续1λ噬菌体的调控.ppt

《续1λ噬菌体的调控.ppt》由会员分享，可在线阅读，更多相关《续1λ噬菌体的调控.ppt（87页珍藏版）》请在装配图网上搜索。

1、噬菌体的基因表达调控,噬菌体的调控有多种形式，有正调节、负调节、自主性的反馈调节、抗终止调节、反义调节及反向调节等。其机制之精巧，过程之复杂，形式之多样堪为集原核生物调控之大成。,噬菌体是一种以大肠杆菌为宿主的温和型噬菌体，它感染大肠杆菌后，通过精确的基因表达调控机制选择裂解周期或溶原周期。 噬菌体长48502nt，共61个基因，其中32个较为重要 .,一、Lamda phage 简介,的整个生命过程都离不开寄主的RNA聚合酶，因为噬菌体没有编码RNA聚合酶的基因。 DNA两条链都有遗传密码，有各自的转录单位向右转录的叫R链；向左转录的叫L链。,Lytic: three phases - im

2、mediate early, delayed early and late （lysis）. Replication of the lambda genome, production of viral coat proteins, and assembly of progeny phage. Lysogenic: three phases - immediate early, delayed early and late （lysogeny）. Repressor protein cI is produced which binds to the two operator regions, O

3、R and OL. Transcription of all lambda genes except cI is stopped. No progeny produced. Phage genome integrates into host genome and becomes prophage. Not disadvantageous for host.,-DNA的基因顺序组织如图所示，按基因组功能共分六大区域:头部编码区、尾部编码区、重组区、控制区、复制区和裂解区. 它们分属四个操纵子结构：阻遏蛋白操纵子、早期左向操纵子、早期右向操纵子以及晚期右向操纵子。,噬菌体的基因组结构,Bacter

4、iophage lambda genome.png,图中OR1、 2 、3的顺序反了?,When contemplating I suggest attention be payed to the following concepts:,时间,空间,量（浓度）, 或然性 temporal，spatial，amount (concentration)，random(to a certain extent),-DNA在宿主细胞环化后，由于DNA分子上没有任何调控蛋白存在，因此宿主的RNA聚合酶便分别结合于PL和PR .,2. immediate early,Both the lytic and l

5、ysogenic pathways require expression of the immediate early and delayed early genes.,When lambda DNA enters a new host cell， the lytic and lysogenic pathways start off the same way.,Figure 12.12 Phage lambda has two early transcription units;in the leftward unit, the upper strand is transcribed towa

6、rd the left; in the rightward unit, the lower strand is transcribed toward the right. . Genes N and cro are separated from the delayedearly genes by the terminators. Synthesis of N protein allows RNA polymerase to pass the terminators tL1 to the left and tR1 to the right. 或许这种抗终止机制的使用是为了保证细胞中一开始就累积一

7、定量的 Cro蛋白。,2.1 Lambda has only two immediate early genes, N and cro. N. N codes for an antitermination factor whose action at the nut sites allows transcription to proceed into the delayed early genes;,Cro. Cro has dual functions: By binding to OR ( OR3 ) it prevents synthesis of the repressor CI wi

8、th PRM ; By interacting with OL (OLl) and OR (ORl)it turns off expression of the immediate early genes (which are not needed later in the lytic cycle （包括自体调控）Cro 浓度较高时。,two replication genes (needed for lytic infection), seven recombination genes (some involved in recombination during lytic infectio

9、n, two necessary to integrate lambda DNA into the bacterial chromosome for lysogeny), and three regulators， cII，cIII， Q(位置较靠后）。,3. delayed early stage,The delayed early genes include,The three regulators have opposing functions: The cII-cIII pair of regulators is needed to establish the synthesis of

10、 repressor. lysogen The Q regulator is an antitermination factor that allows host RNA polymerase to transcribe the late genes. lytic,CII CII is a transcription activator. It activates transcription at three promoters: PRE, PI and PaQ.,活化PRE：产生阻遏蛋白CI。稍后进一步介绍。,活化整合酶启动子PI：由PI起始的mRNA在int基因末端后面约300nt的终点子

11、处停止转录。由PI转录的mRNA较为稳定。,虽然int基因也可以由启动子PL起始转录，但产生的int mRNA会被细胞中的核酸酶所降解，并不能产生整合酶。因为由启动子PL起始转录， RNA聚合酶经过N抗终止蛋白的修饰可以通过终止子tL2，这样RNA得以延伸，形成了核酸酶的靶位点sib。它首先被RNase 剪切，接着核酸外切酶从3-5降解mRNA. sib是int基因下游的负调节位点，降解由下游向上游方向进行，故称其为反向调节（retroregulation）。,活化PaQ ：Transcription from PaQ makes a short 60 nt self-terminating

12、transcript which functions in ANTISENSE control of Q expression.,In order to become established as a lysogen in E.coli, bacteriophage lambda must do two things: repressor must be synthesized the phage must become integrated into the host chromosome,Please Remember:,Conclusion: CII is essential for l

13、ysogeny. Without it - or without enough of it - lysogenic growth is not possible.,CIII: a chaperone protein that protects CII from host proteases CII alone has a half-life of 1 min. In the presence of CIII, CII has a half-life of 5 min. Q Antiterminator of tR4, allows transcription from PR2 (also ca

14、lled PR) to proceed through to tR5, thus transcribing all of the late lamba structural genes required for phage assembly. But at this stage Q may not exist because CII(基因位置在Q基因之前） activates PaQ .维基也解说为No Q.,Lambda immediate early and delayed early genes are needed for both lysogeny and the lytic cyc

15、le,晚早期基因包括2个复制基因（裂解感染所需的），7个重组基因（有的和裂解感染中的重组有关，其中有2个是DNA整合到细菌染色体中所必需的）及3个调节基因（c，c，Q）。启动子PL和操作子OL部分重叠。,4. late lysogeny,CI操纵子 cI基因编码的CI阻遏蛋白，分别作用于早期左向操纵子和早期右向操纵子中的操作子OL和OR，阻止这两个操纵子转录.,CI 阻遏蛋白 CI 阻遏蛋白操纵子与噬菌体溶原状态的确立与维持密切相关。cI基因突变的噬菌体在生长着大肠杆菌的固体培养基上总是形成清晰的噬菌斑，该基因由此得名。,Lysogeny is maintained by repressor

16、protein。,A result of cooperative binding is to increase the effective affinity of repressor for the operator at physiological concentrations. This enables a lower concentration of repressor to achieve occupancy of the operator.,阻遏蛋白操纵子含有一个结构基因(cI基因)，但有两个启动子: PRE (阻遏蛋白建立溶原状态)， PRM (阻遏蛋白维持溶原状态),(The s

17、ubscript RE stands for repressor establishment, RM for repressor maintainment .),由PRE控制转录的mRNA分子中含有一个有效的核糖体结合位点，mRNA的翻译活性比PRM介导的转录产物高7至8倍。 PRE缺少-35区保守序列，-10区序列也不保守.This promoter can be recognized by RNA polymerase only in the presence of cII. （CII蛋白在细胞内很不稳定，极易被宿主蛋白酶HflA降解而失去活性，而CIII蛋白的功能则是保护CII蛋白免遭降

18、解.),PRE,CI is expressed directly. Once the protein starts to accumulate in the cell, it will activate its own transcription as a result of binding to OR to activate PRM. Since the mRNA transcribed from PRE contains an anti-sense copy of the cro mRNA (expressed from PR), the two mRNAs can hybridize w

19、ith one another. This will sequester the cro coding region in a double-stranded RNA structure whcih cannot be translated.,Activation of transcription from PRE serves two functions:,由PRE介导的转录除了能直接表达cI基因外，还有一个间接功能，即同时转录出cro基因的反义RNA，与cro的mRNA互补杂交，从而阻断其翻译，而Cro是 噬菌体进入溶菌状态所必需的。,由PRM启动的cI基因转录以起始密码子AUG开始，转录

20、产物上没有核糖体结合位点，因此cI基因的表达量很低。 To transcribe cI, there must be repressor itself present. 当-DNA分子进入一个新的宿主细胞时，因为没有CI蛋白因子,宿主的RNA聚合酶并不能由启动子PRM转录cI 基因，,PRM,在晚早期基因产物CII的帮助下，cI基因得以表达。 cI阻遏蛋白分别与OL和OR两个操作子结合。 CI通过阻止两个早期操纵子的转录，既关闭了溶菌途径，又通过PRM维持了自身在宿主细胞中的一定水平。,噬菌体溶原状态的建立与维持,Faced with the triplication of binding s

21、ites at each operator, how does repressor decide where to start binding? The repressor always binds first to OL1 and OR1. Lambda repressor binds to subsequent sites within each operator in a cooperative manner. The presence of a dimer at site 1 greatly increases the affinity with which a second dime

22、r can bind to site 2. When both sites 1 and 2 are occupied, this interaction does not extend farther, to site 3. At the concentrations of repressor usually found in a lysogen, both sites 1 and 2 are filled at each operator, but site 3 is not occupied。,A control circuit: so long as the level of repre

23、ssor is adequate, there is continued expression of the cI gene. The result is that OL and OR remain occupied indefinitely. By repressing the entire lytic cascade, this action maintains the prophage in its inert form.,小结：由PRE控制转录产生CI后，PL和PR被抑制，早期基因的表达将受阻，C和C缺乏，CI通过PRE的转录也停止。所以 cI基因转录的起始需要CII，转录的维持需要C

24、I本身。,如果此时第二个噬菌体感染这个溶原化的宿主细胞，由原噬菌体基因组合成的阻遏蛋白便会迅速与新入侵的噬菌体基因组上的OL和OR结合，防止该噬菌体启动溶菌途径，这就是所谓的宿主细胞免疫现象。由OL, OR,cI以及cro基因组成的区域决定了噬菌体的免疫特性，因此也称为免疫区，具有同一免疫区的不同噬菌体存在交叉免疫活性。,5. Late lysisDuring lytic growth, late gene expression depends in part on the action of the Q protein, which is an antiterminator which al

25、lows RNA polymerase to over-ride the termination signal downstream of PR so that it can transcribe the lysis genes (S, R and Rz) and the capsid protein and processing genes (Nu1, A, W etc.).,晚期右向操纵子最大，它包括10个头部蛋白编码基因、11个尾部蛋白编码基因(AJ)以及3个负责裂解宿主细胞的溶菌酶类基因S、R和Rz，其所需启动子为PR,晚期右向操纵子,Lysis plaques of lambda p

26、hage on E. coli bacteria.,The Q protein acts at the level of DNA, unlike the N protein. It binds to the qut site within the PR promoter. RNA polymerase initiating transcription from PR reaches a pause site located 16/17 bps downstream of the transcription start point. Immediately upstream of this pa

27、use site is a second -10 region.,TTGACT -(17)-TAATT-AACGTT ,The lytic cycle depends on antitermination.,When RNA polymerase pauses, Q interacts with the paused complex by binding to the qut site. Q then displaces 70 from the elongating complex and changes its termination properties.,Lysogeny ensues

28、if synthesis of the repressor is established; lytic development follows if the late genes are expressed.,6. 溶源还是溶菌,CI repressor is needed for lysogeny. Cro repressor is needed for lytic infection.,Cro亦是一种负调节因子，作用位点也是OR和OL。Cro与操纵子的结合能关闭两个早期操纵子，但同时也阻止了启动子PRM的转录启动，阻碍溶源建立。,cro mutants usually establish

29、lysogeny, because they lack the ability to switch events away from the expression of repressor.,Cro和CI是噬菌体的两种调节蛋白因子，它们虽然都作用于OR和OL两个操作子，但对每个操纵子中三个不同位点的亲和力次序不同。CI的亲和力次序是OLlOL2OL3，ORlOR2 OR3；Cro恰好相反，为OL3OL2 OLl, OR3 OR2 ORl 。,Cro和CI有何不同？,Cro binds first to OR3. This inhibits RNA polymerase from binding

30、 to PRM. So Cros first action is to prevent the maintenance circuit for lysogeny from coming into play. Then Cro binds to OR2 or OR1. Its presence at either site prevents RNA polymerase from using PR. This in turn stops the production of the early functions (including Cro itself). Because cII is uns

31、table, any use of PRE is brought to a halt. So the two actions of Cro together block all production of repressor.,Cro破坏溶源,So far as the lytic cycle is concerned, Cro turns down (although it does not completely eliminate) the expression of the early genes. Its incomplete effect is explained by its af

32、finity for OR1 and OR2, which is about eight times lower than that of repressor. This effect of Cro does not occur until the early genes have become more or less superfluous, because pQ is present; by this time, the phage has started late gene expression, and is concentrating on the production of pr

33、ogeny phage particles.(Cro开始抑制早期基因时，早期基因的表达产物已经多余了。）,Cro与溶菌,Figure 12.31 The lytic cascade requires Cro protein, which directly prevents repressor maintenance via PRM, as well as turning off delayed early gene expression, indirectly preventing repressor establishment.,Cro蛋白作用于操作子OL，关闭早期左向操纵子，阻断CII和C

34、III蛋白的持续合成。当原先合成的CII和CIII蛋白被降解，导致：CII蛋白因子对PaQ激活作用解除，pQ大量合成。 pQ也是一种抗终止蛋白，促进晚期操纵子在启动子PR 的作用下进行转录，表达出足够数量的头部尾部包装蛋白以及裂解宿主所必需的溶菌酶系，至此噬菌体进入溶菌途径。,噬菌体溶原状态与溶菌状态的选择,噬菌体溶原状态与溶菌状态的确立均依赖于早期基因的有限度表达。 溶原状态建立的起始事件是阻遏蛋白CI与OL1和ORl结合，进而关闭cro基因的表达，并同时启动由PRM介导的CI合成； 进入溶菌途径的首要条件是Cro蛋白因子与OR3结合，打破由PRM介导的溶原维持，然后再作用于ORl或OR2

35、以及OLl或OL2，关闭两个早期操纵子，通过阻断CII和CIII的合成而终止由PRE介导的CI合成。 因此，决定噬菌体建立溶原状态或进入溶菌途径的开关是CI或Cro对操作子OL和OR有利位置的占领。,Cro的合成早于CI。然而，CI对OL和OR的亲和力比Cro蛋白高约8倍，如果CI和Cro两种蛋白同时存在，则Cro必须以分子数的优势才能赢得竞争。 如果CII蛋白有足够的活性，则PRE启动cI基因转录，合成的CI分子使得PRM更有效地表达cI基因,进入溶源；反之，Cro蛋白便依其数量优势占据OL和OR ，噬菌体进入溶菌途径。 可见，任何有利于CII基因表达并使其产物稳定的因素均可导致噬菌体建立和

36、维持溶原状态，这些因素包括营养枯竭以及感染复数等。,The health of the host cell determines the fate of lambda. If the cell is healthy, sufficient energy is available for production of progeny phage and lambda will go lytic. If the cell is unhealthy, lambda will go lysogenic.,当宿主陷入能源枯竭的境地时，细胞内相应发生一系列的生理变化，其中主要的是cAMP的大量合成，cAMP

37、-CAP复合物进而抑制宿主细胞宿主蛋白水解酶基因hfl的表达。hflA和hflB基因又称高频溶原基因，CII蛋白的不稳定性就是这个酶系作用的结果。 因此，能源枯竭的环境条件有助于噬菌体CII蛋白活性的维持，从而保证CI阻遏蛋白的合成，使得噬菌体建立溶原状态，事实上由于能源不足，噬菌体也不可能通过溶菌途径而大量繁殖。上述过程实质上是噬菌体把握环境条件对其自身生长繁殖的一种调节机制。,感染复数指的是在感染时噬菌体数与细菌细胞数的比值，它对溶原状态建立的影响也是通过CII蛋白而起作用的。感染复数与CII蛋白的活性形成有关，CII蛋白的单体没有活性，只有寡聚体才有作用。当感染复数过低时，多个噬菌体同时

38、感染一个宿主细胞的机会也就减少，所表达的CII产物不足以形成有活性的寡聚体，此时噬菌体便进入溶菌途径；当感染复数过高时(通常大于10)，CII能形成足够数量的活性寡聚体，就可促进由PRE介导的cI基因转录启动。该转录产物的翻译活性远高于从PRM转录出的mRNA，因此CI蛋白数量很快占据优势，噬菌体建立起溶原状态。,UV可激活Rec蛋白，活化的Rec蛋白可剪切C蛋白.,其它：,Regulation of int and xis expression,Repressor uses a helix-turn-helix motif to bind DNA,On the role of Cro in

39、lambda prophage induction,Sine L. Svenningsen et al. National Cancer Institute, Bethesda, MD 20892-4255; and National Cancer Institute, Frederick, MD 21702,originally published online Feb 23, 2005;,Abastract The lysogenic state of bacteriophage is exceptionally stable yet the prophage is readily ind

40、uced in response to DNA damage. This delicate epigenetic switch is believed to be regulated by two proteins; the lysogenic maintenance promoting protein CI and the early lytic protein Cro. First, we confirm, in the native configuration, the previous observation that the DNA loop mediated by oligomer

41、ization of CI bound to two distinct operator regions (OL and OR), increases repression of the early lytic promoters and is important for stable maintenance of lysogeny. Second, we show that the presence of the cro gene might be unimportant for the lysogenic to lytic switch during induction of the prophage. We revisit the idea that Cros primary role in induction is instead to mediate weak repression of the early lytic promoters. Key words: CI protein genetic switch transcription,复习思考题,正调节 负调节 抗终止调节 反义调节 反向调节 简述CI蛋白是怎样建立自身的调控循环的。,