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,酶合成调节的遗传机制,操纵子学说,Jacob,和,Monod,在研究大肠杆菌,-,半乳糖苷酶合成的遗传基础上，提出了控制基因表达的操纵子学说,。,The Nobel Prize in Physiology or Medicine 1965,Franois,Jacob,Andr Lwoff,Jacques Monod,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。,酶的诱导,-lac,体系受调控的证据在不含乳糖及,-,半乳糖苷的培养基中，,lac+,基因型每个大肠杆功细胞内大约只有,1-2,个酶分子。如果在培养基中加入乳糖，酶的浓度很快达到细胞总蛋白量的,6%,或,7%,，每个细胞中可有超过,10,5,个酶分子。,科学家把大肠杆菌细胞放在加有放射性,35S,标记的氨基酸但没有任何半乳糖诱导物的培养基中繁殖几代，然后再将这些带有放射活性的细菌转移到不含,35S,、无放射性的培养基中，随着培养基中诱导物的加入，,-,半乳糖苷酶便开始合成。分离,-,半乳糖苷酶，发现这种酶无,35S,标记。说明酶的合成不是由前体转化而来的，而是加入诱导物后新合成的。,乳糖是含有葡萄糖的二糖,葡萄糖状态,乳糖状态,操纵子状态,有,无,关闭,有,有,关闭,无,无,关闭,无,有,开放,lacZ,encodes,-,galactosidase,(LacZ),an intracellular,enzyme,that cleaves the,disaccharide,lactose,into,glucose,and,galactose,.,lacY,encodes,-,galactoside permease,(LacY),a,membrane,-bound transport protein that pumps lactose into the cell.,lacA,encodes,-,galactoside transacetylase,(LacA),an enzyme that transfers an,acetyl group,from acetyl-CoA to,-,galactosides,.,操纵子即基因表达的协同单位，含有结构基因,(,编码,Pr),和控制部位。,控制部分,:,调节基因、操纵基因,一个操纵子的全部基因都排列在一起，控制部分可接受调节基因产物的调节。,OPTG,IPTG,别乳糖,X-gal,具有半乳糖苷的物质都可诱导,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。,catabolite activator protein(CAP;also known as cAMP receptor protein,CRP),cAMP-CAP-DNA,CAP,的效应不应该为乳糖独享,单糖的种类：,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.,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.,We can now distinguish 3 classes of,CRP,-activated promoter,:,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.,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.,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,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。,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,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。,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。,衰减作用,(,弱化作用,),的意义,E.Coli,一个大肠杆菌就是一个完整的生命！,E.coli Bacterium,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,没有粮食吃了才想起种庄稼！,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,弱化作用有没有普遍的意义？,3.The Histidine Operon:An Attenuator,3,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),。,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,。,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。,有葡萄糖无阿拉伯糖,无葡萄糖有阿拉伯糖,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。,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.,Euprymna sco