續(xù)1 λ噬菌體的調(diào)控.ppt

1、噬菌體的基因表達(dá)調(diào)控,噬菌體的調(diào)控有多種形式，有正調(diào)節(jié)、負(fù)調(diào)節(jié)、自主性的反饋調(diào)節(jié)、抗終止調(diào)節(jié)、反義調(diào)節(jié)及反向調(diào)節(jié)等。其機(jī)制之精巧，過(guò)程之復(fù)雜，形式之多樣堪為集原核生物調(diào)控之大成。,噬菌體是一種以大腸桿菌為宿主的溫和型噬菌體，它感染大腸桿菌后，通過(guò)精確的基因表達(dá)調(diào)控機(jī)制選擇裂解周期或溶原周期。 噬菌體長(zhǎng)48502nt，共61個(gè)基因，其中32個(gè)較為重要 .,一、Lamda phage 簡(jiǎn)介,的整個(gè)生命過(guò)程都離不開寄主的RNA聚合酶，因?yàn)槭删w沒有編碼RNA聚合酶的基因。 DNA兩條鏈都有遺傳密碼，有各自的轉(zhuǎn)錄單位向右轉(zhuǎn)錄的叫R鏈；向左轉(zhuǎn)錄的叫L鏈。,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的基因順序組織如圖所示，按基因組功能共分六大區(qū)域:頭部編碼區(qū)、尾部編碼區(qū)、重組區(qū)、控制區(qū)、復(fù)制區(qū)和裂解區(qū). 它們分屬四個(gè)操縱子結(jié)構(gòu)：阻遏蛋白操縱子、早期左向操縱子、早期右向操縱子以及晚期右向操縱子。,噬菌體的基因組結(jié)構(gòu),Bacter

4、iophage lambda genome.png,圖中OR1、 2 、3的順序反了?,When contemplating I suggest attention be payed to the following concepts:,時(shí)間,空間,量（濃度）, 或然性 temporal，spatial，amount (concentration)，random(to a certain extent),-DNA在宿主細(xì)胞環(huán)化后，由于DNA分子上沒有任何調(diào)控蛋白存在，因此宿主的RNA聚合酶便分別結(jié)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. 或許這種抗終止機(jī)制的使用是為了保證細(xì)胞中一開始就累積一

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 （包括自體調(diào)控）Cro 濃度較高時(shí)。,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(位置較靠后）。,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：產(chǎn)生阻遏蛋白CI。稍后進(jìn)一步介紹。,活化整合酶啟動(dòng)子PI：由PI起始的mRNA在int基因末端后面約300nt的終點(diǎn)子

11、處停止轉(zhuǎn)錄。由PI轉(zhuǎn)錄的mRNA較為穩(wěn)定。,雖然int基因也可以由啟動(dòng)子PL起始轉(zhuǎn)錄，但產(chǎn)生的int mRNA會(huì)被細(xì)胞中的核酸酶所降解，并不能產(chǎn)生整合酶。因?yàn)橛蓡?dòng)子PL起始轉(zhuǎn)錄， RNA聚合酶經(jīng)過(guò)N抗終止蛋白的修飾可以通過(guò)終止子tL2，這樣RNA得以延伸，形成了核酸酶的靶位點(diǎn)sib。它首先被RNase 剪切，接著核酸外切酶從3-5降解mRNA. sib是int基因下游的負(fù)調(diào)節(jié)位點(diǎn)，降解由下游向上游方向進(jìn)行，故稱其為反向調(diào)節(jié)（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 .維基也解說(shuō)為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個(gè)復(fù)制基因（裂解感染所需的），7個(gè)重組基因（有的和裂解感染中的重組有關(guān)，其中有2個(gè)是DNA整合到細(xì)菌染色體中所必需的）及3個(gè)調(diào)節(jié)基因（c，c，Q）。啟動(dòng)子PL和操作子OL部分重疊。,4. late lysogeny,CI操縱子 cI基因編碼的CI阻遏蛋白，分別作用于早期左向操縱子和早期右向操縱子中的操作子OL和OR，阻止這兩個(gè)操縱子轉(zhuǎn)錄.,CI 阻遏蛋白 CI 阻遏蛋白操縱子與噬菌體溶原狀態(tài)的確立與維持密切相關(guān)。cI基因突變的噬菌體在生長(zhǎng)著大腸桿菌的固體培養(yǎng)基上總是形成清晰的噬菌斑，該基因由此得名。,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.,阻遏蛋白操縱子含有一個(gè)結(jié)構(gòu)基因(cI基因)，但有兩個(gè)啟動(dòng)子: PRE (阻遏蛋白建立溶原狀態(tài))， PRM (阻遏蛋白維持溶原狀態(tài)),(The s

17、ubscript RE stands for repressor establishment, RM for repressor maintainment .),由PRE控制轉(zhuǎn)錄的mRNA分子中含有一個(gè)有效的核糖體結(jié)合位點(diǎn)，mRNA的翻譯活性比PRM介導(dǎo)的轉(zhuǎn)錄產(chǎn)物高7至8倍。 PRE缺少-35區(qū)保守序列，-10區(qū)序列也不保守.This promoter can be recognized by RNA polymerase only in the presence of cII. （CII蛋白在細(xì)胞內(nèi)很不穩(wěn)定，極易被宿主蛋白酶HflA降解而失去活性，而CIII蛋白的功能則是保護(hù)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介導(dǎo)的轉(zhuǎn)錄除了能直接表達(dá)cI基因外，還有一個(gè)間接功能，即同時(shí)轉(zhuǎn)錄出cro基因的反義RNA，與cro的mRNA互補(bǔ)雜交，從而阻斷其翻譯，而Cro是 噬菌體進(jìn)入溶菌狀態(tài)所必需的。,由PRM啟動(dòng)的cI基因轉(zhuǎn)錄以起始密碼子AUG開始，轉(zhuǎn)錄

20、產(chǎn)物上沒有核糖體結(jié)合位點(diǎn)，因此cI基因的表達(dá)量很低。 To transcribe cI, there must be repressor itself present. 當(dāng)-DNA分子進(jìn)入一個(gè)新的宿主細(xì)胞時(shí)，因?yàn)闆]有CI蛋白因子,宿主的RNA聚合酶并不能由啟動(dòng)子PRM轉(zhuǎn)錄cI 基因，,PRM,在晚早期基因產(chǎn)物CII的幫助下，cI基因得以表達(dá)。 cI阻遏蛋白分別與OL和OR兩個(gè)操作子結(jié)合。 CI通過(guò)阻止兩個(gè)早期操縱子的轉(zhuǎn)錄，既關(guān)閉了溶菌途徑，又通過(guò)PRM維持了自身在宿主細(xì)胞中的一定水平。,噬菌體溶原狀態(tài)的建立與維持,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.,小結(jié)：由PRE控制轉(zhuǎn)錄產(chǎn)生CI后，PL和PR被抑制，早期基因的表達(dá)將受阻，C和C缺乏，CI通過(guò)PRE的轉(zhuǎn)錄也停止。所以 cI基因轉(zhuǎn)錄的起始需要CII，轉(zhuǎn)錄的維持需要C

24、I本身。,如果此時(shí)第二個(gè)噬菌體感染這個(gè)溶原化的宿主細(xì)胞，由原噬菌體基因組合成的阻遏蛋白便會(huì)迅速與新入侵的噬菌體基因組上的OL和OR結(jié)合，防止該噬菌體啟動(dòng)溶菌途徑，這就是所謂的宿主細(xì)胞免疫現(xiàn)象。由OL, OR,cI以及cro基因組成的區(qū)域決定了噬菌體的免疫特性，因此也稱為免疫區(qū)，具有同一免疫區(qū)的不同噬菌體存在交叉免疫活性。,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.).,晚期右向操縱子最大，它包括10個(gè)頭部蛋白編碼基因、11個(gè)尾部蛋白編碼基因(AJ)以及3個(gè)負(fù)責(zé)裂解宿主細(xì)胞的溶菌酶類基因S、R和Rz，其所需啟動(dòng)子為PR,晚期右向操縱子,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. 溶源還是溶菌,CI repressor is needed for lysogeny. Cro repressor is needed for lytic infection.,Cro亦是一種負(fù)調(diào)節(jié)因子，作用位點(diǎn)也是OR和OL。Cro與操縱子的結(jié)合能關(guān)閉兩個(gè)早期操縱子，但同時(shí)也阻止了啟動(dòng)子PRM的轉(zhuǎn)錄啟動(dòng)，阻礙溶源建立。,cro mutants usually establish

29、lysogeny, because they lack the ability to switch events away from the expression of repressor.,Cro和CI是噬菌體的兩種調(diào)節(jié)蛋白因子，它們雖然都作用于OR和OL兩個(gè)操作子，但對(duì)每個(gè)操縱子中三個(gè)不同位點(diǎn)的親和力次序不同。CI的親和力次序是OLlOL2OL3，ORlOR2 OR3；Cro恰好相反，為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破壞溶源,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開始抑制早期基因時(shí)，早期基因的表達(dá)產(chǎn)物已經(jīng)多余了。）,Cro與溶菌,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，關(guān)閉早期左向操縱子，阻斷CII和C

34、III蛋白的持續(xù)合成。當(dāng)原先合成的CII和CIII蛋白被降解，導(dǎo)致：CII蛋白因子對(duì)PaQ激活作用解除，pQ大量合成。 pQ也是一種抗終止蛋白，促進(jìn)晚期操縱子在啟動(dòng)子PR 的作用下進(jìn)行轉(zhuǎn)錄，表達(dá)出足夠數(shù)量的頭部尾部包裝蛋白以及裂解宿主所必需的溶菌酶系，至此噬菌體進(jìn)入溶菌途徑。,噬菌體溶原狀態(tài)與溶菌狀態(tài)的選擇,噬菌體溶原狀態(tài)與溶菌狀態(tài)的確立均依賴于早期基因的有限度表達(dá)。 溶原狀態(tài)建立的起始事件是阻遏蛋白CI與OL1和ORl結(jié)合，進(jìn)而關(guān)閉cro基因的表達(dá)，并同時(shí)啟動(dòng)由PRM介導(dǎo)的CI合成； 進(jìn)入溶菌途徑的首要條件是Cro蛋白因子與OR3結(jié)合，打破由PRM介導(dǎo)的溶原維持，然后再作用于ORl或OR2

35、以及OLl或OL2，關(guān)閉兩個(gè)早期操縱子，通過(guò)阻斷CII和CIII的合成而終止由PRE介導(dǎo)的CI合成。 因此，決定噬菌體建立溶原狀態(tài)或進(jìn)入溶菌途徑的開關(guān)是CI或Cro對(duì)操作子OL和OR有利位置的占領(lǐng)。,Cro的合成早于CI。然而，CI對(duì)OL和OR的親和力比Cro蛋白高約8倍，如果CI和Cro兩種蛋白同時(shí)存在，則Cro必須以分子數(shù)的優(yōu)勢(shì)才能贏得競(jìng)爭(zhēng)。 如果CII蛋白有足夠的活性，則PRE啟動(dòng)cI基因轉(zhuǎn)錄，合成的CI分子使得PRM更有效地表達(dá)cI基因,進(jìn)入溶源；反之，Cro蛋白便依其數(shù)量?jī)?yōu)勢(shì)占據(jù)OL和OR ，噬菌體進(jìn)入溶菌途徑。 可見，任何有利于CII基因表達(dá)并使其產(chǎn)物穩(wěn)定的因素均可導(dǎo)致噬菌體建立和

36、維持溶原狀態(tài)，這些因素包括營(yíng)養(yǎng)枯竭以及感染復(fù)數(shù)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.,當(dāng)宿主陷入能源枯竭的境地時(shí)，細(xì)胞內(nèi)相應(yīng)發(fā)生一系列的生理變化，其中主要的是cAMP的大量合成，cAMP

37、-CAP復(fù)合物進(jìn)而抑制宿主細(xì)胞宿主蛋白水解酶基因hfl的表達(dá)。hflA和hflB基因又稱高頻溶原基因，CII蛋白的不穩(wěn)定性就是這個(gè)酶系作用的結(jié)果。 因此，能源枯竭的環(huán)境條件有助于噬菌體CII蛋白活性的維持，從而保證CI阻遏蛋白的合成，使得噬菌體建立溶原狀態(tài)，事實(shí)上由于能源不足，噬菌體也不可能通過(guò)溶菌途徑而大量繁殖。上述過(guò)程實(shí)質(zhì)上是噬菌體把握環(huán)境條件對(duì)其自身生長(zhǎng)繁殖的一種調(diào)節(jié)機(jī)制。,感染復(fù)數(shù)指的是在感染時(shí)噬菌體數(shù)與細(xì)菌細(xì)胞數(shù)的比值，它對(duì)溶原狀態(tài)建立的影響也是通過(guò)CII蛋白而起作用的。感染復(fù)數(shù)與CII蛋白的活性形成有關(guān)，CII蛋白的單體沒有活性，只有寡聚體才有作用。當(dāng)感染復(fù)數(shù)過(guò)低時(shí)，多個(gè)噬菌體同時(shí)感染一個(gè)宿主細(xì)胞的機(jī)會(huì)也就減少，所表達(dá)的CII產(chǎn)物不足以形成有活性的寡聚體，此時(shí)噬菌體便進(jìn)入溶菌途徑；當(dāng)感染復(fù)數(shù)過(guò)高時(shí)(通常大于10)，CII能形成足夠數(shù)量的活性寡聚體，就可促進(jìn)由PRE介導(dǎo)的cI基因轉(zhuǎn)錄啟動(dòng)。該轉(zhuǎn)錄產(chǎn)物的翻譯活性遠(yuǎn)高于從PRM轉(zhuǎn)錄出的mRNA，因此CI蛋白數(shù)量很快占據(jù)優(yōu)勢(shì)，噬菌體建立起溶原狀態(tài)。,UV可激活Rec蛋白，活化的Rec蛋白可剪切C