the remote control of transcription, dna looping and dna compaction biochimie-1990-1991 m. amouyal

8/14/2019 The remote control of transcription, DNA looping and DNA compaction Biochimie-1990-1991 M. Amouyal

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Aircrinie 1991)j , 12611168O Socirarmdscdebiochimierbiologicmolcuta;e/ lsevier,eis 1261Revierv

The emote ontrol f transcription.D\A looping ndD\A compactionM Amouyal

U^it depbsicochinie des nacrcnatcut.s biotogiqu.s. dpo enenr de bioLaeienatculairc,25. ,e du Doa? Rou,7524 pd,is CJ", t). Fron.e "(Rcceived May 199li accepted9 Seprember 991)

smmry IRNA svnesis c be conL$lled t som. disrece ftom rhe stln of Escoplion in eukaryotesnd prokalyores. I! isscner.llv issumed rh.t the dis.d elementsof the 6ms6pronrl michinery directry inrenciwtur rr:eproimd a""iens, i..e th;9f ll-..hid- il 1.b.q,qN roopomariondEdsdipiior o berrecGdy 1isia"""r.ti,Ln.tr," i"". ;tii;!;J;;.li;:nB. ueu oneniJiron.Uref con(enEronGesponsibteof a .ir- or a farrffeit of rhe DN sequenc*), o oNa --pcti.. tctulmarin. rhe corespondilg n v,ifo ed i iiyo sirullions have been crilicaly ex;,nio r"i i',i,t"i.i'"y.i" ;."ii:;:re-gllatioD ftransoiptio / enh3nceF .epression DNA looping/sliding / chmmatin / DNA compaction distnt eguttionD\-protein interctions

DNA loop formtion was rst considercd or rep.es-siot\of the E coli lactoseoperon l]. This modeiwasbasedoI1physicochemical onsidetions.Th rpres_sor exisring at a low copy nmber in rhe cell wassupposed o be Eappedby rie large amounrof non-specilic D,\-4. Then. nrrural fotding of DNA (DNAIooping) helped ro xnsfer the proiein ro it specificsrte, he a. operrrorOl. Thjs operxtor s localedcloseto rhe stan of ra$cription. Th discovery of remoteoperators by seqencehomology with O I ar.\d.n litroassays seefor erampie [2]) and r}le finding that eteameric /ac repressorneedsonly two subunits!orecogmze the opentot t/| l)if.o led ro a varint of thisview. The prorein wrs now supposed o be shblychelJledro lhe proxim3l 3nd disrnroperalo$ in rh;l^oop o tht dissoci3rion rom the proiimal operarorOl was prevented [3]. But in a -tust analyiis rheconslutile mutauons for tle /d. operon wcre al lfound locared n rhe firsr operaror egion ard repres-sron at J dtstr lce as wel l as DNA looDils $ereprem3ruretydiscuded (for dditionrl historic-delailsabout ac reprcssionat a distance, ee Eview[4]).The contribution of rcote elements,know asnhancers, to mnscriprion and orher biologicalprocesseswas rcally discovered n eukryotes,n theearly 80s, without any referencero DNA looDins.

Erlhance6 are specific DNA sequencesaid the corre-sponding DNA-binding proteins. wlren irvolved intrrnscrption, they ar responsible for rhe high (orlow) Ievel of gene expression hen rhe proximdelemenls of the nrrscriprion machinery iniriste (orrepress) rmscription poorly by emselves.They canluncnon ovec considenble distxnces rom the site ofinitiation, in the normal or inverted orientation f5-71.The first proktryolic counteq)answere found-soonafterwards in the galdctose adarabinose operons and.DNA looping was gdn p(oposed to eipufl rheobserveclong rtnge effecrson repression f theseandother operons (for reviess about prokrrlodc en-hancers, ee 8-10] and or a crirical evaluarionof tevarious modes of remote control in prckaryots ndeukaryotes, ee 1]).Paradoxically, with regard to lhe long distanceeffects sressed above, he need!o prcve the diect inier-acon bet\reen rwo proteinsnormally binding to ad-Jacent itesxlso led lo D\A looping. Upon sepantionof rhesires,D\A flexibiliry w3i inaide;dly found robe responsible for rlile cooperarjviry of bindirg ofph3se .t represson. n ddition ro prolein ffexibllirypreviouslyconsidered 12]-Funhermorc, Jn imporlrnr srep for DNA looping\rrs providedby rhe \lorLs on rhe sponhneousclcli-


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t262zrtion of a DNA fragmen! [l3, 1'1]. The corre-sDondinsk'nowledseof the faclors which affect thispiocessinfluenced mofe or less slrongly severalstudie.. l " inlv, lhe Lieendcncef DNA clcl izt ionon rhe di.r3Jfrcr. Jejiberrtely usedLo prove D\AlooD fofl:nirtion in viro for the first time i! a ldcsvsiem l i l . lnJerend. 'nt iY.h extensivemnsl ionoi thc cyclizarion'studresnto tct.msof D\A loopinglowed to predict some of tlle conditionsnecessaryfr nroiein-DN looD fol.malion r, viro, when thesesrrui lures cre only posiulJLedlol . \ loreover' heuse of ororein-D\A Looos s modcls for D\A circle\lad to in r.tro and in itl,, biophysicalapPlicationsIl7. l3l *hich wi l l nol be dcvelopedn this eviewiesrricrJ o rne unctionrl rspec!sf D\A Iooping.In lhe 6rsl sctionof this paper, he analogywithD\A cycl i , ,r l ion s used as 3 guidel ine o presentsme o[ the requirements tor prolein-D)i loopformrtion in vro. such as the distance between thesiles. heir helicxl positioninS.orienlllion andconcenrl;on, wilh implicit reference o the Lj]own proper'ties ofenhancersand new developments6lst and alsosecondsection). n the following sections, omeof t}lerecent information about their infiuence olr the remolecontrcl of trnscriplion s examinedwilh emphasis nDrokarvoticstudies. The last seciiot discusssmoi;pecifiiaily the possibleoccurrnce f othermodesof

Dissectionofa proteir-D\_A loopThis section s restricted o the works which iliustnteciosclv some of the stnrctunl requkemnB or DNAloop ibrmation observed wilh purilied components.Th E cali la. opertor-repressoroop is a well docu_mented system rom this point of view (t.11, nd thisreview). it is used hete as a convenientpresentationard.DNA clcli.ation and DNA loop fornation are ruledbj DNA lleribilirtFor DNA cyclizxtion this ws indicaledby the exist-ence of n optimal length of lhe fragment around400 bp [13, l3a]. Whe rhe disrancebelween wo ldcoperlorswas close to lhis value, 535 bP precisely,electron microscopy revealed entire fields of DNAloops \rhereas below u6l or above (1873 bP)(l I Amouyal. unpublished observation),only one-thi.d to halfof the proponioo of the 53s-bploops wasobserved with rhe constructions hrt we employed.This is te only k-nown observalion of a.1 optimaldistnce or DNA Drotein-looo ormatiot. This m&\i-mum which reallyioneldes proteit-DNA looping toDNA cyclization indicaled hit DNA fiexibilily is in-hcrcnl nLhe rocess f prolein-DNAooping.

Loop fomJtion carl occur eten \ahenring closureis eenrrllv not tossible. below 150 bp. For lhesesh;d di.r;ce., o\A onty nceas o ben nd no( tocirculrrize for e prolein lo contrct simullsneouslyboth sites,rs first rponed [or cl repressor 5:. and6l-bp loops 2. l9lr. lt was also Eue for ldc rePrcs.sor 61- l+7-bpoops16t. Therefore.ecausefDNA flexibility: DNA loops can form on a wholeranqe of dishnces, from a few ten base pairs tosevral thousand bse pai6. Addilionally to lhe laccaseaiready delailed, long distance DNA looping hasbeen directly observed for the E coli R6K plasmidrepLrcalion nh:rrcer-origin nteraction 201.LtreHin-invenion system of Salmoell\ l2ll, the E caliRepA protein and minj-PL phsmid replic.rtion l22lnd E coli tlpe ll endonuclease Nxel [23].Intermediatedistanceshave been observedwith thebffteri3l transcripdonrl activstor NRI con|3cting rheoso-conldning \A polymersse [2,1]. the DeoRreDressor f the E coli deo operon l5l and for theassociation of DNA-bound progesteronereceptoG[26]; ff regardsDNA looping,DNA is consideledasa flexible tfue.d without any biological content whichobcys the theory of flexibilily of polymen [27]. As aresult. ir is indifferenl lo rny modificroo, even apartial replacement of DNA [23], as long as thetthread'does not become rigid. This property hasbeen used to discriminate DNA looping fi1cm othermecha,'rismssee ast section).SPecificsequeces rproteins inducing D\A bendhg are not required forDNA loop form3don. except when DNA becomesrisid, ie for shon distrnces. n lhis case, e sequence.rhl srucrural defects of DNA or the Droteinswh-ichmodify D\A flebilily belweenthe sites of interestmry f3vor (or disfvor)DNA loop foimauon.Thus a site in a 5 bp TTTAI sequetce between thetvo Id. opemtorsOl aJldOJ is hlpersensitive o theahrck of v$ious chemicalprobes n the Presence fnegrtive supercoiling1291.According. o the aulhors,this sequence becomes Panicul3rly suscePtibleto DNA melting and bending when submined toto6ional sess.severrl biologicrl processes equire the assembly(or disxssembly)of nucleoproteic trucrures 301.andD\A bendins assislsDNA looping in some of iheseproc."ser. Tie )" phege inrelrs protein flnt) isne of the proteins responsible or inlegmtive re-cobination between the phage and bacleril sites andfo. excisive rccombinationbetweenphagesiles. Thebacterial inlegration host factor IIIF prcmotes thesimultaneous inding of Int to its sitesby bending heDNA in the middle of the 50 bp seprating the sites[31]. A tutly bent DNA sequence 3n 'A-Eact')ca.n rcplace successtully he IHF binding site [32].The catabolileactivalorprotein (CAP), known to bendDNA. could rephce IHF t{ith lhe s3me effeci [3:l


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D:!.'A oopin-qor trmscdpticn n p.ok.$otcs md euk.rlotcs l :6iIHF aiso stimuhtes lhe interrction of oJr-R-\A Dolv-m.' rcsc i \rfA. rnc uJn,criprionrlact i raLo_rrnilrogen fixation operons of K/erre11dncumone[33,3.1]. There are also some indications that IHFhelps he i$sembly of p.oteinsat the origin ofpSCl0lplasmid replication 35]. Simil.r functioshave becnproposed or the E coli histone-likeprclein Hu [35]and CAP [17, 32, 37]. The fonnation of a nucleopro-teic assemblybetwecnE coli 3al repressor nd RNApolymensewould lead o gdl repression37,33].DNA c'tcli:ation is also ruled bJ DNA torsional rlgi-droIn other woads, he awo exEemitiesof a short DNAfragmenthave to be exactly in phas, e separated yan integEl number of helical tums for covalentjoining. Howeve.,due !o additiviry of the elemearythermal fluclualions of DNA torsion at the level ofeach bllse pair, this phasing requircment is pro-$essively lost when rhe sizeof the fragmert s incrca-sed 39].when ring closure is mediated by proteins, theproteins may affect the strctu.e of the loop. Contraryto DNA cycliztion, the two sites do not have to bephsedor l DllA to ensure .tlespeciccontacts.But ifmultiples of helical tums are introducedbelween hetwo sites. their initial o.ientation will be restorcd.Thus, NtrC ard osr-tu\A polymense dicdy interact,though ying on different facesofDNA [41,42]-For ldc repressor, the two sites had to be in phase oinduce stable loop formadon, as evidenced by geiretardationassays 6l. k is genelly nor emphasizedthat the sites on the protein are not necessarily aral-lel, though t}lis may be crucial for the nalysis of thedta as in I I 8], or simply for a better knowledge of tllestructurcof the protein. ac reprcssor s composedoffour identical subuniN nd binds as a dimer i3l. ThearTargement of the subunits is not known un-equivocally, due pard], to lhe poor crysrallistion of ldcrcpressor and lack of high rsolution X-ray diffrictionstudies 43]. A parallelan gementofthe two dimersis genelly postulared.as in [14], but leFsedrlzurangementhas also been proposed (reviewd int45l). Wen the two phasedoperato$ wer includedin a rela-\edmini-circle. only the configurationc[awnin figure la was observedby electror microscopy seefi-q 2b of [18]) indicating a parllel fl.]gemem. Atelraedril orgnizadon would have led to an eighlconfiguraiionof the circle (lig 2b).The periodic fot'm ion of stable oopsresuhs romstiffnessof both DNA and protein,a helicalpilch forDNA with a given value. and a unique conngurationof the loop- Dissociatior assays of preformedcomplexes or associalionassays n the presenceofIPIG disphyed an apparenr ack of phasing cquire-

( q ) ( b )Fig 1. Prolein rchitectulend oop oflnation n a rcl&1edcircle: he contacbon the prolein paltel (r), oflho-sonal b).ment in that s!ble loops were formed at any distancebetween 158 and 168 bp on highiy supercoiledDNAwith tac repressoru6l. For these same distaices onthe ftagment, DNA loops were also formed when lhetwo operator siteswerc not in phase,becauseof theamplitude of the natu..al movement of torsion anddetorsion.However, hey were essstable,as ndicatedby the smea.y aspecl and wak density of the bandassigned o the complex.DNA supercoilingampli6esthis phenomenon, since it naturally brings closer thedifferenr pans of DNA. It lso changes he helicalpitch of DNA and the dislxnces, previously in-appropriate or alignmentofthe siteson the frgmen!,becomesuitableon supercoiledDNA [18].Protein DNA loopfomation h'ith t1|.]-mmetricesln large oops such as the 535-bp ac loopspreviouslymentioned,DNA can adopt e two configurarionsa)and (c) drawn in figure 2. Below 150 bp, only oneconfigurrtion(d), equivlent o (a), wasobserved seefigs 7 and 8 of u6l).Both ldc reprcssornd the 'ideal' lrc operalorusedin these experiments are perfectly symmetrical. Ifley werc not symftetrical, the existence of twoconfiguralioswhen DNA has lost ils torsionl ndfiexional rigidities, clerly shows hat DNA looping isinsensitive to the orientalion of the site a! thesedishncesbut might senset over shorl distances.For this to be tme, lhe asyrnmetryat rhe evel ofthebinding sile must be ssocirtedwilh asymmetly ar thelevel of the protein-protein contacts. Enhancerp.oteins are often homodime$ with a two-foldsymmey when they are tiee in the solution.However, the protei! might deviate from symme-yoncebound o its site.


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1261A . . . . .@ Q X i ; l

Fig 2. S,(e invenion ud Ioop formrlion: rhe aow indicates the coding seqence. The asynrmery of the sile issupposed 0be Fansmitred o the prolein.Consequently.heonenution of e s;!e wrr1r cspecLo rhecodin! sequencesindicated b), the asymmeeicri shape of the pteiri. rn *exmple,a loop is fomed when the wo siteshave he sa.neo.ienBdon ( d d). Whcn rhey re in ihe oplosiie di-rccnon, lhe two connsultions of DNA (b) ed (c) possible or long distances d a loop can form (c). Ai shondist.nces.only one DNA confi-qurarions possible e) andthe loop cnnnol fom (f).

DNA c\.li:aIioa depcnds on DNA concentrctionThe cycliztion probabilily or j-factor represents infact the DNA concenEtion such thrt cyclization nddimerizationft]1eragmentreequally robable3,1.11.The scheme of figule 3 tlinslates this situation inrolerms of DNA looping for rhe ldc system. At lowconcenations of DNA and rcpressor, th ina-molecular .eaction for DNA, ie DNA looping. isfavored over the inlermolecuiar reactions and theformtion of 'sandwich'-typespecies.Tandems'arefomed in medium concenEations. All these slructurcshave beenevidencedby gel retirdationajld em assays,on lhis basis 6l .Remotecontrol of transcriptiolDistancesOriginally noticed for their abiliiy to function at adjst3l,]ce.nJr3ncersow frequenliyappear o operatebolh from a few ten to sevral tirodsanas f bases.Some examplesof such a peafoamanceog proka-ryotes are provided by rhe nioge.l regulatory proteit

FtrE I : EtftFig J. Influen.e of D\A Jnd proreir concenrrrronsonD\A loop iomrrion. wirn 1dc etre-or rs e proren.

I

NRI (alsocalled NlrC or clflc, for reviews about rhisansc.iptioni activtor, see [46, 46a]), whichoperrres from 30 to 1,100 p upseemrnd = 2000 bpdownsr.'rm [4], a7l. the ninosen firation proteiiNifA (= 100 o 2150bp [48]), the DeoR fepressor 66to 4600 bp t49l), lhe Hin ehancer for site specicinversion and recombinelion = 100 to 4000 bt [50])and to a lesserextent,by the AraC protein involved inrepressiono[ e a/aB]Doperon 32 to 500 bp [5LI).The lolv affiniry of rhear3c prorein or one of ils siteslimits tIe ringe of operational distances [5 ] I.When both shon and long disnce effects havebeen obsewed, a featruealreadyconsisrentwith DNloop formation, there is geerally some more dkectevidence or DNA looping.Loops could be visualizedby electron microscopy or i\RI [25], DeoR [25] andHin [211. They were detectedelectrophoreticUyncase of arac [52]. NifA has not been purifed yer.However, it is closely relared to NRI. Like NRI, ilfunctions with d5a{ontaining holoenzyme rarher tiunoro-holoenzyme [53, 54]. The protein synrhesized i''rriro is required for activation of a NifHlacz genefusion [53]. Chemical 'footprinting' shows ihat theproposed distant sites are occupid i, ytvo [55] andthat like i\RI, irs presence s required for opencomplex formarion airhe promoter n r}le cell [56. 5?].Lite for some of re above-menlioned enharcels-indirect evidence for D\A looping exisls t33l arwill be presend aler-Prokaryodc enhancershave generally lost the mejorpart of lheir power above 5 kbp (see [58] for DeoR,[48] for NifA, [51] for A-raC, [59] for T]TR represso.and distant rcprcssion of the E coli aroF promoter,160) lor lac rcprcssor). Above this same value, ringclosure probabilily is also very weak, just as low asbelow 150 bp under lhe conditions dened by Shoreetat [13]. An impoftantcosequences !ha! prokaryo-tic DNA is not sikingly differenr from 'naked' lhearDNA in solution.Furthermore, rcgulation of gene exprcssion fromdistntsites s much more equent l1 eukaryotes h3nm prokaryores 61]. Thus, n E co1i. he few ex$plesof repressionor acdvalionst a distancewith a mrur3llocation of the distant sile from 90 to 900 bD. wouldconslituteex,cep(ions- n rhecondrry. in higher euka-ryotes, a t)licai promoter transcribed by fu\A poly-merase tr would irclude an enhancer sequence ocatedor funclioning 't least up to 10 kbp dom rhe RNAstart' [6, 62]. In eukaryotes, the chromatin is highlyorganized inlo nucleosomes and higher orde! struc-tures, while DNA packaging in bacteria is considercdmore labile, ough not much s known [63]. The truedisrnceof rction of a eukrlotic enhaicer m3y thenbe shoner rhen rhsi clcuhred from naked DlA.Along these same lines, the distance effect shouldrcflect the differences in DNA condensation between


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eukal-votcs nd proka4otes and, for me, *ill consti-ttc a tool to detec! such differences. ! might be rel-ated to this poin! that some mammlian enharcerswhich work over a few kb r'nviv, cannotwork over500 bp ln |ro, in lhe absence fchromtin 1281.Helical orientatianof the enhancerseqltenceSdmulation sometimesdependson t}le corfec helicalpoit ioning f thc enh cer *i th re(peci o e proxi.mal elements.This is reverled by itsenions (or de-Ietions)ofsuccessivelyodd and even numbeGof heli-cal tums between the sites of interest, at rcasonablyshon distances.as expected if DNA cqukes sometorsional stiffness (see for example U'7,33, 42, 51,6,1- 61). An exEeme situatron is thtt of adjacentproteinssuch as he I phage epressors 2l .It is generallynot stressed hat a periodic stimu-latio is ot pel .re a prcof for a direct cooperativeinreration belweer the proteins of interesl. Ivlediationby another protein [2], 67], DNA conformationalchanges a! a distrce ol the 'sandwiches' of gule 3may exhibir the sarneproperty.In some caes. on the contnry. tlere is someevidence in favor of DNA looping while thre is no orpoo. helical pe.iodicity for eniancer function. (Il isassumed lhat all rhe length of the spacer belween 0and l0 bp has been explored, since t}le interaclionbetween te t\lo proteins may not be optirnal in thewild tlpe situation.) Thus, the DeoR reprcssor hasthrec or four DNA binding sites and a loop might beformed for different helical positionsof the sites [25,491. Only weal( periodicity was observed for lacrepression n vivo, even arcund 80 bp between an'ideal' /ac oDertor and an ooetor with a constitutivemuixtion ( Kr:imer, PhD thesis, 1988, Cologne:Germany)- DNA is supercoiled in bacleda and thesituarionobserved n virro u8l might be reprcduced

Similarly, a strict alignment is nol required i|t viv,between the cal.t activator and the 'TATA boxelementsof the yeast gxlactokinasegene 68]. This isalso true for some other systems quoted in this work(seealso [69] for a rccent example).According to thauthors, an especially strong teraction between Gal4protein and R-\A polymerase I], forcing DNA totwist, might be responsible or this situation,as wellas the flexibilily of the interactingproteins(seealsot6sl).The degree of periodic stimulation also depends onthe affrnity of the enhancer prctein for iis site- Asong intection in conjunction with the thermalguctuationsof rhe rwisr likeiy stabilizeDNA loopingat ny distance.Such a reducedeffect of phasingwasobsewed r vivo when a high NRI binding site repla-ced a iow affinity one [,12].The use of ideal' lac

DNA looping or rs.riplion it prokaryotesd eukaryor.s 1265operalon insteadof lhc wild type ones had probablythe sxme effecl, as snessedn uSl. The wl operalorsindeed forln less stable oops t}n the 'idexl' ones ?rr, under the sameexperimentl otditions t701.Afortunateconsequences that full inducibility of the/dc operoncan be ensured 18].Orientation bith respect o he coding sequenceSequencenvelsion is genenlly perfomed at the samelime as the site is moved away from its natural Io-cation. It is found that inver:ion has nearly no effec!on stimulaon (numerous ex3mples are given inreviews 5,7.621r. f luenceofsequencenversion nexprcssion over shon distances s less docuented-The schemeof gurc2 suggestsha! the bidirectionalstimulrtion is nor ecessarily mainlained over shor!distmces if the enhancersequences prt of a loop.This is in principle a way to recognizeDNA loopingfrom olher mechanisms r', riro (!t Amouyal md Bvon wilcken-Bergmann,work in progrcss).Intracellular concentrationsof enhancer DNA andThese concenations classically vary for technicalreasons eg multi-copy plasmidsor overproductionofthe protein) or in rcsponse to envircnmental chnges,as n caseof itrogendepivation for NRI [46,46a]. Agiadient of concentration of rcgllatory prcteins canalso be produced naturally as repofted f DrcsophiLtdevelopmen![71]. These concentalions can also, tosome extent,be usd as a physicochemical a.ameterfo. the cell.High amounls of DeoR reprcssor have the sa.rneeffect as distant sites or rcpression n the deo operot[72]. Thus, tie remole oprators ndirectly incresethe local concentnlion of reDresso..a function of theloop sessed y Recordand aoliabolalors 15].The cooperativily of repressionobserved betweenrhe three /dc operrrors s lost wi!h a dimeric repressorunlble to aggregate into lhe tetlameric foam and toinduce DNA loop fomation in the wild-q.-'peiruation[?3]. This result defrnitively stopped he conroversyabout the involvement of the dislaot operatorsitcs inrepression of the /d. operon (/n,rodrcuor). lt could beobtained because he dimeric rcpressorwas deliber-ately used n combintionwith rhe low chromosomalconcentmtions of rcpressor and DNA, cot?ry toprevious rl vilo works. \\lth higher concenEctions frepressor(iqrstrains), e cooperalivily between thethreeoperalorsiteswasgreadyreduced,&1dno differ-ence n the lelel of repressionberseen he teL'3mericand the dimeric repressor wals observed. Therefore,the high amountsof repressor d nduced he predic-ted i/! viva trnsirion lrom DN looping to aoer


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t266mod of reprcssion(pfevious srction and 116l) andlhe three dc operirlorswcre now sepffately occupied('ndem' formation).Concentrationsare lso critical for the c!s/,/dr?reffect ol enhancer sequences. n physicochemicaltelms. this situirtion coven in(a- and ittednolecularreactionsor DNA. Some echanislssuch as slidingimply that the enhancer unctionsexciusively n a c,s-posilion.On the conFary,DNA looping,normaily c,r-acting, cxn be compensated in ,rdrJ. Up to ow' thearans-effectof Eanscdpdonl enhance.s has beenexclusively observedwhen the two siles of interestwere mintained in close proximity, r/ I vi ro, withcatenanes74, 751 or a non{ovalent bridge [28], andin vi!o. in rhe natural phnomenaof tsansvectionnDrosophila li6l. The question has been raised ofwhethe. i! would be possibleto detecrllelic inter-action of the tnisvection type in organisms wheretherc is no apparent somatic paidng of chromosomes[76]. A lrdns-interactioobetween wo sitescarried bytwo unlinked DNA moleculesshould be observedbyra,.rs-complemenlation ith high amountsof DNA inlhe cell, in order to form 's.ndwich'-t?e stluctues.The NifA aperiodic activation of NdH promoter onmulti-copyplasmids s probablyan exarnpleof suchatd,?s-effec! [33]. Along the same lines, acdvation ofthe proka..yotic g/r,4 and NfFl genes is also possiblein le absenceof enhncersite ad the presenceofhigh concenaliors of lhe conesponding rotehs NRI[47,74] and NifA [7?]. The future will tell if equiv-alenrsiluationscan naturally occur.Redun.lanct of infonnat io nEnlnncer sequencesare generally repeated 5,62].They may allow cooperative interactions between thecoresponding proteins and facilitate the contactberween he enhancerprctein ad the components flhe anscriotional machinery at the gromoter. This ishow rhe l repressors tiniulate eiression tom thep@ prcmoEr ll2l and how NRI would also activateexpression [42]. The ennancer protins rnay alsocooperte no! by direcrly louching but by simul-taneously touching some componenl of the tran-scriptionalmachine.y [78, 79] or by aggegating ([5]and refrences therein). ReDeated sircs cxn alsosimply increffe the probability of sigle loop for-malion at one site or favor the fomation of multipieioops,reinforcing 11he functionsof single oops,asobserved or the d"o opercn [25].The other modesaf remotecantrolTr'rppingof the protcin by the distnt site and slidingto the site of interest is ofter viewed s e mairaltemative o DNA looping [5, 80]. Bur retenrior of

enhancerctivity in trarr w|rs obseived n cxse of rtotal or peftial disruption of the DNA Eack [23, 75].Em did not revealseveral unspecific)ocatiosof /dcand deo rcpressoron a frgment (seeall em picru.esin [16] and [25]), though for 1dc repressor, he highaffinity 'ideal' Iac operalorswere used. Such exper-iments rule out sliding as exclusivelyacling undei theprcEise in itro conditions undr which they weaeperformed. 1n vivo, the use of psordlen-modilied DNAbetween the SV:10 enhancer rd tlle humn B-slobingene hibited expression f this gene 8l I. BurF\orr-len ahels DNA propenies [23] Jtd eferenceshe.ein).On the cont ry, Nif activationwas not inhibited bylac repressor binding in the middle of DNA sequencebetween upstream and downsteam elements in ,iva[33]. Also, loss of cooperativity i' l vivo betvr'eerdistant a.ndproximrl sites with prcteins engineered sorhar they becorre unable to aggregateF3,82,831dirccdy suppons he existenceof DNA looping in thecell. This is especially ue when the loop irvolves aunique and multimeric protein such as ldc rcpressor,since otherwise, the qestion of possible intemediatefactors mediating the p.olein-protein contacE is isedir the absenceof an ,n vro proof for direct htenction.Yer, sliding might ssist DNA loopilg. Rece bio-physicalexperimentssuggest t might be the case orlac pressor and wt operlors [8.+].A distantly bound protein can also block Rr\Aelongation [66, 85, 861. The enhancer can also inter-fere with other regulatory proteins and other processesat the same location: the rcpressor hhders CRP ac-tivator binding when located upse"I.n from lhe EarI-scdption start in the E coli galactose operor| 140,81\,while there is no in yr'to evidence for DNA loopingwir this rcprcssor [37]. However, it presumablyexists in rivo. lndeed, a gal to tac opetor conversionin vivo allows efficient repressionof the gdt opercn[88], and this repression s rclieved when a dimeric/@cepressors used 38]. Funhermorc,various nodesof rcmote conEol can be assumedby DNA looping, assEessed y Flashnerand Glla [85]. in caseofbiock-age of RNA elongation.Finally, in somes cirses, environmental chrngesinduce the rradsirion from DNA loopig to anothermechanisrn. Variations of concentations lead to scha situarionn6, 731.Vadalionsof DNA superhelicity,due to an osmotic shock or anaerobiosis [89] andreferences therein) or DNA trinscription [90-92],rrlight also favor such rnsitions with low f6nityoperirtors o ensure oop breakage sl.AcknowledgentDavidPenin s aclnowledgedor cful re.ding of thep.esen!anicle n 1990.


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D\.\ looping or scripllor i!1prhrlores .t ekrores t26'1

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the remote control of transcription, dna looping and dna compaction biochimie-1990-1991 m. amouyal

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