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1、8.1 Sigma Factor Switching8.2 The RNA Polymerase Encoded in Phage T7 8.3 Infection of E.coli by Phage(antitermination),Chapter 8 Major Shifts in Bacterial Transcription,8.1 Sigma Factor Switching,Phage infection of bacterium subverts host transcription machinery In process,establishes a time-depende
2、nt program of transcriptionFirst early phage genes are transcribedThis is followed by the later genesLate in the infectious cycle there is no longer transcription of the host genes,only phage genesChange in what genes transcribed is caused by a change in transcription machinery,in RNA polymerase its
3、elf,Chapter 6 established that is the key factor in determining specificity of T4 DNA transcriptionTo shift the transcription process is a likely candidateStudy of the process done in B.subtilis and its phage,SPO1,Phage Infection,Temporal Control of Transcription,Like T4,SPO1 has a large genomeTempo
4、ral transcription program:First 5 minutes:expression of early genesDuring 5 10 minutes:expression of middle genesAfter 10 minutes to end:late genes expressed,Polymerases were separated by chromatography and subjected to SDS-PAGE to display their subunits.Enzyme B(first lane)contains the core subunit
5、s(,and),as well as subunit IV(gp28).Enzyme C(second lane)contains the core subunits plus subunits V(gp34)and VI(gp33).The last two lanes contain separated and subunits,respectively.,gp28,gp34,gp33,A,This switching is directed by a set of phage-encoded factors that associate with the host core RNA po
6、lymeraseThese factors change the host polymerase specificity of promoter recognition from early to middle to lateThe host factor is specific for the phage early genesPhage gp28 protein switches the specificity to the middle genesPhage gp33 and gp34 proteins switch to late specificity,Transcription S
7、witching,Sporulation,During infection,phage SPO1 changes specificity of host RNA polymeraseSame type of mechanism applies to changes in gene expression during sporulationBacteria can exist indefinitely in vegetative state if nutrients are availableUnder starvation conditions,B.subtilis forms endospo
8、res,tough dormant bodies,Two types of B.subtilis cells.(a)vegetative cells and(b)a sporulating cell,with an endospore developing at the left end.,Bacteria can exist indefinitely in vegetative state if nutrients are availableUnder starvation conditions,B.subtilis forms endospores,tough dormant bodies
9、,Sporulation involves the differentiation of a vegetative bacterium into a mother cell that is lysed and a spore that is released.8 hrs,40%RNA,spoOA,H,43,PO4,F triggers synthesis of the next sigma factor in the forespore and turns on SpoIIR which causes SpoIIGA to cleave pro-E.,The crisscross regula
10、tion of sporulation coordinates timing of events in the mother cell and forespore.,Sporulation Switching,During sporulation,a whole new set of genes is turned on,and vegetative genes are turned offSwitch occurs largely at the level of transcriptionSeveral new s-factors displace the vegetative s-fact
11、or from the polymerase coreEach s-factor has its own preferred promoter sequence,Genes with Multiple Promoters,Some sporulation genes must be expressed during 2 or more phases of sporulation when different s-factors predominateGenes transcribed under different conditions are equipped with two differ
12、ent promotersEach promoter is recognized by one of two different s-factorsThis ensures their expression no matter which factor is presentAllows for differential control under different conditions,The B.subtilis spoVG Gene One of the sporulation genes with two promoters is spoVG,which is transcribed
13、by both EB and EE(holoenzymes bearing either B or E).,The heat shock response is a defense by cells to minimize damageMolecular chaperones are proteins:Bind proteins partially unfolded by heatingHelp these proteins refold properly Genes encoding proteins that help cells survive heat are called heat
14、shock genes,Bacterial Heat Shock,Other s Switches,Result of heat shock on transcription in E.coli.Top:At normal temperature,H(green)is somehow sequestered so it cannot bind to the core polymerase.As a result,the normal A(blue)associates with core,directing it to normal vegetative promoters(pA).As a
15、result,vegetative transcripts(blue)are produced.Bottom:On heating,the isolated H is released.It competes with A for binding to core and directs the polymerase to heat shock promoters pH.Consequently,heat shock transcripts(green)are made.,Map of the DNA region used to assay the transcription initiati
16、on from the E.coli glnA gene.The bold red region contains the glnA promoters glnA P1 and glnA P2.Downstream from these promoters,in the blue region of the DNA,lies the T7 phage terminator(tT7).Transcripts initiating at glnA P1 or glnA P2 and terminating at tT7 produce truncated transcripts of 415 an
17、d 300 nt,respectively.,The E.coli glnA Gene,70,54,-NH4+strong-carbon weak,Phage like T7,T3,and 11 have small genomes and many fewer genesThese phage have 3 phases of transcription:classes I,II,and IIIOf 5 class I genes,gene 1 is necessary for class II and class III gene expressionIf gene 1 is mutate
18、d,only class 1 genes are transcribedGene 1 codes for a phage-specific RNA polymerase of just one polypeptide,8.2 The RNA Polymerase Encoded in Phage T7,Host polymerase transcribes the class I genesOne of class I genes is the phage polymeraseThe phage polymerase then transcribes the class II and III
19、genes,Temporal Control of Transcription,Gene 1 RNA polymerase transcribes only T7 class II and III genes,not class I genesRNA polymerase of phage T7 is unusually specificThis polymerase will transcribe virtually no other natural templateA similar polymerase has been isolated from phage T3.It is spec
20、ific for T3,rather than T7 genes.,Gene 1 RNA Polymerase,8.3 Infection of E.coli by Phage l,Virulent phage replicate and kill their host by lysing or breaking it openTemperate phage,such as l,infect cells but dont necessarily killThe temperate phage have 2 paths of reproductionLytic mode:infection pr
21、ogresses as in a virulent phageLysogenic mode:phage DNA is integrated into the host genome,repressor cI gene,lysogen,prophage,Lysogenic Phase,Cro,Two Paths of Phage Reproduction,Lytic Reproduction of Phage l,Lytic reproduction cycle of phage l has 3 phases of transcription:Immediate earlyDelayed ear
22、lyLate Genes of these phases are arranged sequentially on the phage DNA,12 nt,Genetic Map of Phage l,DNA exists in linear form in the phageAfter infection of host begins the phage DNA circularizesThis is possible as the linear form has sticky ends,194,One of 2 immediate early genes is crocro codes f
23、or a repressor of cI gene that allows lytic cycle to continueOther immediate early gene is N coding for N,an antiterminator,Antitermination and Transcription,Antitermination,Antitermination is a type of transcriptional switchA gene product serves as antiterminator that permits RNA polymerase to igno
24、re terminators at the end of the immediate early genesSame promoters are used for both immediate early and delayed early transcriptionLate genes are transcribed when another antiterminator permits transcription of the late genes from the late promoter to continue without premature termination,Geneti
25、c sites surrounding the N gene include:Left promoter,PLOperator,OLTranscription terminatorWhen N is present:N binds transcript of N utilization site(nut site)Interacts with protein complex bound to polymerasePolymerase ignores normal transcription terminator,continues into delayed early genes,N Anti
26、termination Function,Effect of N on leftward transcription,Host proteins,Five proteins collaborate in antitermination at the l immediate early terminatorsNusA and S10 bind RNA polymeraseN and NusB bind to the box B and box A regions of the nut site N and NusB bind to NusA and S10 probably tethering
27、the transcript to the polymeraseNusA stimulates termination at intrinsic terminator by interfering with binding between upstream part of terminator hairpin and core polymerase,Proteins Involved in N-Directed Antitermination,Protein complexes involved in N-directed antitermination,Weak,nonprocessive
28、complex,Strong,processive complex,?,?,?,In vitro,In vitro,(in vitro),Model for the Function of NusA and N in Intrinsic Termination,Demonstration of protein-RNA contacts in the paused EC,with and without NusA and N,Antitermination and Q,Antitermination in the l late region requires QQ binds to the Q-
29、binding region of the qut site as RNA polymerase is stalled just downstream of late promoterBinding of Q to the polymerase appears to alter the enzyme so it can ignore the terminator and transcribe the late genes,Map of the PR region of the genome.The PR promoter comprises the 10 and 35 boxes.The qu
30、t site overlaps the promoter and includes the Q binding site upstream of the 10 box,the pause signal downstream of the transcription start site,and the pause site at positions+16 and+17.,A 27-kD phage protein(l repressor,CI)appears and binds to 2 phage operator regionsCI shuts down transcription of
31、all genes except for cI,gene for l repressor itselfLysogen is a bacterium harboring integrated phage DNAThis integrated DNA is called a prophage,Lysogenic Mode,Establishing Lysogeny,Phage establish lysogeny by:Causing production of repressor to bind to early operatorsPreventing further early RNA syn
32、thesisDelayed early gene products are usedIntegration into the host genomeProducts of cII and cIII allow transcription of the cI gene and production of l repressorPromoter to establish lysogeny is PRE,Model of Establishing Lysogeny,Delayed early transcription from PR produces cII mRNA translated to
33、CIICII allows RNA polymerase to bind to PRE and transcribe the cI gene,resulting in repressor,Phage establishes lysogeny by causing production of enough repressor to bind to the early operators and prevent further early RNA synthesis.The promoter used for establishment of lysogeny is PRE,which lies
34、to the right of PR and cro.Transcription from this promoter goes leftward through the cI gene.The products of the delayed early genes cII and cIII also participate in this process:CII,by directly stimulating polymerase binding to PRE and PI;CIII,by slowing degradation of CII.,SUMMARY,Autoregulation
35、of the cI Gene during Lysogeny,As l repressor appears,binds as a dimer to l operators,OR and OL results in:Repressor turns off further early transcriptionInterrupts lytic cycleTurn off of cro very important as product Cro acts to counter（对抗）repressor activityStimulates own synthesis by activating PR
36、M,Maintaining Lysogeny,PRE,Three of the preceding assertions:1)the repressor at low concentration can stimulate transcription of its own gene 2)the repressor at high concentration can inhibit transcription of its own gene 3)the repressor can inhibit cro transcription,Map of the DNA fragment used to
37、assay transcription from cI and cro promoters.The red arrows denote the in vitro cI and cro transcripts,some of which terminate prematurely,110 nt,300 nt,The repressor clearly inhibited cro transcription,but it greatly stimulated cI transcription at low concentration,then inhibited cI transcription
38、at high concentration.,Repressor protein A dimer of 2 identical subunitsEach subunit has 2 domains with distinct rolesAmino-terminal is the DNA-binding end of moleculeCarboxyl-terminal is site of repressor-repressor interaction that makes dimerization and cooperative binding possible,Repressor Prote
39、in,Model of Involvement of OL in Repression（阻遏）of PR and PRM,Involvement of OL in Repression,Repressor binds to OR1 and OR2 cooperatively,but leaves OR3RNA polymerase to PRM which overlaps OR3 in such a way it contacts repressor bound to OR2Protein-protein interaction is required for promoter to wor
40、k efficientlyHigh levels of repressor can repress transcription from PRMProcess may involve interaction of repressor dimers bound to OR1,OR2,and OR3Repressor dimers bound to OL1,OL2,and OL3 via DNA looping,RNA Polymerase/Repressor Interaction,?,Principle of Intergenic Suppression,Direct interaction
41、between repressor and polymerase is necessary for efficient transcription from PRMMutant with compensating amino acid change in RNA polymerase subunit restores interaction with mutant repressorIn intergenic suppression,a mutant in one gene suppresses a mutation in another,Selection for intergenic su
42、ppressor of cI pc mutation,(Arg596His),wild-type mutant versions-irrelevant mutation,Activation Via Sigma,Promoters subject to polymerase-repressor activation have weak-35 boxesThese boxes are poorly recognized by sActivator site overlaps-35 box,places activator in position to interact with region 4
43、,Intergenic suppressor mutation studies show that crucial interaction between repressor and RNA polymerase involves region 4 of the s-subunit of the polymerasePolypeptide binds near the weak-35 box of PRM placing the s-region 4 close to the repressor bound to OR2Repressor can interact with s-factor
44、helping to compensate for weak promoterOR2 serves as an activator siteRepressor Cl is an activator of transcription from PRM,SUMMARY,Balance between lysis or lysogeny is delicatePlace phage particles on bacterial lawnIf lytic infection occursProgeny spread and infect other cellsCircular hole seen in
45、 lawn is called plaqueInfection 100%lytic gives clear plaquePlaques of l are usually turbid meaning live lysogen is presentSome infected cells suffer the lytic cycle,others are lysogenized,Determining the Fate of a l Infection,What determines whether a given cell infected by will enter the lytic cyc
46、le or lysogeny?The balance between these two fates is delicate,and we usually cannot predict the actual path taken in a given cell.The cells within a plaque are all genetically identical,and so are the phages,so the choice of fate is not genetic.Instead,it seems to represent a race between the produ
47、cts of two genes:cI and cro.,Battle Between cI and cro,The cI gene codes for repressor,blocks OR1,OR2,OL1,and OL2 so turning off early transcriptionThis leads to lysogenyThe cro gene codes for Cro that blocks OR3 and OL3,turning off cI transcriptionThis leads to lytic infectionGene product in high c
48、oncentration first determines cell fate,What determines whether cI or cro wins the race?The most important factor seems to be the concentration of the cII gene product,CII.It activates PRE and helps turn on the lysogeny program.What controls the concentration of CII?CIII protects CII against cellula
49、r proteases,but high protease concentrations can overwhelm CIII,destroy CII,and ensure that the infection will be lytic.Such high protease concentrations occur under good environmental conditions-rich medium,for example.,When lysogen suffers DNA damage,SOS response is inducedInitial event is seen in
50、 a coprotease activity in RecA proteinRepressors are caused to cut in half,removing them from operatorsLytic cycle is inducedProgeny phage can escape potentially lethal damage occurring in host,Lysogen Induction,When a lysogen suffers DNA damage,it induces the SOS response.The initial event in this
