转录水平的调控

,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,*,二、转录水平的调控,（一）原核生物的操纵子模型,1.乳糖操纵子,1,1.乳糖操纵子,大肠杆菌生长需要碳源, 常见的是糖类, 最方便利用的是葡萄糖, 但有些条件下培养基中并无葡萄糖，仅有半乳糖等糖类，这时分解它们的酶类必须合成，才能利用半乳糖。,2,3,4,5,6,7,8,当葡萄糖与乳糖共存，情况比较复杂，分解它们的酶类必须不合成,。,降解物基因活化蛋白CAP（catabolic gene activate protein, CAP), 与cAMP形成复合物结合于启动子部位，引起DNA构象的变化，促进RNA聚合酶与启动子结合，使转录的起始更加频繁，是一种正调控。当有葡萄糖存在时，其分解代谢产物可抑制腺苷酸环化酶活性，激活磷酸二酯酶活性，cAMP含量下降，使CAP失活。,9,10,cAMP-CAP-DNA,11,12,Ever since the function of,CRP,as an activator of transcription was established, a great deal of work has been done to try and understand exactly how this occurs.,13,Does,CRP,activate transcription directly? In other words, does it assist,RNA polymerase,to recognize the promoter by means of direct protein-protein contacts between,CRP,and,RNA polymerase,?,OR,Does,CRP,activate transcription indirectly? Since,CRP,bends and thus distorts the DNA double helix when it binds, is it possible that increased RNA polymerase binding is a result of structural changes in the DNA?,A third possibility is that some combination of both direct and indirect effects might be responsible for activation at individual promoters.,14,We can now distinguish 3 classes of,CRP,-activated promoter,:,15,CLASS I,Class I,CRP,-activated promoters require only,CRP,for activation and the,CRP,binding site is located upstream of the promoter.,The prototype,CLASS I,CRP,-activated promoter is the,lac,operon promoter (,lacP1,). This,CRP,-binding site is centred 61.5 bp upstream of the startpoint of transcription.,16,17,18,19,20,21,CLASS II,CLASS II,CRP,-activated promoters require only,CRP,for activation and the,CRP,binding site overlaps the promoter. In these promoters, the,CRP,binding site appears to replace the usual -35 region of the promoter.,The prototype,CLASS II,CRP,-activated promoter is the,gal,operon promoter (,galP1,). This,CRP,-binding site is centred 41.5 bp upstream of the startpoint of transcription.,22,CLASS III,CLASS III,CRP,-activated promoters require additional regulator proteins as well as,CRP,for activation. The location of the,CRP,binding site can be quite variable though it is typically more than 90 bp upstream of the startpoint of transcription.,There is no prototype,CLASS III,CRP,-activated promoter since they all have different requirements by way of additional regulator proteins. Examples of,CLASS III,CRP,-activated promoters are the,araBAD,promoter and the,malK,promoter,23,2.色氨酸操纵子,大肠肝菌培养在只含无机盐及单一碳源的培养基中，大肠杆菌细胞内可以测出色氨酸合成的酶系, 如果在培养基中加入色氨酸, 大肠杆菌中色氨酸合成的酶系就明显降低。色氨酸存在时, 阻止了色氨酸合成酶系的形成, 细菌可直接利用色氨酸, 而不用自己合成, 这种减少酶量的现象称为酶合成的阻遏。,24,25,26,翻译过程对转录的调节衰减作用(弱化作用),E. Coli的色氨酸合成,27,翻译过程对转录的调节衰减作用(弱化作用),色氨酸mRNA的5端有162核苷酸的前导序列，当RNA的合成启动后除非缺乏色氨酸，否则大部分mRNA仅合成140核苷酸即停止。前导肽能编码一小段14肽，其终止区具有潜在的茎环构象和成串的U，表现出转录终止位点的特征。前导RNA链有4个区域彼此互补，可形成奇特的二级结构，有些情况下出现终止子结构。,28,29,30,31,32,33,转录与翻译偶联是原核生物的特征。,原核生物翻译结束mRNA上有特殊的终止子结构。,34,35,衰减作用(弱化作用) 的意义,E. Coli,36,E. coli Bacterium,37,弱化子系统主要是对外源色氨酸浓度作出反应，维持浓度的恒定。,色氨酸浓度,t,38,AUG-AAA-CGC-GUU-CAA-UUU-AAA-CAC-CAC-CAU-CAU-CAC-CAU-CAU-CCU-GAC,Met-Thr-Arg-Val-Gln-Phe-Lys-His-His-His-His-His-His-His-Pro-Asp-,a leader coding region,3.The Histidine Operon: An Attenuator,39,40,2 阿拉伯糖操纵子,阿拉伯糖(arabinose)是另一个可以为代谢提供碳源的五碳糖。在大肠杆菌中阿拉伯糖的降解需要3个基因：araB、araA和araD，分别编码3个酶：araB基因编码核酮糖激酶(ribulokinase)，araA编码L-阿拉伯糖异构酶(L-arabinose isomerase)，araD编码L-核酮糖-5-磷酸-4-差向异构酶(L-ribulose-5phosphate-4epimerase)。,41,42,与araBAD相邻的是一个复合的启动子区域和一个调节基因araC，这个AraC蛋白同时显示正、负调节因子的功能。AraBAD和araC基因的转录是分别在两条链上以相反的方向进行的。在标准的遗传学图谱上，araBAD基因簇从启动子P,BAD,开始向左进行转录，而araC基因则是从P,c,向右转录,。,43,44,45,AraC蛋白以C,rep,与araO1的结合阻遏自身基因的调节并控制着自身的合成。,46,有葡萄糖无阿拉伯糖,47,无葡萄糖有阿拉伯糖,48,阿拉伯糖和葡萄糖都很低或都很高的情况下，araBCD的转录都被阻遏。,49,5.Quorum Sensing: An Activator,(群体意识）,There exists a species of squid （鱿鱼）,the squid has evolved a light organ in which it cultures a very pure, very dense population of a bacteria called,Vibrio fischeri,. This bacteria produces a substance called luciferase, which glows with the same intensity as the moon.,50,Euprymna scolopes,swimming,51,Euprymna scolopes,hatching,52,Euprymna scolopes,ventral view of light organ,53,In this ventral cross-section of Euprymna Scolopes, the symbiotic light-emitting photophore is the black and silver, two-lobed structure in the center. Courtesy of Margaret McFall-Ngai.,bobtail squid,54,This Hawaiian bobtail squid (Euprymna scolopes) has silvery tissue around and across its eye. These reflective, silvery tissues of the eye and skin are made of the same unusual proteins that help make the squids flashlight work.,55,This Hawaiian bobtail squid (Euprymna scolopes) is a night active predator that buries in the sand during the day and comes out at night to forage. This adult specimen, which is preparing to bury for the day, has silvery tissues around its eyes and blue-silver tissues in its skin due to proteins called reflectins.,56,Protein-based reflectors appear to be unique to squid, octopus and other members of the cephalopod class of marine animals. Most cephalopods have big heads, large eyes, grabbing tentacles and sacs filled with ink.,57,Close-up of the eye of a Hawaiian bobtail squid (Euprymna scolopes,).,58,When,Vibrio fischeri,is not in the squids light organ, it does not need to be making luciferase, since glowing will not help it or anything else.,On the other hand, when inside the light organ, it is to the bacterias advantage to glow, because then the squid will not get eaten and will feed it, away from competition from any other kinds of bacteria. So how can the bacteria know that it is in a light organ in order to turn on synthesis of luciferase?,59,The Hawaiian bobtail squid has a built-in flashlight on its underside which is beamed downward by stacks of silvery reflector plates which are made from an unusual family of proteins, according to new research.,60,The squids light producing organ is powered by glowing bacteria with stacks of the reflector plates surrounding the light organ. This glowing flashlight serves as a spotlight during feeding in the dark ocean,.,61,62,Vibrio fischeri,grown for 24 hours on,Photobacterium,sea water agar,63,64,Transmission Electron micrograph of,Vibrio fischeri,65,quorum sensing,Each bacterium is continuously secreting a unique small molecule called VAI (,Vibrio fischeri,autoinducer) that can diffuse readily through the cell membrane. Thus, there is a declining concentration of the small molecule in a growing circumference around the bacterium. When there are many bacteria around, the local concentration of the small molecule will be very high,.,66,The genes for making luciferase are contained in the lux operon. A DNA binding site (luxO) near the lux promoter (luxP) binds a protein called luxR. This protein somehow calls RNA polymerase over when it is bound to the DNA, thus increasing transcription of the DNA and making more polymerase. Thus, luxR is a transcriptional activator of the lux operon.,67,68,It is important to note that LuxI is the gene that encodes for the enzyme that synthesizes VAI（,autoinducer,）. When a bacterium undergoes the transition from not making luciferase to making luciferase, it needs to have the autoinducer around in order to promote binding of LuxR to the operator.,69,70,Serine,71,N,-acyl-L-homoserine lactones (AHLs),酰化高丝氨酸内酯,72,发光的的好处,对于弧菌：,稳定的食物来源和生活环境。,73,对于,squid：,诱捕,消除阴影,吓阻,通讯,74,Margaret McFall-Ngai, a biologist at the University of Hawaii. She says that while symbiotic relationships between luminescent bacteria and animals are nothing new, theres some evidence that the bacteria are getting a raw deal.,75,So is this may be less a case of symbiosis than of slavery. The squid, she observes, inhibits the growth of the bacteria to enhance their luminescence. And the bacterium could make a better living drifting in the ocean, or in the gut of another marine animal, she observes.,76,细菌的许多行为,包括,:,生物发光、共生现象、生物膜形成、抗生素生产、群体移动性,(Swarming motility),、孢子形成、基因交换以及发病机理等均受到群体效应的调节。,77,例子,铜绿假单胞菌,(,Pseudomonas aeruginosa,),是肺囊肿性纤维化病人患慢性或致命性感染的最为常见的病原,并会感染烧烫伤或癌症接受化疗的病患。,78,79,含有铜绿假单胞菌的痰液的革兰染色 囊性纤维变性病人痰液显示葡萄球菌和铜绿假单胞菌、细的革兰阴性杆菌。（革兰染色，放大1000倍）,80,铜绿假单胞菌 培养于麦康培养基，铜绿假单胞菌为非乳糖发酵菌，经常产生稍带绿色的绿脓菌素色素，其氧化酶阳性。（麦康基琼脂，18小时，37）,81,82,铜绿假单胞菌生物膜的形成受到群体效应的调控,这种生物膜对包裹其中的铜绿假单胞菌起到一定的保护作用,(,避免了暴露于人体免疫反应中,减少了与抗菌药物的直接接触,) ,使得感染持久而且难以治愈。在感染早期通过基因手段切除了铜绿假单胞菌中的信息素合成酶后发现感染症状明显减轻了。因此,自诱导剂的生物合成酶及自诱导剂感应部位为新的抗菌药物的开发提供了有潜力的靶点,83,真核生物转录的调节,84,1转录因子,顺式作用成分与反式作用成分,顺式作用元件：,转录其始点,上游30bp处的TATA序列,上游几百bp的CCAAT序列或GGGCGG序列(GC box),在基因上或下游远端或内含子内增强子(enhancer)序列,负调控序列抑制子(silencer),其它特异调控序列,以上各种特异的核苷酸序列都是反式作用因子蛋白质的靶位点。,85,反式作用因子：,通用转录因子： 结合在TATA附近的：TFA、TFB、TFD、TFE.,转录调控因子：结合在上游特异核苷酸序列上的因子如SPI、CTF、AP-1、,CREB,还有些蛋白质因子，它们自身并不与DNA相结合，却能激活基因的转录，可能在两种不同的调控因子中起桥梁作用。,86,2. 转录因子的功能域(domain),a. 转录因子DNA结合区,Helix-turn-helix(or helix-loop-helix) motif,87,最早在 噬菌体CI和Cro蛋白及大肠杆菌的CAP蛋白中发现。虽然在氨基酸序列上有很大的可变性，但在高级结构上是高度保守的。在两个helix中，一个为识别螺旋，它的氨基酸残基直接与靶DNA大沟的碱基专一结合。,真核生物中最早在控制果蝇早期发育的同源域（homeodomain)蛋白中发现。,88,CAP-cAMP,89,CAP-cAMP-DNA,90,Zinc finger motif,H2C2最早发现于TFIIA 的DNA结合区，是RNA聚合酶转录5SRNA基因的必需的因子。含有碱性和极性氨基酸的的区域是识别特异DNA序列的位点。哺乳细胞的SP-1 有类似的结构。,C4：酵母细胞的GAL-4,哺乳动物甾体激素受体，这一类蛋白质中的锌指对DNA的结合是必需的，但对结合的专一性并不重要，在锌指附近的其他氨基酸残基与DNA直接作用形成专一结合。,91,Zinc finger(H2C2) motif,92,Zinc finger(C4) motif,93,Zinc finger(C4),94,Zine finger-DNA,95,Zine finger-DNA,96,Zine finger DNA,97,P53-DNA(example of zinc finger),98,Leu zipper motif,99,酵母的GCN4，哺乳动物的fos,jun,mys等蛋白质DNA结合区都含有45个Leu， 它们之间相距7个氨基酸，在helix 上每两圈出现一个Leu, 它们排成了一行。这类蛋白质因子都是以双体形式与DNA靶位点结合，两个蛋白质分子相应的helix之间靠Leu残基的疏水作用形成一条拉链，对双体的形成十分重要，但参与于DNA作用的使该区域以外的氨基酸残基。在Leu重复区N端的邻近的30个氨基酸片段中有许多碱性氨基酸。当Leu 拉链使亚基形成双体时，碱性区处于适当位置，决定了与DNA序列的专一结合。,100,GCN4-DNA,101,Trp repressor,102, sheet motif,103,Met repressor,104,b. 转录因子激活基因转录的功能区,酵母的GAL4,GCN4：GAL4有两个激活转录的功能区，分别在147-196和768-881aa的范围内。GCN4的在106-125aa的肽段内。共同特点是含有很多带负电荷的螺旋，但在aa序列上很少有同源性，aa的替换实验表明激活转录的水平与净负电荷变化有关，增加激活区的负电荷数能提高激活转录的水平。,哺乳动物的AP-1、fos、jun、及糖皮质激素受体也有类似的酸性激活区。,酵母和哺乳动物的酸性激活区可以互换。用GAL4的DNA结合区与fos的酸性激活区融合成的杂蛋白具有激活GAL基因表达的功能，同样用fos的DNA结合区与GAL4酸性激活区融合也具有促进基因转录的功能。,105,SP1：结合于GC box上，其中在N端的两个主要结合区含有25%的谷氨酰胺。,类似的有：,果蝇的Antennapedia和Zeste蛋白,酵母的HAP-1、HAP-2和GAL11,哺乳动物的Oct-1、Oct-2、jun、fos、SRF等。,106,CTF-1、CTF-、CTF-3和AP-2,蛋白质羧基端具有激活基因转录的功能，这一区域含有20-30%的脯氨酸残基。,107,3.转录因子的作用规律,一种蛋白质因子可以结合多个顺式作用元件。,一种顺式作用元件可以作为多种蛋白质因子的作用靶区。,有些蛋白质因子经物理或化学诱导后才具有活性。,磷酸化作用对一些蛋白质因子的功能作用是很重要的 。,蛋白质因子与特异DNA序列相互作用涉及蛋白质因子之间的反应。,108,4.转录因子与生物的性状 同源异型突变和同源域,l,指发育过程中躯体的一部分发育成另外一部分，最早在果蝇中发现，说明不同蛋白质或者几种调节蛋白质的不同组合可以控制细胞的发育。,109,110,111,112,现已证明，有两类8个同源异形基因控制胚胎最早期的空间方位，这些基因从果蝇到人类都是基本相同的。这些基因都含有180个核苷酸的序列，称homeobox（同源域），高度保守，编码蛋白质羧基末端的60个氨基酸。,113,这些同源异型基因编码的都是区域特异性的转录因子。,动物的发育过程开始由少数几个主基因(master gene)控制胚胎分化的方向性，然后由不同的器官特异性基因决定器官的发生。基因与基因间存在明显的等级关系。,114,同源域-DNA（ Helix-turn-helix）,115,5.真核生物转录调控的例子：,Galactose Metabolism Is Regulated by Specific Positive and Negative Control Factors in Yeast,116,Metabolism of galactose,117,Metabolism of galactose,118,Even when functionally related genes are clustered, they usually give rise to separate transcripts. Three of the four genes (GAL7:transferase,GAL10:epimerase,GAL1;kinase)associated with galactose utilization are clustered on chromosome XI, whereas the fourth, for galactose transport, is specified by a gene(GAL2) located on chromosome XII.,119,Expression of the four structural genes is by specific postive and negative controls.,Each of the structural genes is associated with a distinct mRNA. The GAL7 and GSL10 genes are transcribed from the same DNA strand, whereas the GAL1 gene, approximately 600bp from GAL10, is transcribed from the complementary DNA strand.,120,Transcription of the GAL1,GAL7, and GAL10 genes are increased over 1,000-fold when galactose is present, suggesting that galactose is an inducer.,Two tran-acting gene products that regulate expression: GAL4 and GAL80.,121,The active GAL4 gene produce is a postive control protein(an activator) like CAP in the,lac operon.,The GAL4 protein bind to a site upstream of the gene(s) it regulates and promotes RNA polymeraseII-dependent transcription of these genes.,122,Site of binding of GAL4 protein,GAL7,GAL10,GAL1,GAL80 protein,GAL4 protein,No,transcription,No inducer,Inducer present,GAL1,GAL1,XI,123,Most,gal80,mutants also give rise to constitutive expression of the GAL genes, which suggest that GAL80 normally acts as a negative control protein(a repressor) of GAL gene expression. The wild-type GAL80 protein binds the inducer galactose and that this binding converts the protein to an inactive form. Induction by galacose results from the sugar to the GAL80 protein, which changes its structure so that the GAL4-GAL10 complex is altered.,124,酵母GAL4的激活转录功能区在有葡萄糖条件下被另一负调控因子GAL80结合覆盖，失去激活转录的功能；经半乳糖诱导，GAL4和GAL80的结合解离，GAL4的酸性激活区才促进基因的转录。这是酵母半乳糖代谢酶基因在有葡萄糖条件下表达受阻遏，在有半乳糖时被诱导的原因。,125,Site of binding of GAL4 protein,GAL7,GAL10,GAL1,GAL80 protein,GAL4 protein,No,transcription,No inducer,Inducer present,GAL1,GAL1,XI,126,GAL4 protein is Separated into Domains with Different Functions,GAL4 activation domain,GAL4 DNA-binding domain,Yeast GAL1 gene,DNA,RNA,GAL4 binding site on DNA,lexA DNA-binding domain,No transcription,lexA DNA-binding site on DNA,RNA,127,128,129,