Molecular biology - Chapter 8: Major shifts in bacterial transcription

Molecular BiologyFourth EditionChapter 8Major Shifts in Bacterial TranscriptionLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.18.1 Sigma Factor SwitchingPhage infection of bacterium subverts host transcription machinery In process, establishes a time-dependent, or temporal, 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 itself2Phage InfectionChapter 6 established that s is the key factor in determining specificity of T4 DNA transcriptionTo shift the transcription process s is a likely candidateStudy of the process done in B. subtilis and its phage, SPO13Temporal Control of TranscriptionLike T4, SPO1 has a large genomeTemporal transcription program:First 5 minutes: expression of early genesDuring 5 – 10 minutes: expression of middle genesAfter 10 minutes to end: late genes expressed4Transcription SwitchingThis switching is directed by a set of phage-encoded s factors that associate with the host core RNA polymeraseThese s factors change the host polymerase specificity of promoter recognition from early to middle to lateThe host s factor is specific for the phage early genesPhage gp28 protein switches the specificity to the middle genesPhage gp33 and gp34 proteins switch to late specificity5SporulationDuring 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 endospores, tough dormant bodies6Sporulation SwitchingDuring 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-factor from the polymerase coreEach s-factor has its own preferred promoter sequence7Genes With Multiple PromotersSome 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 different 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 conditions8Bacterial Heat ShockThe 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 shock genes9Other s-SwitchesHeat shock response is governed by an alternative s-factor, s32 or sHDirects RNA polymerase to the heat shock gene promotersAccumulation of sH with high temperature is due to: Stabilization of sHEnhanced translation of the mRNA encoding sHResponses to low nitrogen and starvation stress also depend on genes recognized by other s-factors108.2 The RNA Polymerase Encoded in Phage T7Phage 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 mutated, only class 1 genes are transcribedGene 1 codes for a phage-specific RNA polymerase of just one polypeptide11Gene 1 RNA PolymeraseGene 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 template12Temporal Control of TranscriptionHost polymerase transcribes the class I genesOne of class I genes is the phage polymeraseThe phage polymerase then transcribes the class II and III genes138.3 Infection of E. coli by Phage lVirulent phage replicate and kill their host by lysing or breaking it openTemperate phage, such as l, infect cells but don’t necessarily killThe temperate phage have 2 paths of reproductionLytic mode: infection progresses as in a virulent phageLysogenic mode: phage DNA is integrated into the host genome14Lysogenic ModeA 27-kD phage protein (l repressor, CI) appears and binds to 2 phage operator regionsCI shuts down transcription of all genes except for cI, gene for l repressor itselfLysogen is a bacterium harboring integrated phage DNAThis integrated DNA is called a prophage15Two Paths of Phage Reproduction16Lytic Reproduction of Phage lLytic reproduction cycle of phage l has 3 phases of transcription:Immediate earlyDelayed earlyLate Genes of these phases are arranged sequentially on the phage DNA 17Genetic Map of Phage lDNA exists in linear form in the phageAfter infection of host begins the phage DNA circularizesThis is possible as the linear form has sticky ends18AntiterminationAntitermination is a type of transcriptional switchA gene product serves as antiterminator that permits RNA polymerase to ignore 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 termination19Antitermination and TranscriptionOne of 2 immediate early genes is crocro codes for a repressor of cI gene that allows lytic cycle to continueOther immediate early gene is N coding for N, an antiterminator20N Antitermination FunctionGenetic 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 genes21Proteins Involved in N-Directed AntiterminationFive 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 the transcript to the polymeraseNusA stimulates termination at intrinsic terminator by interfering with binding binding between upstream part of terminator hairpin and core polymerase22Protein Complexes Involved in N-Directed Antitermination23Model for the Function of NusA and N in Intrinsic Termination24Antitermination and QAntitermination in the l late region requires QQ binds to the Q-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 genes25Establishing LysogenyPhage establish lysogeny by: Causing production of repressor to bind to early operatorsPreventing further early RNA synthesisDelayed 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 PRE26Model of Establishing LysogenyDelayed early transcription from PR produces cII mRNA translated to CIICII allows RNA polymerase to bind to PRE and transcribe the cI gene, resulting in repressor27Autoregulation of the cI Gene During LysogenyAs l repressor appears, binds as a dimer to l operators, OR and OL results in:Repressor turns off further early transcriptionInterrupts lytic cycleTurnoff of cro very important as product Cro acts to counter repressor activityStimulates own synthesis by activating PRM28Maintaining Lysogeny29Repressor ProteinRepressor 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 possible30Model of Involvement of OL in Repression of PR and PRM31Involvement of OL in RepressionRepressor 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 work 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 looping32RNA Polymerase/Repressor InteractionIntergenic 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 helping to compensate for weak promoterOR2 serves as an activator siteRepressor l is an activator of transcription from PRM33Principle of Intergenic SuppressionDirect interaction 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 another34Selection for Intergenic Suppressor35Activation Via SigmaPromoters 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 436Determining the Fate of a l InfectionBalance between lysis or lysogeny is delicatePlace phage particles on bacterial lawnIf lytic infection occursProgeny spread and infect other cellsCircular hole seen in 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 lysogenized37Battle Between cI and croThe 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 transcriptionThis leads to lytic infectionGene product in high concentration first determines cell fate 38Lysogen InductionWhen lysogen suffers DNA damage, SOS response is inducedInitial event is seen in a coprotease activity in RecA proteinRepressors are caused to cut in half, removing them from l operatorsLytic cycle is inducedProgeny phage can escape potentially lethal damage occurring in host39