阻断其靶基因的表达。,Antisense RNA can be generated by reversing the orientation of a gene with respect to its promoter,and can anneal with the wild-type transcript to form duplex RNA.,At what level does the antisense RNA inhibit expression?,It could in principle prevent:,1),transcription of the authentic gene,2),processing of its RNA product,or 3),translation of the messenger,.,The inhibition depends on,formation of RNARNA duplex molecules,but this can occur either in the nucleus or in the cytoplasm.,十八、小RNA分子可以调节翻译,调节因子RNA通过与靶RNA形成双链区域来行使功能。,双链体可以阻遏翻译起始，引起转录终止，或产生一个核酸内切酶的作用靶点。,A protein that binds to a single-stranded region in a target RNA could be excluded by a regulator RNA that forms a duplex in this region.,By binding to a target RNA to form a duplex region,a regulator RNA may create a site that is attacked by a nuclease.,The secondary structure formed by base pairing between two regions of the target RNA may be prevented from forming by base pairing with a regulator RNA.In this example,the ability of the 3 end of the RNA to pair with the 5 end is prevented by the regulator.,十九、细菌内含有调节因子RNA,细菌中的调节因子RNA是短的分子，统称为sRNA。,大肠杆菌包含至少17种不同的sRNA，有一些sRNA是普遍的调节因子，可以影响许多靶基因。,Oxy R（可被H,2,O,2,刺激表达的基因）,Oxy S（靶基因之一）,Oxy S RNA,（,一种在转录后水平影响基因表达的反式作用调节因子）,超过10个靶基因,转录,氧化应急反应展示了RNA作为调节因子的通用控制系统的一个有趣例子。,oxyS,RNA inhibits translation of,flhA,mRNA by base pairing with a sequence just upstream of the AUG initiation codon.,二十、在许多真核生物中，微小RNA是调节因子,动物和植物基因组编码许多短的RNA分子（约22个碱基），称为微小RNA（,microRNA，miRNA).,。,微小RNA通过与靶mRNA 序列互补配对原则来调节基因的表达。,lin4,RNA,regulates expression of,lin14,by binding to the 3 nontranslated region.,The,lin4,RNA is an example of a microRNA(miRNA).There are,80 genes,in the,C.elegans,genome,coding for microRNAs of 21-24 nucleotide length,.They have varying patterns of expression during development and are likely to be regulators of gene expression.,Many of the,C.elegans,microRNAs have homologues in mammals,so the mechanism may be widespread.They are also found in plants.,Of 16 microRNAs in,Arabidopsis,8 are completely conserved in rice,suggesting widespread conservation of this regulatory mechanism.,二十一、RNA干扰与基因沉默有关,dsRNA,由ATP依赖的切割而被降解为由21-23个碱基组成的寡核苷酸，这种短的RNA有时被称为小干扰RNA（short interfering RNA,siRNA),可以导致宿主基因的沉默。,RNA干扰能触发与短双链RNA（dsRNA）中任何一链互补的mRNA的降解。,siRNA that mediates RNA interference is generated by cleaving dsRNA into smaller fragments.The cleavage reaction occurs 21-23 nucleotides from a 3 end.The siRNA product has protruding bases on its 3 ends.,enzyme,基因沉默依赖于RNA-RNA的相互作用，同样有可能的是，dsRNA也与DNA相互作用从而抑制基因的表达。,RNAi occurs when a dsRNA is cleaved into fragments that direct cleavage of the corresponding mRNA.,RNAi:RNA interferance,二十二、小结,1）基因表达可由基因的激活剂正向调控，也可由基因的抑制物负向调控。最常见的控制水平是在转录起始水平，当然在转录终止水平也可控制。,2）翻译可由那些与mRNA相互作用的调节因子调控。调节产物可能是蛋白质，这常常受控于应答环境变化时出现的变构作用；或者是RNA，这主要通过与靶RNA的碱基配对并改变其二级结构来发挥作用。,3）把调控因子互相联系起来就形成了调控网络，如此一个调控因子的产生或者活性会被其他调控因子所调控。,