第六章-色氨酸(trp)操纵子3.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,色氨酸（,trp,）,操纵子,lac,和,ara,操纵子是编码,分解代谢,途径酶系的操纵子，负责碳源（例如乳糖和阿拉伯糖等）的分解利用，这些操纵子的表达受相应碳源的诱导。,在细菌中还有负责一些物质,合成代谢,的操纵子，例如色氨酸操纵子（,tryptophan operon,trp operon,）,就是负责色氨酸合成的操纵子。,trp,操纵子是由一个启动子和一个操纵基因区组成，该操纵基因区控制一个编码色氨酸生物合成需要的5种蛋白的多顺反子,mRNA,的表达。,由于,trp,体系参与生物合成而不是降解,，它不受,葡萄糖或,cAMP,-CRP,的调控。,色氨酸的合成主要分5步完成，有7个基因参与整个合成过程。,trpE,和,trpG,编码邻氨基苯甲酸合酶，,trpD,编码邻氨基苯甲酸磷酸核糖转移酶，,trpF,编码异构酶，,trpC,编码吲哚甘油磷酸合酶，,trpA,和,trpB,则分别编码色氨酸合酶的,和,亚基。,trpE,基因是第一个被翻译,的基因，和,trpE,紧邻的是,启动子区和操纵区,。另,外，,前导区和弱化子区分别定名为,trpL,和,trpa,（,不是,trpA,）。,trp,操纵子中产生阻遏物的基因是,trpR,，,该基因距,trp,基因簇很远。后者位于大肠杆菌染色体图上25分钟处，而前者则位于90分钟处。在位于65分钟处还有一个,trpS,（,色氨酸,tRNA,合成酶），它与携带有色氨酸的,tRNA,Trp,共同参与,trp,操纵子的调控作用。,弱化子,在,trp mRNA,5,端,trpE,基因的起始密码前有一个长,162,bp,的,mRNA,片段被称为前导区，其中,123,150,位碱基序列如果缺失，,trp,基因表达可提高,6-10,倍，而且无论是在阻遏细胞内还是在永久性突变的细胞内都是这样。当,mRNA,合成起始以后，除非培养基中完全没有色氨酸，转录总是在这个区域终止，产生一个仅有,140,个核苷酸的,RNA,分子，终止,trp,基因转录，这就是,123,150区序列缺失会提高,trp,基因表达的原因。因为转录终止发生在这一区域，并且这种终止是被调节的，这个区域就被称为弱化子。,研究引起终止的,mRNA,碱基序列,，,发现该区,mRNA,通,过自我配对可以形成茎,-,环结构，有典型的终止子特点。,前 导 肽,实验表明衰减作用需要负载,tRNA,Trp,参与，这意味着前导序列的某些部分被翻译了。分析前导序列发现，它包括起始密码子,AUG,和终止密码子,UGA,；,如果翻译起始于,AUG,，,应该产生一个含有,14,个氨基酸的多肽。这个假设的多肽（还未实际观察到）被称为前导肽。,前导序列具有一个非常有意义的特点，在其,第10和第11位,上有相邻的,两个色氨酸密码子,。这一点很重要，因为组氨酸操纵子中，也具有弱化子，也具有一个类似的能编码前导肽的碱基序列，此序列中含有7个相邻的组氨酸密码子。苯丙氨酸操纵子中同样存在弱化子结构，其前导序列中也有7个苯丙氨酸密码子。这些密码子参与了,trp,及,其他操纵子中的转录弱化机制。,mRNA,前导区的序列分析,trp,前导区的碱基序列已经全部测定，引人注目的是其中4个分别以1、2、3和4表示的片段能以两种不同的方式进行碱基配对（图6-22），有时以1-2和3-4配对，有时只以2-3方式互补配对。,RNaseT1,降解实验（此酶不能水解配对的,RNA）,表明，纯化的,trp,前导序列中确有1-2和3-4的配对方式，由此定位的3-4配对区正好位于终止密码子的识别区，当这个区域发生破坏自我配对的碱基突变时有利于转录的继续进行,。,转录弱化作用,转录的弱化理论认为,mRNA,转录的终止是通过前导肽基因的翻译来调节的。,因为在前导肽基因中有两个相邻的色氨酸密码子，所以这个前导肽的翻译必定对,tRNA,Trp,的浓度敏感。,当培养基中色氨酸的浓度很低时，负载有色氨酸的,tRNA,Trp,也就少，这样翻译通过两个相邻色氨酸密码子的速度就会很慢，当4区被转录完成时，核糖体才进行到1区（或停留在两个相邻的,trp,密码子处），这时的前导区结构是2-3配对，不形成3-4配对的终止结构，所以转录可继续进行，直到将,trp,操纵子中的结构基因全部转录。,而当培养基中色氨酸浓度高时，核糖体可顺利通过两个相邻的色氨酸密码子，在4区被转录之前，核糖体就到达2区，这样使2-3不能配对，3-4区可以自由配对形成茎-环状终止子结构，转录停止，,trp,操纵子中的结构基因被关闭而不再合成色氨酸（图6-24）。所以，弱化子对,RNA,聚合酶的影响依赖于前导肽翻译中核糖体所处的,位置。,The,trp,operon is negatively regulated,by the Trp repressor,Trp repressor binds its target DNA sequence only when it itself is bound by its co-repressor,tryptophan.,The,trp,operator is a palindromicDNA sequence,trp,操纵子转录的调控是通过,Trp,阻遏物,实现的，它结合于,trp,操纵基因序列,，但,Trp,阻遏物的,DNA,结合活性直接受色氨酸调控，色氨酸结合,Trp,阻遏物，并起着一个效应分子的作用（也称之辅阻遏物）。,在有,高浓度色氨酸,存在时，,Trp,阻遏物-色氨酸复合物形成一个同源二聚体，并且紧密结合于,trp,操纵基因序列，因此可以阻止转录。然而当,色氨酸水平低,时，缺少色氨酸的,Trp,阻遏物以一种非活性形式存在，不能结合,DNA。,在这样的条件下，,trp,操纵子被,RNA,聚合酶转录，同时色氨酸生物合成途径被激活。,trp,操纵子的另一种转录调控是称之衰减作用（,attenuation,）,的调控机制，这是一种将翻译与转录联系在一起的新的转录调控形式。细胞内,Trp,-,tRNATrp,浓度决定核糖体是否停留在,trp mRNA,中的前导序列内的两个连续的色氨酸密码子处。当色氨酸水平高和,Trp,-,tRNATrp,可利用时，起转录终止作用的发卡环结构（由区3和区4之间）形成,，,RNA,聚合酶刚好在一个聚尿嘧啶的下游处脱离,DNA,模板，转录终止。,当由于细胞内色氨酸有限，,Trp,-tRNATrp,水平低时，核糖体就停留在,RNA,中连续的一对色氨酸密码子处。核糖体这一瞬间的停留给出时间使得一种替换的发卡结构在新的,RNA,中（由区2和区3之间）形成，是一种抗终止的,RNA,结构，它破坏了转录终止信号，使得,RNA,聚合酶能够继续沿着,DNA,模板滑动完成转录。,The,trp,operon,P=promoter,T=terminator,O=operator,trpR,trpA,P O,P,T,T,Polycistronic,mRNA,(encodes 5 proteins),mRNA,TrpR,(repressor),5 separate proteins that were synthesized from one,mRNA,trpB,trpC,trpD,trpE,Attenuator,TrpR,Attenuation in the,trp,operon,Effectively adds a fine tuning to the regulation of the,trp,operon,.,Several key points,:,Transcription&translation are tightly coupled in bacteria(attenuation requires this).,Synthesis of a leader sequence rich in,Trp,influences whether transcription of the,trp,operon,is complete.,If,Trp,is adequate transcription is terminated before the,trp,operon,.,If,Trp,is,inadequate,transcription is completed.,Termination of transcription is determined by leader,mRNA,sequence.,Attenuation a,transcriptional,form of control,mRNA,leader sequence,Attenuator,110,140,trpE,Leader polypeptide,14,aa,with 2,Trp,aa,1,162,Typical stem loop,of Termination site,Attenuation,1,2,3,4,4,2,3,1,mRNA,Trp,codons,mRNA,sections,base pairs with 2,base pairs with 4,ONLY 3+4 generate,the termination site,Attenuation Inadequate,Trp,1,2,3,4,mRNA,Trp,codons,2,3,1,4,Ribosome,stalls,due to low,Trp,This large stem loop of 2+3 does NOT act as a terminator.,Transcription continues!,RNA,polymerase,Attenuation Adequate,Trp,1,2,3,4,4,3,1,Ribosome moves,Rapidly along,mRNA,mRNA,sections,base pairs with 4 to,form a termination site,such that,RNApolymerase,prematurely falls off the,mRNA,and aborts further,transcription.,mRNA,Attenuation works by have the RNA,polymerase,stop(terminate)before the transcription of the structural genes,but AFTER it has already started to make an RNA.,The key is the region of the,operon,called,trpL,(see the figures above).,The,trp,operon,of,E.coli,In,tryptophan,-poor medium,In,tryptophan,-rich medium,Operon,Summary,We have considered in detail three,operons,:the,lac operon,the,ara operon,and the,trp operon,.The first two are,operons,concerned with the control of catabolic processes(utilization of energy substrates)while the third is concerned with anabolic processes(synthesis of a molecule the cell needs).,All three share negative control features,using repressor proteins binding to operators to place the,operon,in the off state.The first two have positive control features that increase transcription in response to low glucose(CAP-,cAMP,binding to the CAP site).This is not the case for the,tryptophan operon,.The,tryptophan operon,however,has the additional negative control feature of attenuation.These features are summarized in the following table:,consensus,TATA(,Pribnow,)box,E.,coli,Promoters,Allosteric,Effectors,Binding can also be required for binding of repressor(e.g.,Trp,)or can block an activator.,Genomic Organization of the,Trp Operon,The,Trp Operon,Note that the order of the genes follows the order of the biosynthetic pathway!,Control of Gene Expression in the,Trp OperonThe enzyme catalyzing the first step in the pathway is inhibited by,Trp,(feedback control).,In the presence of,Trp,a repressor protein binds to an operator upstream of the,Trp operon,and shuts off transcription,Attenuation.There is a 160 base pair region in the,Trp,mRNA,that causes transcription to terminate prematurely if,Trp,is present,.,Feedback Control,Transcriptional Control,Attenuation Control,Attenuation Control,High,Trp,LevelsRibosome proceedsLoop 3/4 forms Transcription terminates,Low,Trp,LevelsRibosome stallsLoop 2/3 formsTranscription continues,Trp Operon,Controlled by Attenuation,trpR,P,O,1 2 3 4,trpE trpD trp,C,trp,B,trpA,attenuator,Leader,attenuation can form,under certain conditions,base-pairing can occur,between,1-2 2-3 3-4,Attenuation,58,High levels of,Tryptophan,in Cell,transcription&translation occur simultaneously in Prokaryotes,leader transcript(1)has 2,trp,codons,(UGGUGG),ribosomes,moves fast along transcript,stem-loop 3-4 forms,poly Us after,early termination of transcription,translation stops,(only leader peptide forms,-has no function),59,Low Levels of,Tryptophan,in Cell,ribosome stalls at UGGUGG in leader transcript(1),stem-loop 2-3 forms,no poly U after,transcription continues,60,Low Levels of other Amino Acids,ribosome stalls way early,stem-loops 1-2&3-4 form,poly U after,early termination of transcription,61,The,Trp,Operon,:,When,Tryptophan,Is Present,STOP,Right there,Polymerase,Trp,Trp,Repressor,Repressor,Repressor,Promo.,trpD,trpB,Lead.,Operator,trpA,trpC,trpE,Aten,.,RNA,Pol.,Foiled,Again!,Repressor,mRNA,Hey man,Im,constitutive,3,5,5,3,Transcription And Translation In Prokaryotes,Ribosome,Ribosome,5,mRNA,RNA,Pol.,Met-,Lys,-Ala-,Ile,-,Phe,-Val-,AAGUUCACGUAAAAAGGGUAUCGACA-AUG-AAA-GCA-AUU-UUC-GUA-,Leu,-,Lys,-,Gly,-,Trp,-,Trp,-,Arg,-,Thr,-SerSTOP,CUG-AAA-GGU-,UGG-UGG,-CGC-ACU-UCC,-UGA-AAC,GGGCAGUGUAUU,CACCA,UGCGUAAAGCAAUCAG,AUACCCAGCCCGCC,UAAU,GAGCGGGCUU,UU,Met-,Gln,-,Thr,-,Gln,-,Lys,-Pro,UUUU,-GAACAAAAUUAGAGAAUAACA-AUG-CAA-ACA-CAA-AAA-CCG,trpE,.,Terminator,The,Trp,Leader and Attenuator,4,1,2,3,The,mRNA,Sequence Can Fold In Two Ways,4,1,2,3,Terminator,haripin,4,1,2,3,3,5,5,3,The Attenuator When Starved For,Tryptophan,4,1,2,3,RNA,Pol,.,Ribosome,Help,I need,Tryptophan,3,5,5,3,The Attenuator When,Tryptophan,Is Present,4,1,2,3,RNA,Pol,.,Ribosome,RNA,Pol,.,