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UIUC MCB 250 - 28-lambda 2

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Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Propagation of bacteriophage X ,Part IIBacteriophage life cyclesThis is a 2-way genetic switch - i.e. during infection, each phage must commit to either lysogeny or lysis.Lysogenic CycleLysogenic CycleLytic CycleLytic CycleWatson, Fig. 18-20Last Lecture•After infection, w phage adopts one of two alternative patterns of gene expression: one leads to lysis; the other to lysogeny.•The critical control gene for the lytic pathway is cro:-Expressed by a strong promoter, cro is activated immediately following infection.-If cro expression continues unabated, lysis will ensue.•The critical control gene for the lysogenic pathway is cI:-Transcription of cI is initially activated through the weak PRE promoter with the help of the cII transcription factor.-Once expressed, the cI protein can repress cro and simultaneously activate its own expression via PRM promoter.-If cI expression continues unabated, lysogeny will ensue.•The choice between lysis and lysogeny is determined by the physiological state of the cell. Lysis is favored in rapidly growing cells; lysogeny is favored in slowly growing cells.LYSOGENYFour phases:•Commitment•Site-specific recombination of phage DNA into the bacterial chromosome•Maintenance of the lysogenic prophage during bacterial growth and fission•Exit from lysogeny (called “induction”), and return to the lytic cycleWatson, Fig. 12-2During lysogeny, insertion of the phage DNA into the bacterial chromosome is accomplished by a specific enzyme, w integrase, that is encoded by the phage genome.INTEGRASENote how this differs from homologous recombination, which can occur ANYWHERE in regions of DNA that share >50 bp of sequence similarity.Integrase unites the phage and bacterial chromosomes by a mechanism known as conservative site-specific recombination.Watson, Fig. 12-3First, the enzyme physically aligns short recognition sequences on the two chro-mosomes. The enzyme then recombines the two DNA molecules at a specific position within the intervening crossover region:•double-stranded breaks in the DNA produce blunt ends;•the enzyme religates the blunt ends in an altered arrangement, e.g. red-to-blue in the example shown.recognition sequencesOnce the phage undergoes integration, it is replicated and passed along with the host cell's DNA. Hence, during lysogeny the survival and propagation of the phage genome depends on the survival and propagation of the bacterium.Maintenance of lysogenyIf the host bacterium is in danger, so is the phage. To augment its survival, w phage has evolved an escape mechanism:•When it experiences DNA damage, the bacterium initiates its own disaster plan called the SOS response. (DNA damage can result from UV radiation, chemical mutagens, or genetic attack by transposons.)•One of the earliest events in SOS is biochemical activation of the RecA protein to help repair double-stranded DNA breaks.•The w phage has evolved such that its repressor protein cI undergoes autocleavage (i.e. destroys itself) when it comes into contact with activated RecA. •Thus, in bacteria undergoing an SOS response the cI protein becomes unstable and its concentration rapidly drops.When the prophage is in maintenance phase:• there is abundant cI protein;• the cI gene is actively transcribed (positive autoregulation);• the cro gene is repressed.<<<<<<<<<<>>>>>>>>>>>>>>>>>>>>>>OFFON= cI proteinPRMPRWatson, Fig. 18-27<<<<<<<<<<<<<<<<<<>>>>>>>>>>>>>>>>>>>>>OFFPRPRMOFFDuring the SOS response:•cI protein is degraded.•without cooperative binding to cI protein, RNA polymerase can only associate weakly with the PRM promoter.•transcription of the cI gene decreases dramatically.degradation of CI protein<<<<<<<<<<<<<<<<<<>>>>>>>>>>>>>>>OFFONWatson, Fig. 18-27Exit from lysogeny involves a resumption of the lytic cycle:•In the absence of the cI repressor protein, the PR promoter is unblocked, and transcription of the cro gene resumes.•Integrase reverses its prior function, excising the prophage from the bacterial chromosome.•The lytic cycle ensues.Maintenance of lysogenyExit from lysogenyUV radiationLytic cycleDNA excisedWatson, Fig. 18-21Genome map: bacteriophageUltimately the decision between lysis and lysogeny is determined by the transcriptional activity at 4 promoters in this 5 kb control region.Circularization siteExcess CI proteinInsufficient CI proteinProphage may exit from lysogeny spontaneously even though the host cell is healthy. Prophage will be slow to exit from lysogeny, and may be destroyed if the host cell dies rapidly.During maintenance phase, the prophage synthesizes a concen-tration of cI protein that is optimizes its chance of survival.To ensure the optimal level of protein expression, the CI protein autoregulates its own transcription.Excess CI proteinInsufficient CI proteinProphage may exit from lysogeny spontaneously even though the host cell is happily growing. Prophage will be slow to exit from lysogeny, and will be destroyed if the host cell dies rapidly.Optimal CI conc. Negative autoregulation Positive autoregulationAffinity forCI proteinLow (1X) Low (1X) High (10X)XBlocks crotranscriptionEven if the concentration of w repressor (cI) protein is low, it will bind at the high affinity OR1 operator …Affinity forCI proteinLow (1X) Low (1X) High (10X)cI dimers at OR1 and OR2 can bind to one another to form a tetramerXBlocks crotranscriptionEven if the concentration of w repressor (cI) protein is low, it will bind at the high affinity OR1 operator …… which will in turn recruit another cI dimer at OR2<<<<<<<<<<>>>>>>>>>>>>>>>>>>>>>>OFFON= cI proteinPRMPRWatson, Fig. 18-27Even if the concentration of w repressor (cI) protein is low, it will bind at the high affinity OR1 operator …… which will in turn recruit another cI dimer at OR2, followed by the recruit of RNAP to the PRM promoter.If the cell is synthesizing cI protein at a rate greater than it is being turned over, the concentration of cI will rise.Affinity forCI proteinLow (1X)


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UIUC MCB 250 - 28-lambda 2

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