分子生物學(xué)MajorShiftsinBacterialTranscription

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 Chapter 8 Major Shifts in Bacterial Transcription Major Shifts in Bacterial Transcription 8.1 Sigma Factor Switching Phage infection of bacterium subverts host transcript

2、ion machinery In process, establishes a time-dependent program of transcription First early phage genes are transcribed This is followed by the later genes Late in the infectious cycle there is no longer transcription of the host genes, only phage genes Change in what genes transcribed is caused by

3、a change in transcription machinery, in RNA polymerase itselfPhage InfectionTemporal Control of TranscriptionnLike T4, SPO1 has a large genomenTemporal transcription program: First 5 minutes: expression of early genes During 5 10 minutes: expression of middle genes After 10 minutes to end: late gene

4、s expressedPolymerases were separated by chromatography and subjected to SDS-PAGE to display their subunits. Enzyme B (first lane) contains the core subunits (, , 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

5、 lanes contain separated and subunits, respectively. gp28gp34gp33ASporulationnDuring infection, phage SPO1 changes specificity of host RNA polymerasenSame type of mechanism applies to changes in gene expression during sporulationnBacteria can exist indefinitely in vegetative state if nutrients are a

6、vailablenUnder starvation conditions, B. subtilis forms endospores, tough dormant bodiesTwo types of B. subtilis cells. (a) vegetative cells and (b) a sporulating cell, with an endospore developing at the left end. Sporulation involves the differentiation of a vegetative bacterium into a mother cell

7、 that is lysed and a spore that is released. 8 hrs, 40% RNAspoOAH43PO4F triggers synthesis of the next sigma factor in the forespore and turns on SpoIIR which causes SpoIIGA to cleave pro-E.The crisscross regulation of sporulation coordinates timing of events in the mother cell and forespore.Sporula

8、tion SwitchingnDuring sporulation, a whole new set of genes is turned on, and vegetative genes are turned offnSwitch occurs largely at the level of transcriptionnSeveral new s-factors displace the vegetative s-factor from the polymerase corenEach s-factor has its own preferred promoter sequence Gene

9、s with Multiple Promoters The B. subtilis spoVG Gene One of the sporulation genes with two promoters is spoVG, which is transcribed by both EB and EE (holoenzymes bearing either B or E). nThe heat shock response is a defense by cells to minimize damagenMolecular chaperones are proteins: Bind protein

10、s partially unfolded by heatingHelp these proteins refold properlyn Genes encoding proteins that help cells survive heat are called heat shock genes Bacterial Heat ShockOther s SwitchesResult of heat shock on transcription in E. coli. Top: At normal temperature, H (green) is somehow sequestered so i

11、t 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 result, vegetative transcripts (blue) are produced. Bottom: On heating, the isolated H is released. It competes with A for binding to core and directs t

12、he polymerase to heat shock promoters pH. Consequently, heat shock transcripts (green) are made.Map of the DNA region used to assay the transcription initiation 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 bl

13、ue 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 and 300 nt, respectively. The E. coli glnA Gene7054- NH4+ strong- carbon weak 8.2 The RNA Polymerase Encoded in Phage T7Temporal Control

14、of TranscriptionnA similar polymerase has been isolated from phage T3. It is specific for T3, rather than T7 genes.Gene 1 RNA Polymerase8.3 Infection of E. coli by Phage l lnVirulent phage replicate and kill their host by lysing or breaking it opennTemperate phage, such as l, infect cells but dont n

15、ecessarily killnThe temperate phage have 2 paths of reproductionLytic mode: infection progresses as in a virulent phageLysogenic mode: phage DNA is integrated into the host genome repressorrepressor cI cI gene lysogenlysogen prophageprophage Lysogenic PhaseCroTwo Paths of Phage ReproductionLytic Rep

16、roduction of Phage l l12 ntGenetic Map of Phage l lnDNA exists in linear form in the phagenAfter infection of host begins the phage DNA circularizesnThis is possible as the linear form has sticky ends194Antitermination and TranscriptionN Antitermination FunctionEffect of N on leftward transcriptionH

17、ost proteinsProteins Involved in N-Directed AntiterminationProtein complexes involved in N-directed antiterminationWeak, nonprocessive complexStrong, processive complex?In vitro In vitro (in vitro)Model for the Function of NusA and N in Intrinsic TerminationDemonstration of protein-RNA contacts in t

18、he paused EC, with and without NusA and NAntitermination and Q Antitermination in the l l late region requires Q Q binds to the Q-binding region of the qut site as RNA polymerase is stalled just downstream of late promoter Binding of Q to the polymerase appears to alter the enzyme so it can ignore t

19、he terminator and transcribe the late genesMap of the PR region of the genome. The PR promoter comprises the 10 and 35 boxes. The qut 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

20、positions +16 and +17. Lysogenic Mode Establishing LysogenyModel 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 repressorPhage establishes lysogeny by causing production

21、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 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 a

22、nd cIII also participate in this process: CII, by directly stimulating polymerase binding to PRE and PI; CIII, by slowing degradation of CII.SUMMARYAutoregulation of the cI Gene during Lysogeny PREThree of the preceding assertions: 1) the repressor at low concentration can stimulate transcription of

23、 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 assay transcription from cI and cro promoters. The red arrows denote the in vitro cI and cro transcripts, some of which termin

24、ate prematurely 110 nt300 ntThe repressor clearly inhibited cro transcription, but it greatly stimulated cI transcription at low concentration, then inhibited cI transcription at high concentration. Repressor protein A dimer of 2 identical subunits Each subunit has 2 domains with distinct roles Amin

25、o-terminal is the DNA-binding end of molecule Carboxyl-terminal is site of repressor-repressor interaction that makes dimerization and cooperative binding possibleModel of Involvement of OL in Repression（阻遏） of PR and PRMInvolvement of OL in Repression Repressor binds to OR1 and OR2 cooperatively, b

26、ut leaves OR3 RNA polymerase to PRM which overlaps OR3 in such a way it contacts repressor bound to OR2 Protein-protein interaction is required for promoter to work efficiently High levels of repressor can repress transcription from PRM Process may involve interaction of repressor dimers bound to OR

27、1, OR2, and OR3 Repressor dimers bound to OL1, OL2, and OL3 via DNA looping?Principle of Intergenic SuppressionnDirect interaction between repressor and polymerase is necessary for efficient transcription from PRMnMutant with compensating amino acid change in RNA polymerase subunit restores interact

28、ion with mutant repressornIn intergenic suppression, a mutant in one gene suppresses a mutation in anotherSelection for intergenic suppressor of cI pc mutation(Arg596His)wild-type mutant versions- irrelevant mutationActivation Via SigmaSUMMARY Balance between lysis or lysogeny is delicate Place phag

29、e 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 present Some infected cells suffer the lytic cycle, others are lysogeni

30、zedDetermining the Fate of a l l Infection What determines whether a given cell infected by will enter the lytic cycle 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 iden

31、tical, and so are the phages, so the choice of fate is not genetic. Instead, it seems to represent a race between the products of two genes: cI and cro. Battle Between cI and croWhat determines whether cI or cro wins the race ?The most important factor seems to be the concentration of the cII gene p

32、roduct, CII. It activates PRE and helps turn on the lysogeny program.What controls the concentration of CII ?CIII protects CII against cellular proteases, but high protease concentrations can overwhelm CIII, destroy CII, and ensure that the infection will be lytic.Such high protease concentrations o

33、ccur under good environmental conditions-rich medium, for example.Lysogen InductionWhen a lysogen suffers DNA damage, it induces the SOS response. The initial event in this response is the appearance of a coprotease activity in the RecA protein. This causes the repressors to cut themselves in half,

34、removing them from the operators and inducing the lytic cycle. In this way, progeny phages can escape the potentially lethal damage that is occurring in their host. SUMMARYMajor Shifts in Prokaryotic Transcription8.1 Sigma Factor Switching8.2 The RNA Polymerase Encoded in Phage T7 8.3 Infection of E

35、. coli by Phage (antitermination)Questions：13You are studying a gene that you suspect have two different promoters, recognized by two different sigma-factors. How would you prove your hypothesis? Why is it an advantage for a gene to have two different promoters?14The immediate early/delayed early/late transcriptional switching in the lytic cycle of phage lambda is controlled by