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Molecular Biology of the Gene

by: Jasmin Roob Sr.

Molecular Biology of the Gene B M B 400

Jasmin Roob Sr.
Penn State
GPA 3.58


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Date Created: 11/01/15
B M B 400 Part Four III 2 Chpt 17 Transcriptional regulation in bacteriophage lambda B M B 400 Part Four Gene Regulation Section III Chapter 17 TRAN SCRIPTIONAL REGULATION IN BACTERIOPHAGE LAMBDA Not all bacteriophage lyse their host bacteria upon infection Temperate phage reside in the host genome and do not kill the host whereas lytic phage cause lysis of their hosts when they infect bacteria The bacteriophage A can choose between these two lifestyles The molecular basis for this decision is one of the best understood genetic switches that has been studied and it provides a fundamental paradigm for such molecular switches in developmental biology This chapter reviews some of the historical observations on lysogeny in bacteriophage A covers the major events in lysis and lysogeny and discusses the principal regulatory proteins and their competition for overlapping cis regulatory sites We will examine one of the common DNA binding domains in regulatory proteins the helix turn helix which was first identified in the A Cro protein Also the use of hybrid genes to dissect complex regulatory schemes was pioneered in studies of bacteriophage A and that approach will be discussed in this chapter A Lysis versus lysogeny 1 Lytic pathway a Leads to many progeny virus particles and lysis of the infected cell b Have extensive replication of A DNA formation of the viral coat head and tail proteins with packaging of the A DNA into phage particles cell lysis and release of many progeny phage 2 Lysogenic pathway a The infecting phage DNA integrates into the host genome and is carried passively by the host b Have repression of A lytic functions integration of A DNA into the host chromosome at the alt site The bacterial cell carrying the integrated prophage is called a lysogen the A DNA is replicated passively along with the E coli genome The host cell is not killed and is immune to further infection by A phage 3 Early Observations on Lysogeny Lysogeny is the hereditary ability of a bacterium to produce phage Bacteriophage that can bring about the lysogenic state in bacteria are called temperate phage those that only lyse cells are called virulent Studies on lysogeny started in the 1920 s and continued through the 1940 s particularly from the laboratories of Eugene and Elizabeth Wollman and Andre Lwoff examining a lysogenic strain of Bacillus megaterium This system was particularly amenable to studies of lysogeny because an indicator strain was available ie a related nonlysogenic strain that is sensitive to the phage produced by the lysogenic strain upon induction and because the cells of B megaterium are very large and could be isolated as single cells by micromanipulation B M B 400 Part Four III 2 Chpt 17 Transcriptional regulation in bacteriophage lambda Examination of single cells and other studies showed that 1 All cells of a lysogenic culture are lysogens 2 The lysogenic character persists after repeated passage of a culture through an antiserum specific for the phage ie no free phage are required to maintain the lysogenic state 3 Lysogenic bacteria can adsorb the phage they produce but they are not infected they are immune to the phage 4 After the phage infect a sensitive host one can isolate bacteria resistant to the phage which can now produce phage identical to the original ie infection of a sensitive host leads to the formation of new lysogens Figure 431 Temperate phage can either lyse host cells or generate lysogens E coli cell E 00quot 09 E coli chromosome xphage Q lylic growth of phage i cellundergoeslysogeny 9 x prophage 00 90 99 90 Q20 09 X 09 Dr M tants of phage that have lost the capacity to Temperate ha ge generate turbid plaques ogenlze form clear lysogenized cells lysed cells lysed cells uninfected cells The specific hereditary structure within lysogens needed for the production of phage was called a prophage In contrast to the random spontaneous lysis of a small fraction of lysogens eg about 1 1000 Lwoff discovered by irradiation with UV would induce lysis of virtually all bacteria in a culture of lysogens BMB400 Part Four III 2 Chpt 17 Transcriptional regulation in bacteriophage lambda Three basic phenomena were discovered Figure 432 9 Lysogeny hereditary ability to produce phage 9 Induction stimulation of lysis of a whole population of lysogens O Immunity or resistance lysogens are resistant to superinfection with the phage produced by the lysogen Induction and immunity of lysogens A lysogen 9 Lysogens are immune to Spontaneously 11000 lysogens will induceyie Ihel UV treatment leads Prop Will to Induction of edee replicate and virtually all lysogens wg 39onigtglhogrgg39m s39mllar W391 0911 in a culture 09 W W W W O P Zygotic induction of a lysogen Hfrr donor 9 W W Hfr xrecipient Lysogeny in E coli zygotic induction about 1951 Joshua and Esther Lederberg studying conjugation worked with E coli strain K12 for many years without realizing that it was a 2 lysogen They had no indicator strain to reveal the presence of 2 as a prophage In describing these experiments I will refer to the original strain as K120 to denote its lysogenic state even though it was not recognized as such until after these experiments Some UV generated mutants of K120 showed an unusual behavior referred to as zygotic induction Although these mutants would grow normally in culture when used as recipients in conjugation experiments with male Hfr strains of wild type K120 as the donor the cells would lyse BMB400 Figure 433 P1 C Part Four III 2 Chpt 17 Transcriptional regulation in bacteriophage lambda Zygotic induction Mate with Hfr nonmutant K12 lambda Not lyse Lyse Zyg otic induction E coli K12 lambda quotwild typequot Not lyse mutant 3 Lederberg called the phage released by induction of E coli K120 lambda or 2 since it was found just after the K factor from Paramecium Infection of the 2 sensitive strain E coli C with 2 produced turbid plaques Most infected cells did lyse but some lysogenized generating colonies of 2 resistant cells in the midst of an otherwise clear area ie turbid plaques from these and other exneriments39 1 The original E coli K12 was a 2 lysogen ie K120 It carried a 2 prophage integrated into the E coli chromosome at an 2 The prophage confers the heritable ability to produce 2 ie lysogenicity 2 Lysis can be induced either spontaneously about 1 in 10001ysogens or by UV induction essentially all lysogens Induction requires rec4 3 Lysogens are immune to further infection by the same phage Other lambdoid phage can infect eg 2 lysogens can be infected by phage 434 4 Some of the mutants of E coli K120 had lost the 2 prophage and hence they are not longer lysogens Fig 432 mutant 2 When the 2 prophage is donated to these nonlysogenic recipients by conjugation zygotic induction occurs That is the 2 prophage in the Hfr strain is induced when it enters the nonlysogenic strain This indicates that some negative factor is present in the lysogen that is absent in the nonlysogen that prevents induction Alternatively the converse is possible a B M B 400 Part Four III 2 Chpt 17 Transcriptional regulation in bacteriophage lambda positive factor present in the nonlysogen But as we will see later the negative factor or A repressor is present in the lysogen and prevents lysis 4 Regulatory mutants of A a Clear plague mutants Dale Kaiser 1957 Wt required for establishment Wt required for maintenance of lysogeny of lysogeny 01 yes yes 011 yes no 0111 yes no Act in trans b virulent or vir lyse host cells do not lysogenize Act in cis are double mutants in OR andor 0L B M B 400 Part Four 7 III Chpt 1397 Transcriptional regulation in bacteriophage lambda B Map of 7 Figure 434 Latecontrol Virushead Recombination Controlregion Replication Lysis amptai gam Int red att XIS cm N cl are all I I I I I I I Pm tn PL 0L PRM PR thPRE tR2 OR R8 origin P promoter 390 operator I terminator 1 DNA from a 7 phage particle is linear but the ends are complementag cohesive ends cos Thus when the phage DNA is in39ected into a cell upon infectio e ds a neal to form a circle Maps of DNA are frequently drawn fro the left ms site to the right one but the map in Fig 4 4 is the linear map 7 opened at the all site This presentation shows the clustering of functions on the genome The map is not to scale N Genetic functions are clustered in 7 including both Iransracting proteins and cisracting sites a Control region 1 Control at PR PL and PRM I encodes the repressor that turns off lytic functions are encodes the IIantirepressorII that turns off the re sor Both of these act at operators OR and OL that control promoters PR PL and PR Note the proximity between the genes and the sites at which the gene product acts 2 Control at PRE39 cll encodes a positive regulator of transcription a tPRE CIquot encodes a protein that is needed to stabilize the product of all 3 N is an antiterminator that allows trancriptional readrthrough at tLl t Rl and tRZ BMB400 9 0 P Part Four III 2 Chpt 17 Transcriptional regulation in bacteriophage lambda Replication 0 and P encode proteins required to initiate replication The product of 0 is analogous to DnaA forming a complex at the origin of replication which is Within the coding region of 0 The P protein brings in DnaB to the origin to initiate replication in a mechanism similar to that at oriC Late control The product of Q is an antiterminator that prevents termination at tgs which is just downstream of the Q gene Recombination 1 The product of the int gene is required for integration into the host chromosome using the an attachment site that is adjacent to int The products of the xis and int genes are required for excision of the prophage again using the adjacent an site 2 The products of red and gum gamma are needed to convert from 6 form replication to rolling circle during the viral replication pathway Late genes The products of several genes A through J are the protein components of the viral head and tail needed for make phage particles Nonessential region The 172 region named for a large deletion that leaves the phage still viable is not needed This is a substantial part of the region that is replaced when A is used as a cloning vector B M B 400 Part Four 7111 cnpt 1 7 Transcriptional regulation in bacteriophage lambda c Lytie easeade 1 Early delayed early and late genes a Early genes are expressed before DNA neplimu39on initjaues 1 Immediate early genes are nansen39bed by the host RNA polymerase and include 39 39 diene t etin 39 39 2 a regulatnrtn be expressed The delayed early genes make a regulator required forlaue gene expression h lane genes are expressed amr DNA neplimh39on initiates 39 l A 39 genes for the 39 39 39 lysis Figure 435 Transcription and translation orininiediate early genes Transcription by E coli RNA polymerase initiates promo 39 ters PR PR and PL andterminates a gam t I I I I I I I I I Pint 1L1 PL 0L PRM PR 1R1 PRE tRZI PR 165 l Rl a dds 1 es RNA 2 N encodes an andterminawr at strong PRand PR39 a 39 39rquot quot h E 39DNA withno Uulcl i i i c promins 1 710 and 735 boxes PL from which DNA a leftvmd direction B MB 400 PartFour 7 III Chpt 1397 Transcriptional regulation in bacteriophage lambda c The product of N called pN or N protein prevents RNA polymerase from stopping at the pedependent terminators IL leftward transcription from FL and IR and IR2 rightward transcription from PR Fig 436 Antitermination by N protein leads to early gene expression I I I I Pint tLl PL PRM PR tRl PRE tRZI PR 165 R8 E l as RNA N protein Cro Clll Cll Qprotein Recombination proteins Repncation proteins 3 Cro antirepressor a are is the first gene transcribed from PR from which RNA polymerase transcribes in a rightward direction F Early in the infection the protein Cro binds to 0R3 to prevent transcription from PRM the promoter for repressor maintenance Hence it acts against the repressor so it is called the antirepressor and it helps prevent lysogeny c As the Cro increases later in the infection it also binds to the other sites in the leftward and rightward operators to turn off immediate early transcription after the products of these genes are no longer needed B M B 400 Part Four 7 III Chpt 1397 Transcriptional regulation in bacteriophage lambda Fig 437 Lytic cascade Cro turns off cl Q protein action leads to late gene expression t gam an I I I I I Pint tLl PL 0L PRM PR tRl PRE 1R2 PR 155 l R l l a E Repncation proteins Lytlc functions Viral head amp tail proteins 4 Products of leftward transcription recombination and integration a Action of pN at 1L allows readrthrough transcription of red and gum which are required for a recombination event during replication so they are involved in lysis b The cIII gene which is required for lysogeny is also transcribed as a result of the ack of termination at 1L c The m and xix genes are also transcribed but this readrthrough transcription extends past the pedependent terminator 11quot because of antitermination by N protein Transcripts that extend into the b2 region form a secondary structure that is recognized y an ase w ich degrades the transcript from the 339 end thereby removing m from the transcri t 5 Products of rightward transcription replication and Q a Action of pN at 1R allows readthrough transcription of the 0 and P genes required for replication initiation as well as all required for lysogeny b Action of pN at 1R2 allows further readthrough transcription of the Q gene B M B 400 Part Four 7 III Chpt 1397 Transcriptional regulation in bacteriophage lambda 6 The protein pQ is also an antiterminator a Acts on transcription initiating at PR to prevent termination at 155 b Allows expression of late genes S and R for lysis A through J for head and tail proteins c Expression of Q commits the infected cell to the lytic pathway Fig 438 Late stage of lytic cascade High concentrations of Cro turn off PR and P L Abundant expression from P R gam Int red att XIS cm N cl cro oil A I I I I I I I Pm tn PL 0L PRM PR 1R1 PRE tR2 P QR 1R8 l a 165 Lytic functions Viral head amp tail proteins BMB400 D E Fig 439 Re uilesne ressor Pan Four 7111 Clipt 1 7 Tianseriptional regulan39on in bacteriophage lambda Lysogeny roductof e1 ene ando eratois Repressor binds at 0L and OR to block transcription fmm PL and PR Muizn39onal analysis a Clear plague mumtions cl on cm is cixracu39ng vir mutations No lysogeny in these mutants Sites 0R1 0R2 0L1 and 0L2 in the opeiatois are alteied to prevent binding of repressor in vir mumnts CI and CI nes encode ositive ie ulatois of and Pit lysogenie pathway is quiue similar to that of the lytie pathway Arm 39 39 mm a i areexpressed CH and cm stimulate expression of cl to make repressor A J gam red XIS cm N cl are A I I I I I I I I I I tlnl Pint 1L1 PL 0L PRM PR 1R1 PRE tF12 PR 165 l 0R 1R8 S a PRE promoter for repression establishment i i Repressor a I 7 7 m a b RNA polymeiase alone The 710 and 735 boxes aie Veiy poor matelies to the consensus for E coli promoters The pmtein pmduet of the CI gene will enhance B M B 400 Part Four 7111 Chpt 17 Transcriptional regulation in bacteriophage lambda the binding of RNA polyrnerase to PRE and hence s mulam initiation of transcription from this promoter d The en product is an unstable promin A protease encoded by the h A gene on the E coli chromosome will degrade the en pmmin Mum ons in h A cause a high frequency oflysogeny do you see why hence the acronyrn for its narne The protein encoded by the cm gene will inmrfere with degradation of the en protein by H A e 39 39 39 39 39 n m DNA 1 n 39 39 I 39 39 39 39 39 39 the Aiepressor f Th quot 39 39 39 39 39 39 P l he integrase gene Production of inmgmse allows itto camlyze the inmgration of the A 39 L E 39 39 Thisoccuisby 39 39 quot 39 L L 39 39A and u A 390 oh 7 39 I 4 Binding of repressor to operators a Binding to 0L1 and 0L2 blocks leftward hanscnption horn PL and binding to 0R1 and 0R2 blocks rightward hanscnption from PR This mms off transcription of the genes required for phage multiplication and cell lysis n h p ann by p 39 39 39 quott lysogeny Fig 4310 Lysogeny Repressor turns off transcription r 9T2 r att xii cm N I I I I Pint 1L1 PL 0L PRM PR 1R1 PRE tRZI PR 165 R8 l R PRM promoter for repression maintenance Repressor B M B 400 Part Four III 2 Chpt 17 Transcriptional regulation in bacteriophage lambda 9 Binding to OR and 0122 also enhances transcription from PRM PRM is the promoter for repressor maintenance It is adjacent to PR and directs transcription leftward through 01 After lysogeny the concentration of repressor in the cell will decrease as the cells multiply Transcription from PRM allows the repressor to be maintained at an adequate level to prevent transcription from PR and PL Table 431 Gene Products and Sites Involved in the Different Pathways of A Lysis or Lysogeny Lysis Lysogeny Both Cro represses cI Repressor cl cII cHI establish lysogeny N antiterminator O P Red Gam replication cI Repressor maintains lysogeny Q antiterminator Int integrates A DNA S R lysis Xis with Int excises A prophage A through J are head and tail proteins PR PRE P int PRM PL PR 0L 0R qut alt nut tRZ t6S tint IL IR oriL B M B 400 Part Four 7 III Chpt 1397 Transcriptional regulation in bacteriophage lambda E Opa39ator structure 1 3 binding sites a 0L and 0L2 and 01 comprise 0L b OR and 052 and OR comprise 0R 2 Had symmetg a Each of the binding sites is 17 hp with an imperfect dyad centered on the 9th bp b Although 39 quot l h the 39 39 and as we will see shortly the affinitites of repressor and Cro differ for each site Figure 4311 2 operators overlap with prmnoters OR I 0R3 0R2 0R1 PR 5 10 TTGACT GATAAT V cm TTAGAT 5 ATAGAT 5 10 35 PRM 3 These operators overlap the promoters a OR and 0R2 overlap with the 710 and 735 boxes respectively ofPR Binding of repressor to these sites should block access of RNA olymerase to these sites Note that this is the steric interference mo el again Even t ough we saw with ac that this model does not hold the lac operator is centered at10 and polymerase can bind even w en lac repressor is oun However for 0R as wel as for 0L the repressor and polymerase are in direct competition for the same sites 5 Similarly 0L and 0L2 overlap with the 710 and 735 boxes respectively ofPL Binding of repressor to these sites should block access of RNA polymerase to these es c 0R3 overlaps PRM so when Cro binds to this site transcription from PRM is blocked BMB400 Figure 4312 Affinities of Repressor and Cro for A operators Part Four III 2 Chpt 17 Transcriptional regulation in bacteriophage lambda PR 35 10 0 IOL1 0L2 013 IOR3I 0R2 IOR1I CI N 10 35 10 35 PL PRM 0L1 OLZ 0L3 High High Low Low Low High 0R3 0R2 0R1 Low High High High Low Low Af nity for Repressor Cro B M B 400 Part Four III 2 Chpt 17 Transcriptional regulation in bacteriophage lambda F Repressor protein 1 Protein structure a Functions as a dimer each monomer of which is 236 amino acids in sequence Note the symmetrical protein binding to a dyad motif in the DNA One monomer binds to one half site of the dyad binding site eg a dimer binds to OR b The monomers have an N terminal DNA binding domain amino acids 1 92 a connector and a C terminal protein interaction domain for dimerization A web tutorial on lambda cro and repressor binding to DNA is at http WWW bimcore emory edu home Kins bimcoretutorials Mrobbin protein dnamodlefthtml Figure 4313 Lambda Re ressor roduct of cl ene Dimer 2 functional domains monomer is 236 amino acids Cterminal domain proteinprotein interaction dimerization and oooperativity Connector Nterminus DNA binding HelixTurnHelix motif operator operator 0R2 0R Induction RecA coprotease cleaves in connector lose cooperativity and dimerization gt 66 8 8 Repressor remnants dissociate from El operator operator operator operator operator 0R2 0R1 0R2 0R1 BMBAOO pm we of 17 2 DNA bmdmg domam hehxemmehehx The a a r DNA brrrdrrrg sues has been defgrmmed by Xeray crystallography ermlardata are ayarlable for coecrystals of Cro prorerrr and operator DNA helroes orre othth hehx 3 m the smromre rs m the major groove contacung makes contacts wrur bases In the major groove loonnected L A h r Lhedesr rrauorr othxsscrucLuralmoufashehxemmehehwalD HthZhes asmde e phosphodxesLerbackbone and makes speorno contacts wrur 1L LAMBDA REPRESSOCOMFLEXED WITH DNA OPERATOR 1 LL Figure 4314 mehnp waw rm nken go JpJouhoulrm gednarpmtemall WILN 1m glf d form two Hebondsthh a G atposmon 4 for Arepressor or arr atposmon 3 of r of Hebonds to formZ Hebonds wah adenme In COanSL a guanme can formZ H u h a m mm on n e y rrr nuClEODdES for ths structural mouf Thxs IS well xllusLmtEd by the example of the In thscasa B M B 400 Part Four III 2 Chpt 17 Transcriptional regulation in bacteriophage lambda the protein will interact with different nucleotides depending on whether it is in the A repressor or in Cro e Networks of interactions play important roles in determining the specificity of proteins binding to DNA The combination of interactions at the different half sites probably contributes to the different affinities eg A repressor binding much more avidly to OR than to 0R3 Figure 4315 Sequencespecific binding of A Cro and Repressor to operator sites Tyr26 Gn27 Cro bound 539 C A AVGVGVG A T A tooRS 339 63 A9 A GTTCCCTAT gt llll iigtgtlll IIIIgt w I IIIG Jgt0 Ser28 Ly532 Arg 38 Zeonsensus 539 1 A T A halfsites T A T Gn33 Gn44 Ala49 v vE v v v Repressor539TATCACCGCCAGAGGTA boundto 12345678 0R1 339ATAgTAgAGACGGTCTCCAATA Ser45 Asn55 Lys4 V Phosphate protected from ethylation by protein binding G Guanine protected from methylation by protein binding Amino acid sequences of helix 2 turn and helix 3 of Cro and Repressor 16 26 27 28 Cro 1 T J 1 quot 39IaTquot n T 39IaT39ItaTh39 A39IaC39IvArng Rep GlnGluSerVa39lA39l 1 39quotvA39 r 39IvIleAsnAli 33 44 45 49 5 B M B 400 Part Four 7 III Chpt 17 Transcriptional regulation in bacteriophage lambda 3 Protein interaction domain The Citerminal domain is required for dimerization between 2 monomers The two oths m the dimer fit mcely into adjacent major groove in the DNA ie the two halfisites of the A operator 4 Differential affimties for operators a Repressor binds with greatest affinity to OR then with 107fold less affimty for 0R2 The affimty for 0R3 is quite low b Repressor has similar differential affimties at 0L 5 Cooperativity a Once a repressor dimer binds to OR it facilitates subsequent binding of an additional repressor dimer to 0R2 so in fact this cooperatiVity means that both OR and 0R2 are occupied when repressor is expressed b This preVents transcription from PR c Similar cooperatiVity occurs at 0L and 0L2 to turn off transcription from PL d The same Citerminal domain that is needed for dimerization is also needed for interactions between dimers to produce the cooperatiV1ty Figure 4316 Binding of repressor blocks transcription from pR but activates pFWI PR 3935 3910 2 dimers of Repressor boun cooperatively 0 0 E M E 400 Part Four In Chpt 17 Tmnscnpuoml regulzuonln bactenophzge lambda UMUD39 BASED MODEL OF A LAMBDA REPRBSDR T TRAMER TWO DIMERSD EDUND To TWO ADJACENT OPERATOR SITES lGFX sltes From http waw rte nleen go JpJoulloulmagednaeprolemallsmallm l gx glf 5 Acumen of msmphon al PM u m F zmyfrom the DNA b An aspanale same and glutamate eompnse an zadlc surface mal ls required to sumulale llansenpuen by RNA polymease from PRM e ms ls most lllmlyz duect lntemcuon between RNA polymease and mls pan of hellx 2 a Subsequently several more examples of aeldle sequences servmg as acumtors of nansenpuon have been dlscovered e g GALA m yeast VP15 m mammallan eells lnfected wth Herpes vlns B M B 400 Part Four III 2 Chpt 17 Transcriptional regulation in bacteriophage lambda G Cro protein 1 Mutations in cm lead toahi her fre uenc of 1 so en Cro is needed for lytic infection It blocks expression of the repressor and in fact competes with it for the same operators Fig 4317 Cro protein has a single domain and functions as a dimer Cro Dimer one domain monomer is 66 amino acids DNA binding HelixTurnHelix motif also dimerization operator 0R3 2 Small protein only 66 amino acids that functions as a dimer It still has a DNA binding domain and a dimerization domain The crystal structure shows that the Cro monomer consists of three anti parallel S sheets and three oc helices The Cro dimer is stabilized by pairing between Glu54 Va155 Lys56 from each monomer in the S sheet region This provides two electrostatic interactions the negatively charged Glu with the positively charged Lys and one hydrophobic interaction W DNA binding domain helix turn helix The 3 D structure of the HTH is similar to that of A repressor and the overall interactions are similar ie helix 3 in the major groove with helix 2 above it and alongside the phosphodiester backbone 4 Affinities for operators opposite to those of repressor 9 Cro binds with highest affinity to 0133 and to 051 This turns off PRM thus blocking production of A repressor Binding to 013 has little effect on PL 9 At higher Cro it will also bind to OR and 0R2 as well as 0L and 012 thus turning off transcription from both PR and PL At later stages of the lytic infection early gene expression is not needed only late trascription from PR39 with transcription reading through the t6 terminator to allow expression of S R and A through J 0 The amino acid sequence in the HTH region differs in some residues from that of the repressor and the actual contacts in the major groove differ from repressor operator interactions in some cases This gives Cro a different affinity for these operator sites in fact the opposite affinities compared to repressor 01 Competition between repressor and Cro for the same sites will determine the decision between lysis and lyso geny B M B 400 Part Four 7 III Chpt 17 Transcriptional regulation in bacteriophage lambda H Use of hybrid reporter genes to dissect regulatory schemes 1 Although the genetic analysis has resolved different regions in the operator it was necessary to design an artificial system to test the effects of each region individually This can be done conveniently with hybrid reporter genes Fquot Ptashne and his colleagues decided to let the promoter operator regions of A regulate expression of the 162 gene in an E coli strain a The activity of the enzyme encoded by the 102 gene egalactosidase can be measured quickly and accurately with high sensitivity In this case 102 is the reporter gene b Other examples of reporter genes in widespread use are those encoding 57 glucuronidase chloramphenicol acetyl transferase and luciferase L The production of either repressor or Cro can be regulated by driving expression of C or cm wi e fat in a cell that has WildetVDe lac ie that has the lac re ressor Figure 4318 Mlac hybrid genes Place A cgene under ac control Use acZas a reporter acpo lc lngUq aCZ 5 7 321 Control amount of A ltVgt gt repressor byPTG E co39with ac repressor no CZ See effect of Arepressor by Bgalactosidase activity a This allows one to use IPTG to induce expression of the desired A regulatory protein E7 In eukaryotic cells one would use an appropriate regulated promoter e g a heat shock promoter or a hormonally inducible promoter e g MMTV promoter which is activated by glucocorticoids B M B 400 Part Four 111 Chpt 17 Transcriptional regulation in bacteriophage lambda 4 A few illustrative results a Consider an E coli strain carrying two plasmids The lacZ reporter is driven by wildtype A PR 0 R and the A CI gene is driven by the lac promoteroperator 1 Increasing concentrations of the repressor generated by increasing IPTG cause a cooperative decrease in Sgalactosidase activity 2 One concludes that A repressor will turn off expression from PR in a cooperative manner Fig 4219 A repressor will turn off expression from PR amp PL acpo Ac ApHUq aCZ Bgalactosidase A repressor I PTG A repressor acts cooperatively b In a similar strain except that 0R1 has been mutated one sees that a higher repressor is needed to turn off expression from A PR One concludes that 0R has the highest af nity for the repressor and that the remaining two sites will still show cooperativity in binding repressor They just need a higher A repressor to bind Fig 4220 Mutation of 0R1 decreases affinity for A repressor acp0 Ac Apm AacZ LOF mutation at 0H1 Bgalactosidase A repressor IPTG BMB400 Fig 4221 c Part Four 7 III Chpt 17 Transcriptional regulation in bacteriophage lambda Consider a strain carrying the same regulator construct A C driven by ac p11 but the lacZ reporter gene is driven by the A PROR fragment in the reverse orientation In this case the reporter gene is driven by PRM 1 In this case the increasing A repressor causes an increase in Eigalactosidase activity Although it is not shown in this figure as the A repressor increases further the amount of Eigalactosidase now decreases 2 One concludes that the A repressor can activate transcription from PRM at low concentrations but represss at higher concentrations By testing mutants of the individual operator sites sineg and in combination one can show at it is occupancy of OR an 0 at stimulates transcription from PRM but occupancy of 0R3 will turn off transcription Repressor will stimulate transcription from PRM acp 0 A c 1E7MUH acZ g 123 Bgalactosidase A repressor IPTG A repressor at 0 R1 and o R21stimulates transcription from pm 5 The cm gene was placed under control of lacp to test the effects of Cro on these 1 1 1 1 Lh same cons c s discussed prevrously u e opposite affinities for operator sites as BMB400 Part Four III 2 Chpt 17 Transcriptional regulation in bacteriophage lambda 1 Decision between lysis and lysogeny 1 9 5 The initial pathways for both lysis and lysogeny are identical expression of immediate early and delayed early genes via production of pN All the players needed for the quotcommittedquot steps for each pathway are present The competition between repressor and Cro for sites in the leftward and rightward operators will be key determinants in the decision betwen the two pathways The CH ie the concentration of the product of the CH gene will in turn determine the initial repressor by its stimulation of transcription from PRE Two environmental factors will cause an increase in CH and thereby favor lysogeny a A high multiplicity of infection MOI will generate more CII because there are more templates producing it 1 The M01 is just the ratio between infecting phage particles and host cells At an MOIgt10 the C11 is high enough to favor lysogeny 2 In a way the phage are sensing that it is too crowded and they are better off just being carried along with the bacterium as the prophage of the lysogen b When E coli is starving poor medium the glucose is low and the cAMP increases 1 The increase in cAMP will repress expression of the h A so that the CH will be higher and lysogeny will be favored 2 Again the environment is not favorable for a lytic infection and the phage lysogenizes the host Genetic factors E g h A39 mutations cause a high frequency of lysogeny J Induction of lysogenic prophage 1 N 9 When SOS functions are induced recall this pathway from the section on DNA repair RecA converts to an activated conformation RecA a co protease Just as RecA activates the protease activity in LexA it also activates a protease in the A repressor which cleaves the connector region between the N and C terminal domains of repressor See Figure 4313 The loss of the dimerization domain of the repressor leaves only the DNA binding domains Their affinity for the operator sites is substantially less than that of the intact repressor and they dissociate This leaves the operator sites empty and transcription can begin from PR and PL thus starting the lytic cascade The activity of RecA will keep the intact repressor low and the induced prophage will proceed along the pathway to lysis B M B 400 Part Four III 2 Chpt 17 Transcriptional regulation in bacteriophage lambda Questions on Chapter 17 Transcriptional regulation in bacteriophage lambda 171 POB Bacteriophage A Bacteria that become lysogenic for bacteriophage A are immune to subsequent A lytic infections Why 172 2 cm protein 1 binds preferentially to 0R3 2 turns off transcription from PRM 3 binds to 0R1 and 0R2 at high concentration to turn off transcription from PR and from FL by analogous activity at OL Which of the above statements is are correct 173 The A mutants 01 and CH produce no lysogens so they make clear plaques If they are coinfected into E coli will they produce turbid plaques and if so which phage will be found in the resulting lysogen Occupancy of the A operator by repressor and Cro next 5 problems This gives you some practice with the equations and analyses in Chapter 16 and hopefully provides some insights into the competitions of repressor and Cro as well as the effects of cooperativity These questions are based on a discussion in Appendix One of M Ptashne39s book A Genetic Switch Gene Control and Phage A Let s imagine a stage after infection of E coli with A where there are 100 molecules of Cro dimer per cell and 100 molecules of A repressor dimer per cell The A phage has not yet replicated so there is one copy of the A genome per cell These problems were designed and answered when the estimate of the E coli genome was about 42 X 106 bp you can use the value of 46 gtlt106 if you wish Assume that there is only one genome per cell The volume of the cell is 1 X 10 15 L Binding of the A repressor to an operator a specific site or a nonspecific site is described by the following equations Similar equations apply to binding of Cro to DNA The following values for Ks are based on binding to an operator like OR to which repressor has a higher affinity than does Cro Let R A repressor dimer O A operator site D a nonspecific binding site in the genomic DNA C Cro dimer R Olt gtRO eqn 1 Ksr 2 1011 M 1 eqn 2 B M B 400 Part Four III 2 Chpt 17 Transcriptional regulation in bacteriophage lambda Knsr 105 M 1 eqn 3 C O lt gtCO eqn 4 Ksc 2 1010 M 1 eqn 5 Knsc 2 E1333 2 105 M 1 eqn 6 174 If the equilibrium constant for binding of the A repressor to an operator site call it Ks r is 1 X 101 1 M4 and the equilibrium constant for the binding of A repressor to a nonspecific non operator site on the DNA call it Knsr is 1 x105 M l what fraction of the repressor molecules are free ie not bound to either the operator or any nonspeci c site on DNA For simplicity calculate how much free repressor would be present for a A phage that had only a single operator not the 6 each with different affinities that are present in vvild type A 175 Using the same values for Ksr and Knsr and the same simplification of considering a single operator site as given in the previous problem calculate the fraction of operator sites not bound by A repressor For this problem ignore the effects of Cro ie ignore the competing equilibria of Cro for O 176 If Cro has a 10 fold lower affinity for this single operator site but is also present at 100 dimers per cell what fraction of the operator sites would be bound by Cro Again for simplicity ignore the competing effects of A repressor 177 The results from the two previous problems suggest that the A repressor would quotwinquot in a competition with Cro for the operator given its ability at a given concentration to fill more of the operator sites This fits with the 10 fold higher value for Ks that we are using for repressor compared to Cro To take another look at this divide eqn 2 by eqn 5 and derive an expression for ROCO What do you calculate for the ratio of repressor bound to operator to Cro bound to operator 178 The binding of A repressor to the operator sites 0R1 and 0R2 as well as 0L1 and 0L2 is cooperative ie the binding of the first repressor dimer increases the affinity of a second repressor dimer for the adjacent site This can be modeled quantitatively as follows Given that repressor binds to a single site with Ksr 101 1 M l that means that the free energy AG for binding to DNA is about 15 kcal per mole you may recall that AG 2 RT In K The protein protein interactions of the repressor dimers will add a AG 2 2 kcal per mole to B M B 400 Part Four III 2 Chpt 17 Transcriptional regulation in bacteriophage lambda the affinity of binding two repressors to adjacent sites so the effects of cooperativity increases the apparent Ksr to 3 X 1012 M4 How much more repressor is needed to fill 99 of the operators for non cooperative binding than for cooperative binding to adjacent sites Let s consider an in Vitro situation where you are adding increasing amounts of repressor protein to a short DNA fragment that has the operator site this allows you to ignore the effects of binding to nonspecific sites Calculate the R at which ROO 99 Since in the case of cooperativity the two adjacent sites will be filled almost simultaneously consider these adjacent sites to be equivalent to a single larger binding site for repressor


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