week_8_lecture_2 - Week 8 Lecture 2 1st Hour phage genetic...

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Unformatted text preview: Week 8 Lecture 2 1st Hour: phage genetic switches, T7/T4 lytic cycles, lambda phage, lysogeny Break 2nd Hour: lambda phage lysogeny (continued), repressor-operator interactions, enhancers RECAP RECAP Lac OPERON: •Negative Control: Repressor LacI binds to the operator as tetramer, keeps RNAP Repressor from transcribing the three lac genes (Z, Y, A) lac When no glucose, lactose available, allolactose acts as an inducer When by binding to LacI, causing a conformational shift that leads to dissociatation of LacI from the operator transcription proceeds • Positive Control: Mediated by a factor called catabolite activator protein (CAP), which, Mediated with cyclic AMP (cAMP) stimulates transcription. The CAP-cAMP complex stimulates transcription by binding to an activator-binding site adjacent to the promoter and helping RNAP to bind site [glucose] down [cAMP] up cAMP-CAP activated transcription transcription lacZYA induction: repressor + inducer & cAMP + CAP RECAP2 RECAP2 Ara Operon Regulation by Looping: -arabinose: AraC induced repression loop formed between O2 and I1 -arabinose: +arabinose: repression loop broken, AraC binds I1 and I2 +arabinose: araC__araO2___________araO1__araPc__araI1_araI2____araPBAD__ The ara operon is controlled by the AraC protein. AraC represses the The ara operon by looping out the DNA between two sites, araO2 and araI1, operon araO2 araI1 that are 210 bp apart. Arabinose can derepress the operon by causing AraC to loosen its attached to araO2 and bind to araI2 araO2 araI2 instead. This breaks the loop and allows transcription of the operon. CAP and cAMP further stimulate transcription by binding to a site upstream of araI. araI MODEL GENETIC SWITCHES: PHAGE MODEL • • • • • Lysis vs lysogeny lysogeny Lysis: phage genes transcribed in set order, phage genome is replicated & packaged, the host is lysed replicated Lysogeny: phage inserted into host genome, the latent prophage is Lysogeny: inherited with bacterial genome (phage λ not T7 or T4) Prophage released from lysogeny by induction Prophage induction Phage use elegant control systems that rival bacterial operons TYPICAL LYTIC DEVELOPMENT DEVELOPMENT • Two regulatory cascades specific to the Two lysis cycle: lysis • (1) early infection: induction of genes (1) for DNA replication for • (2) late infection: DNA replication (2) continues, but synthesis of phage particle proteins induced particle • Lytic phage genomes often cluster Lytic genes with related function, reflecting progression of lytic development progression • Transcription control in form of Transcription cascade in which gene expressed at one stage required for expression of genes in next stage genes Lewin Genes VII 2000 Oxford University Press Temporal control of transcription in phage SPO1 infected B. subtilis LYTIC CONTROL IN PHAGE T7 LYTIC • Phage T7 (38kb): 3 gene Phage classes expressed sequentially sequentially • Class I early genes: Class includes T7 RNAP includes • Class II genes: DNA Class replication genes transcribed by T7 RNAP transcribed • Class III genes: assembly Class genes for mature particle genes • Phage T4 (165kb): larger Phage phage with many gene groups groups Weaver 2002 Molecular Biology Ed. 2 PHAGE T4 GENE EXPRESSION PHAGE Lewin Genes VII 2000 Oxford University Press • • • • • T4 numbered genes – ESSENTIAL T4 ESSENTIAL T4 genes with 3-letter abbreviation - nonessential T4 nonessential T4 early genes: transcribed by host RNAP --> products include MotA T4 & AsiA AsiA Middle genes: lack -35 promoter consensus but transcribed by host Middle RNAP aided by MotA & AsiA RNAP MotA binding sites at -30 in middle gene promoters & AsiA coactivator interacting with E. coli σ 70 factor at -10 consensus activator E. PHAGE T4 GENE EXPRESSION PHAGE N antiterminator (works at nut site, permits replcation of delayed early genes) cro (control of repressor and other things) represses transcription, like the Lac repressor cI gene encodes λ repressor O and P code for proteins necessary for phage DNA replication Q is another antiterminator (works at qut site, permits replication of late genes) λ REGULATORY CONTROL: LYSIS vs. vs LYSOGENY LYSOGENY • When λ enters host, lysis When & lysogeny start off together the same way: immediate early & delayed early genes expressed early • LYSIS: if late genes LYSIS: expressed expressed • LYSOGENY: if λ repressor LYSOGENY: synthesis established synthesis λ IMMEDIATE IMMEDIATE EARLY GENES • 1. When λ enters a host, the 1. immediate early genes N & cro are transcribed cro independently by host RNAP independently • N: antitermination factor acts N: at nut (Nutilization) sites & nut ilization) enables transcription to proceed into delayed early genes genes • cro: blocks lysogeny by (i) blocks preventing λ repressor synthesis synthesis λ REGULATORY REGION REGULATORY • 2 transcription units for immediate early genes, N and transcription cro, transcribed in opposite directions from promoters PL & PR • RNAP stops at Rho-dependent terminators tL1 & tR1 in RNAP L1 absence of the N gene product, pN N ANTITERMINATION • in absence of pN, RNAP in transcript terminates at tL1 and makes N mRNA only product makes • in presence of pN, RNAP can in read through tL1 & tR1 terminators into distal delayed early genes early – pN binds transcribed nut pN nut region and also to NusA protein complex; together the proteins alter RNAP so it reads through terminators reads Weaver 2002 Molecular Biology Ed. 2 λ DEVELOPMENT DEVELOPMENT Weaver 2002 Molecular Biology Ed. 2 • Immediate early genes: N and cro cro • Cro protein promotes the lytic cycle by Cro preventing cI repressor synthesis cI • Delayed early genes: expressed expressed because of pN - includes 2 replication, 7 recombination, and 3 regulatory genes with opposing functions genes (i) cII/cIII involved in λ repressor (i) cII synthesis synthesis (ii) Q, which encodes pQ (ii) which antitermination factor antitermination pQ binds at qut & enables RNAP to pQ qut read through terminator (from PR’ ) into late genes late So, transcription of late genes is induced by a delayed early regulatory proteins by λ LYSOGENY: AUTOGENOUS CIRCUIT LYSOGENY: PL & PR control gene expression through the entire early period with antitermination enabling transcription of delayed early genes early operators associated operators with both promoters repressed when λ repressor bound - RNAP transcription blocked & lytic cycle inhibited lytic cI mutants cannot mutants establish lysogeny (constitutive lytic) (constitutive λ “CLEAR” MUTANT GENES: cI, cII & cIII CLEAR” cI cII cIII • Mutants in cI & either of its cII /cIII positive regulators form clear plaques, not wildtype cloudy plaques - all mutants cannot establish lysogeny • wild-type phage produce cloudy plaques because some cells have established lysogeny rather than dying • almost all mutant phage in lytic state means all host cells lysed by infection leading to clear plaques MAINTENANCE OF LYSOGENY MAINTENANCE Weaver 2002 Molecular Biology Ed. 2 λ repressor is transcribed from PRM - promoter “repressor maintenance” ∀ λ repressor protein produced by cI binds as dimer to OL cI & OR independently, preventing transcription from PL & PR promoters • positive autogenous circuit: λ repressor binding to OR positive The λ repressor monomer Various surfaces are involved in different activities carried out by the λ repressor. N indicates the amino domain, C the carboxyl domain. “Tetramerization” denotes the region where two dimers interact when binding cooperatively to adjacent sites on DNA (more later) Repressor bound at OR2 contacts RNAP at PRM, activating expression LYSOGENIC CELL IMMUNITY LYSOGENIC • cI repressor protein confers immunity to lysogenic cI bacterial cells from re-infection by other λ phage • When new phage DNA enters prophage-containing When bacterium, the new phage DNA immediately bound by λ repressor at OL & OR • Lytic cascade cannot be initiated • If transcription from PRM requires binding of λ repressor itself, then how is cI synthesis cI established in the first place? established • cI mRNA synthesis is first established from another promoter: PRE another ESTABLISHMENT OF LYSOGENY ESTABLISHMENT Weaver 2002 Molecular Biology Ed. 2 • • • • if transcription from PRM requires binding of repressor itself, how if is λ repressor synthesis established in the first place? is when λ DNA enters new host, immediate early & then delayed early when genes expressed - among products of delayed early genes are CII & CIII proteins CIII between cro & cII genes is “repressor establishment” promoter PRE between cro cII cI repressor gene transcribed from PRE but PRE does not have conserved -10 and -35 core --> RNAP recognizes PRE if bound by CII protein protein RNAP BINDS PRE ONLY WITH CII • Although –35 and –10 Although PRE regions not conserved relative to conventional bacterial operons, they are bound by RNAP as shown by both footprinting and mutational analysis mutational ESTABLISHMENT OF LYSOGENY ESTABLISHMENT • conventional DNase I conventional footprinting reveals that CII binds PRE between –21 & –44 (around –35 region) (around • because of large size of DNase, because footprint generated with this enzyme is not high resolution as it is difficult for DNase to gain access to “nooks and crannies”--> gaps in detection of protein-DNA interaction protein-DNA • by DMS footprinting, more by precise analysis of CII contacts with promoter made with Weaver 2002 Molecular Biology Ed. 2 DMS FOOTPRINTING OF PRE & CII or RNAP • DMS footprinting: endlabeled DNA bound by labeled protein protected from protected methylation • methylate with DMS methylate such that about one methylation occurs per DNA fragment DNA • however, if protein however, causes melting of DNA duplex it creates sites more sensitive to more methylation methylation • after methylation, after protein removed,DNA cleaved at methylated sites and run on gel sites Weaver 2002 Molecular Biology Ed. 2 DMS FOOTPRINTING OF PRE & CII or RNAP • detect CII binding to PRE detect RE promoter, protection of bases in the –35 region confirmed confirmed • CII + RNAP footprints CII show RNAP also binds in –35 region but uses different bases • RNAP also binds near – 10 Weaver 2002 Molecular Biology Ed. 2 cII DNASE I -46 -36 -26 -41 -21 RNAP λ LYSOGENY: SUMMARY SUMMARY - - - CII/RNAP binding to PRE CII/RNAP results in “backwards” transcription through cro cro gene and into cI gene, cI thus producing repressor thus CIII protects CII from CIII degradation (CII unstable degradation backwards transcription backwards thru cro gene enroute to cro the cI gene produces cro antisense transcripts that hybridize to cro mRNA, cro inhibiting its translation inhibiting cro inhibition aids lysogeny since cro cro protein necessary for lytic cycle cycle Figure 18-31 λ REPRESSOR - OPERATOR GEOMETRY REPRESSOR N-terminal N-terminal DNA binding domain domain C-terminal C-terminal dimerization domain domain • • • • • Weaver 2002 Molecular Biology Ed. 2 detailed studies of λ detailed repressor: pioneering work on transcriptional regulators inspired work in all organisms (now > 10,000 papers a year on regulation of gene expression!) expression!) 27 kDa repressor subunit = 2 27 domains joined by connector domains N-terminal domain = operator N-terminal binding binding C-terminal domain = C-terminal dimerization dimerization Lytic cycle initiated by Lytic cleavage of dimeric repressors in connector region region λ REPRESSOR - OPERATOR GEOMETRY REPRESSOR N-terminal N-terminal DNA binding domain domain C-terminal dimerization domain domain • • Weaver 2002 Molecular Biology Ed. 2 DNA-binding sequences of DNA-binding repressor exhibit a structure found repeatedly in DNAfound binding proteins from bacteria binding to eukaryotes: short stretches of α -helix N-terminal domain has 5 α N-terminal helices, two of which are used helices, to bind DNA and constitute the helix-turn-helix motif helix-turn-helix λ REPRESSOR - OPERATOR GEOMETRY REPRESSOR • helix turn helix α -helices 2 & 3 in each -helices subunit form H-bonds with operator DNA; in dimer, two helix-3 regions lie 34Å apart & fit into successive DNA major grooves on same side of helix same • α -helix 3 = RECOGNITION HELIX: it recognizes HELIX: specific target sequence specific • helix-turn-helix found in helix-turn-helix other DNA-binding proteins • one can change the DNAbinding specificity of a binding repressor by substituting its recognition helix with that from a different repressor repressor HYDROGEN BONDS BETWEEN λ REPRESSOR & OPERATOR DNA REPRESSOR OL1 Weaver 2002 Molecular Biology Ed. 2 • • • Cl & Cro are both helix-turn-helix repressors In both CI & Cro, 2 helix-3 amino acids are conserved (neighbouring In helix-3 Gln & Ser) that bond to specific bp in DNA Gln Helix-2 mediates H-bonding with phosphate backbone (for example Gln 33 in λ repressor) but doesn’t control specificity of target Gln ’t HYDROGEN BONDS BETWEEN λ REPRESSOR & OPERATOR DNA REPRESSOR OL1 Weaver 2002 Molecular Biology Ed. 2 • in addition to hydrogen bonding, helix-2 undergoes ionic in interactions with phosphate backbone due to fact that its amino end, which has a net positive charge, is pointing directly towards negatively charged DNA backbone directly OPERATOR/REPRESSOR-BINDING SITES: COOPERATIVE BINDING SITES: • • • -Repressor highest affinity for OL1 & OR1 Repressor R1 -Cooperative binding: binding at site 1 Cooperative increases repressor affinity for site 2; when OL1-2 & OR1-2 bound by repressor, PL & R1-2 PR transcription blocked • each operator contains each three 17bp repressorthree binding sites with binding partial symmetry about central base pair central central bp divides each central site into 2 half-sites, which show similarity to consensus sequence each half-site binds each one monomer in repressor dimer repressor site 1 in each operator site has a 10X greater affinity for λ repressor than other sites than λ LYSOGENY: AUTOGENOUS CIRCUIT LYSOGENY: -we we end up with 4 monomers of repressor at each operator operator • Unlike PL & PR , Unlike repressor binding stimulates stimulates transcription from PRM • mutations identified in mutations repressor that prevent transcription from PRM; known as positive control mutations & identify region of repressor that may directly interact with RNAP Figure 18-25 Figure 18-26 Figure 18-28 DNA looping is involved in repression of PRM by cI • The 4 monomers of CI The repressor bound to each operator can form an octamer, causing formation of DNA loop formation • When cI reaches high When levels this loop can enable binding of cI at OR3 and OL3, leading to and 3, repression of PRM RM INTERGENIC SUPPRESSION BETWEEN λ REPRESSOR & RNAP • Compensatory intergenic Compensatory suppressor mutations in RNAP can suppress repressor mutants suppress defective in PRM transcriptional activation - confirms that RNAP & cI repressor directly interact at PRM THE BATTLE OF cI vs. cro THE cI vs. cro • • • • • Weaver 2002 Molecular Biology Ed. 2 • • If cI establishes control then If cI lysogeny lysogeny If cro establishes control then lysis If cro cro wins by blocking PRM transcription & by repressing early genes dispensable for lytic cycle genes cro same operator as cI (λ repressor) but with reverse affinity: cro affinity OR3 > OR2 > OR1 cro Cro binds OR3 first and thereby Cro blocks RNAP binding at PRM - no RM maintenance of λ repressor repressor Cro subsequently “fills up” OR & Cro OL preventing cII & cIII gene cII cIII expression - no λ repressor expressed from PRE expressed all routes for synthesis of λ all repressor are blocked and lytic cycle assured cycle THE BATTLE OF cI vs. cro: THE cI vs. DETERMINED BY CII? DETERMINED • as [CII] increases, lysogeny increases • CII sensitive to proteases and protected from CII these by CIII these • Cellular proteases that degrade CII are higher Cellular when cells grown in rich medium, lower during starvation starvation • CII degradation & lytic cycle likely when cells CII grown in rich medium grown • CII more stable & lysogeny likely when cells CII under starvation conditions - allows phage to wait until effective resources available for replication & packaging replication λ LYTIC CASCADE: SUMMARY SUMMARY -Cro binds OL and OR Cro and inhibits cI, cII, & cI cII cIII expression, and cIII turns-off early genes not needed for lysis not - pQ permits RNAP transcription of late genes that begin the lytic cascade lytic INDUCING λ PROPHAGE • λ induction dependent Weaver 2002 Molecular Biology Ed. 2 on the SOS response and on RecA on • Like its action on LexA, Like RecA also co-protease for λ repressor • RecA promotes λ RecA repressor self-cleavage releasing repressor from PL & PR and cro cro expressed expressed • Thus, DNA damage & cell Thus, stress “tell” λ to leave host cell before host cell dies dies RECALL THE SOS RESPONSE RESPONSE (error-prone bypass repair) • RecA also RecA involved in SOS involved response response • metabolic alarm metabolic system activated in response to DNA damage (UV, alkylating agents) or inhibition of replication (eg. mutations in dna dna genes) genes) • response response manifested as increased capacity to deal with damaged DNA Weaver 2002 Molecular Biology Ed. 2 Summary 1 • The immediate early/delayed early/late transcriptional switching in the lytic cycle of phage λ is controlled by antiterminators. • One of the two immediate early genes is cro, which codes for a repressor of the cI gene that allows the lytic cycle to continue • The other, N, codes for an antiterminator, that overrides the terminators after the N and cro genes. Transcription then continues into the delayed early genes. • One of the delayed early genes, Q, codes for another terminator (Q) that permits transcription of the late genes from the late promoter, PR’, to continue without premature termination Summary 2 • Phage λ 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 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 to binding to PRE and PI; CIII, by slowing degradation of CII. Summary 3 • The promoter that is used to maintain lysogeny is PRM. It comes into play after transcription from PRE makes possible the burst of repressor synthesis that establishes lysogeny. This repressor binds to OR1 and OR2 cooperatively, but leaves OR3 open. • RNAP binds to PRM, which overlaps OR3 in such a way that it contacts the repressor bound to OR2. This protein-protein interaction is required for this promoter to work efficiently. • High levels of repressor can repress transcription from PRM via DNA looping when repressor dimers are bound to OR1, OR2, and OR3 Summary 4 • Whether a given cell is lytically or lysogenically infected by phage λ depends on the outcome of a race between the products of the cI and cro genes. • The cI gene codes for a repressor, which blocks OR1, OR2, OL1, and OL2, turning off all early transcription, including transcription of the cro gene. This leads to lysogeny • On the other hand, the cro gene codes for Cro, which blocks OR3 (and OL3), turning off cI transcription. This leads to lytic infection. • Whichever gene product appears first in high enough concentration to block its competitor’s synthesis wins the race and determines the cell’s face. The winner of this race is determined by the CII concentration, which is determined by the cellular protease concentration, which is in turn determined by environmental factors such as richness of the medium ...
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  • Spring '11
  • DAVIDSON
  • DNA, cII, CrO, lysogeny

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