Aircrinie(1991)

7j, 12611168
O Sociérairmçdscdebiochimie peis
erbiologicmolécuta;e/Elsevier, 1261

Revierv

Theremotecontrolof transcription.
D\A loopingand D\A compaction

M Amouyal
U^ité de pbsicochinie des nacrcnatécut.s biotogiqu.s. dëpo enenr de bioLaeienatéculairc,
"
25.t,e du Doa?ù Rou,7524 pd,is CèJ", t). Fron.e
(Rcceived7 May 199li acceptedt9 Seprember1991)

sùmmâry ûIRNA svnùesis cù be conL$lled ât som. disrece ftom rhe stln of Eùscoplion in eukaryotesând prokalyores.
I! is
scner.llv issumed rh.t the disÎ.d elements of the 6ms6pronrl michinery directry inrenciwtur rr:e proimd a""iens, i..ùe th;
9f hid- il 1.b.q,qNÀ roopromarion
aÎd Edsdipiiorco beârrecGd
byt1àisia""" r.ti,Ln.tr,".i"". ù;tii;!;J;;.li;:
nB. ll-..
ueu oneniJiron. Uref con(enErùon Gesponsibte fof a .ir- or a farræffeit of rhe DNÀ sequenc*),
ctulmarin. rhe corespondilg n v,ifo ed iÀ iiyo sirullions have been crilicaly ex;,nio r"i i',i,àt"i.i'"y.i"Â -o oNa --pâcti.. t
;."iîËi:ô;:

re-gllatioDoftransoiptioû / enh3nceF/.epression / DNA looping/sliding / chmmatin / DNA compaction/

D\À-protein interâctions distânt .egutâtion /

DNA loop formâtion was ôrst considercdfor rep.es-
siot\ of the E coli lactose operon [l]. This modeiwas
basedoI1physicochemicalconsideÉtions.Th€ r€pres_
sor exisring at a low copy nùmber in rhe cell was
supposedto be Eapped by rie large amounr of non-
specilic D,\-4. Then. nrrural fotding of DNA (DNA
Iooping) helped ro ùxnsfer the proiein ro it specific
srte,the la. operrror Ol. Thjs operxtoris localedclose
to rhe stan of ra$cription. Thè discovery of remote
operators by seqùence homology with O I ar.\d. in litro
assays(see for erampie [2]) and r}le finding that ùe
teûameric /ac repressorneeds only two subunits !o
recogmze the opentot t/| l)if.o led ro a variânt of this
view. The prorein wrs now supposed to be shbly
chelJled ro lhe proxim3l 3nd disrânr operalo$ in rh;
l^oopso thât dissoci3rion from the -tust
proiimal operaror
Ol was prevented [3]. But in a analyiis rhe
consùlutile mutauons for tle /d. operon wcre all
found locared in rhe firsr operarorregion ard repres-
sron at J dtstrlce as well as DNA looDils $ere
prem3ruretydiscuded (for âdditionrl historic-delails
about /ac reprcssionat a distance,seerEview [4]).
The contribution of rcûote elements, knowû as
€nhancers, to mnscriprion and orher biological
processeswas rcally discoveredin eukâryotes,in the
early 80s, without any reference ro DNA looDins.
Erlhance6 are specific DNA sequencesaid the corre-
sponding DNA-binding proteins. wlren irvolved in
trrnscrption, they ar€ responsible for rhe high (or
low) Ievel of gene expression \Àhen rhe proximd
elemenls of the nrrscriprion machinery iniriste (or
repress)trmscription poorly by ùemselves. They can
luncnon ovec considenble distxncesfrom the site of
initiation, in the normal or inverted orientation f5-71.
The first proktryolic counteq)answere found- soon
afterwards in the galdctose aÂdarabinose operons and.
DNA looping was âgdn p(oposed to eipufl rhe
observecllong rtnge effecrson repressionof theseand
other operons (for reviess about prokrrlodc en-
hancers,see [8-10] and for a crirical evaluarionof tÏe
various modes of remote control in prckaryot€s ând
eukaryotes,see [1]).
Paradoxically, with regard to lhe long distance
effects sressed above, lhe need !o prcve the diect inier-
acùon bet\reen rwo proteins normally binding to ad-
Jacentsites xlso led lo D\A looping. Upon sepantion
of rhe sires,D\A flexibiliry w3i inaide;dly found ro
be responsible for rlile cooperarjviry of bindirg of
ph3se i.t represson. in âddition ro prolein ffexiblliry
previously considered[12]-
Funhermorc, Jn imporlrnr srep for DNA looping
\rrs provided by rhe \lorLs on rhe sponhneousclcli-
t262
zrtion of a DNA fragmen! [l3, 1'1]. The corre-
sDondins k'nowledseof the faclors which affect this
'influenced
piocess mofe or less slrongly several
studie..\l"inlv, lhe LieÊendcnce of DNA clclizâtion
on rhe di.r3Jfrcôsr. Jejiberrtely used Lo prove D\A
looD fofl:nirtion in viro for the first time i! a ldc
svsiemllil. lnJerend.'ntiY. lhù extensivemnsliÛon
oi thc cyclizarion'studresinto tct.msof D\A looping
lowed to predict some of tlle conditions necessary
fôr nroiein-DNÀ looD fol.malion ir, viro, when these
srruiluresucre only posiulJLed llol. \loreover' the
use of ororein-D\A Looos1s modcls for D\A circle\
lèad to in r.tro and in itl,, biophysical apPlications
Il7. l3l *hich will nol be dcveloped in thisreview-
iesrricrËJto rne lunctionrl rspec!sôf D\A Iooping.
In lhe 6rsl s€ction of this paper, the analogy with
D\A cycli,,rlion is used as 3 guidelineto present
sùme o[ the requirements tor prolein-D)iÀ loop
formrtion in vùro. such as the distance between the
siles. their helicxl positioninS.orienlllion and concen
Ûrl;on, wilh implicit referenceto the Lj]own proper'
ties ofenhancersand new developments(6lst and also
secondsection).ln the following sections,someof t}le
recent information about their infiuence olr the remole
contrcl of trènscriplion is examinedwilh emphasison
Drokarvotic studies. The last seciiot discuss€smoi€
;pecifiiaily the possibleoccurr€nceof other modesof
Dissection ofa proteir-D\_A loop
This section is restrictedio the works which iliustnte
ciosclv some of the stnrctunl requkem€nBfor DNA
loop ibrmation observed wilh purilied components.
Thé E cali la. operâtor-repressor loop is a well docu_
mented system from this point of view (t.11,and this
review). it is used hete as a convenientpresentation
ard.
DNA clcli.ation and DNA loop fornation are ruled
bj DNA lleribilirt
For DNA cyclizxtion this wâs indicaled by the exist-
ence of ân optimal length of lhe fragment around
400 bp [13, l3a]. Wheû rhe disrancebelweentwo ldc
operâlors was close to lhis value, 535 bP precisely,
electron microscopy revealed entire fields of DNA
loops \rhereas below u6l or above (1873 bP)
(lI Amouyal. unpublished observation), only one-
thi.d to halfof the proponioo of the 53s-bploops was
observed with rhe constructionsthrt we employed.
This is tÏe only k-nown observalion of a.1 optimal
distÂncefor DNA Drotein-loooformatiot. This m&\i-
mum which reallyioneldes proteit-DNA looping to
DNA cyclization indicaled thit DNA fiexibilily is in-
hcrcnlrn Lheprocessof prolein-DNAlooping.
Loop fomJtion carl occur eten \ahen ring closure
is eenèrrllv not tossible. below 150 bp. For lhese
sh;d di.r;ce., o\A onty nceas to benù ând no( to
circulrrize for ùe prolein lo contrct simullsneously
both sites, rs first rèponed [or Àcl repressor(5:. and
6l-bp loops 2. l9lr. lt was also Eue for ldc rePrcs.
because
sor(61-tô l+7-bploops[16t. Therefore. of
DNA flexibility: DNA loops can form on a whole
ranqe of dishnces, from a few ten base pairs to
sevéral thousand bâse pai6. Addilionally to lhe lac
case aiready delailed, long distance DNA looping has
been directly observed for the E coli R6K plasmid
repLrcalionenh:rrcer-origininteraction t201. LtreHin-
invenion system of Salmoûell\ l2ll, the E cali
RepA protein and minj-P L phsmid replic.rtion l22l
nd E coli tlpe ll endonuclease Nxel [23].
Intermediate distanceshave been observed with the
bffteri3l transcripdonrl activstor NRI con|3cting rhe
oso-conldning È\A polymersse [2,1]. the DeoR
reDressorof the E coli deo operon l5l and for the
association of DNA-bound progesterone receptoG
[26]; ff regardsDNA looping, DNA is consideled as
a flexible tfue.d without any biological content which
obcys the theory of flexibilily of polymen [27]. As a
result. ir is indifferenl lo rny modificrûoo, even a
partial replacement of DNA [23], as long as the
tthread'does not become rigid. This property has
been used to discriminate DNA looping fi1cm other
mecha,'risms(see last section). SPecific sequeûcesor
proteins inducing D\A bendhg are not required for
DNA loop form3don. except when DNA becomes
risid, ie for shon distrnces.ln lhis case,làe sequence.
rhl srucrural defects of DNA or the Droteinswh-ich
modify D\A fleùbilily belween the sites of interest
mry f3vor (or disfâvor) DNA loop foimauon.
Thus a site in a 5 bp TTTAI sequetce between the
tvo Id. opemtorsOl aJldOJ is hlpersensitive to the
ahrck of v$ious chemical probes in the Presenceof
negrtive supercoiling 1291.According.!o the aulhors,
this sequence becomes Panicul3rly suscePtible
to DNA melting and bending when submined to
to6ional sùess.
severrl biologicrl processesrequire the assembly
(or disxssembly)of nucleoproteicstrucrures[301. and
D\A bendins assislsDNA looping in some of ihese
proc."ser. Tie )" phege inrelrsè protein flnt) is
àne of the proteins responsible for inlegmtive re-
coûbination between the phage and bacleriâl sites and
fo. excisive rccombinationbetween phage siles. The
bacterial inlegration host factor IIIF prcmotes the
simultaneousbinding of Int to its sitesby bending lhe
DNA in the middle of the 50 bp sepârating the sites
'A-Eact')
[31]. A ûtuËtly bent DNA sequence(3n
ca.n rcplace successtully the IHF binding site [32].
The catabolileactivalorprotein (CAP), known to bend
DNA. could rephce IHF t{ith lhe s3me effeci [3:l
 for trmscdpticnin p.ok.$otcs md euk.rlotcs
D:!.'Aloopin-q
IHF aiso stimuhtes lhe interrction of oJr-R-\A Dolv-
m.'rcsc \ iù \rfA. rnc uJn,criprionrl actiraLo_r fôr
nilrogen fixation operons of K/eàrre11dpncumonûe
[33,3.1]. There are also some indications that IHF
helps the i$sembly of p.oteins at the origin ofpSCl0l
plasmid replication [35]. Simil.r functioûs have becn
proposedfor the E coli histone-like prclein Hu [35]
and CAP [17, 32, 37]. The fonnation of a nucleopro-
teic assemblybetwecn E coli 3al repressorand RNA
polymense would lead to gdl repression[37,33].
DNA c'tcli:ation is also ruled bJ DNA torsional rlgi-
dro
In other woads, the awo exEemities of a short DNA
fragmenthave to be exactly in phas€,ie separatedby
an integEl number of helical tums for covalent
joining. Howeve., due !o additiviry of the elemeûÎary
thermal fluclualions of DNA torsion at the level of
each bllse pair, this phasing requircment is pro-
$essively lost when rhe size of the fragmert is incrca-
sed[39].
when ring closure is mediated by proteins, the
proteins may affect the strûctu.e of the loop. Contrary
to DNA cyclizâtion, the two sites do not have to be
phâsedorl DllA to ensuret.tlespeciûccontacts.But if
multiples of helical tums are introducedbelween the
two sites. their initial o.ientation will be restorcd.
Thus, NtrC ard osr-tu\A polymense diæcdy interact,
though lying on different facesofDNA [41,42]-
For ldc repressor, the two sites had to be in phase !o
induce stable loop formadon, as evidenced by gei
retardationassaysn6l. k is geneÉlly nor emphasized
that the sites on the protein are not necessarilyparal-
lel, though t}lis may be crucial for the ânalysis of the
dâta as in I I 8], or simply for a better knowledge of tlle
structurcof the protein. tac reprcssoris composedof
four identical subuniN ând binds as a dimer i3l. The
arTargement of the subunits is not known un-
equivocally, due pard], to lhe poor crysrallisâtion of ldc
rcpressor and lack of high r€solution X-ray diffriction
studies[43]. A parallel an gementofthe two dimers
is geneûlly postulared. as in [14], but â leFsedràl
zurangementhas also been proposed (review€d in
t45l). Wàen the two phased operato$ wer€ included
in a rela-\edmini-circle. only the configurationc[awn
in figure la was observedby electror microscopy(see
fi-q 2b of [18]) indicating a parâllel Àfl.]gemem. A
telraedril orgânizadon would have led to an eighl
configuraiionof the circle (lig 2b).
The periodic fot'm ion of stableloops resuhsfrom
stiffnessof both DNA and protein, a helical pilch for
DNA with a given value. and a unique connguration
of the loop- Dissociatior assays of preformed
complexes or associalion assays in the presenceof
IPIG disphyed an apparenriack of phasingrcquire-
l:6i
(q) ( b)
Fig 1. Proleinarchitectule
andloop foflnationon a rcl&1ed
circle: the contacbon the proleinæ pùaltel (r), oflho-
sonal(b).
ment in that s!âble loops were formed at any distance
between 158 and 168 bp on highiy supercoiledDNA
with tac repressoru6l. For these same distaices on
the ftagment, DNA loops were also formed when lhe
two operator sites werc not in phase,becauseof the
amplitude of the natu..al movement of torsion and
detorsion.However, they were lessstable,as indicated
by the smea.y aspecl and w€ak density of the band
assignedto the complex. DNA supercoilingampli6es
this phenomenon, since it naturally brings closer the
differenr pans of DNA. It âlso changes the helical
pitch of DNA and the dislxnces, previously in-
appropriatefor alignment ofthe siteson the frâgmen!,
becomesuitableon supercoiledDNA [18].
Protein DNA loopfomation h'ith t1|.]-mmetric
sûes
ln large loops such as the 535-bp /ac loops previously
mentioned,DNA can adopt tàe two configurarions(a)
and (c) drawn in figure 2. Below 150 bp, only one
configurrtion (d), equivâlentto (a), was observed(see
figs 7 and 8 of u6l).
Both ldc reprcssorând the 'ideal' lrc operalorused
in these experiments are perfectly symmetrical. If
làey werc not symftetrical, the existence of two
configuralioûs when DNA has lost ils torsionâl ând
fiexional rigidities, cleârly showsthat DNA looping is
insensitive to the orientalion of the site a! these
dishnces but might senseit over shorl distances.
For this to be tme, lhe asyrnmetryat rhe level ofthe
binding sile must be âssocirtedwilh asymmetly ar the
level of the protein-protein contacts. Enhancer
p.oteins are often homodime$ with a two-fold
symmeûy when they are tiee in the solution.
However, the protei! might deviate from symmeù-y
once bound to its site.
1261
A .....
@QXi;l
Fig 2. S,(e invenion ud Ioop formrlion: rhe Àaow in
dicates the coding seqùence. The asynrmery of the sile is
supposed10be Fansmitredto the prolein. Consequently.the
onenution of ùe s;!e wrr1rrcspecLro rhe codin! sequenceis
indicated b), the asymmeeicri shape of the pËteiri. rn û*
exâmple,a loop is fomed when the lwo siteshavethe sa.ne
o.ienBdon (â ùd d). Whcn rhey re in ihe oplosiie di-
rccnon, lhe two connsultions of DNA (b) ed (c) æ
possiblefor long distancesed a loop can form (c). Ai shon
dist.nces. only one DNA confi-qurarionis possible(e) and
the loop cnnnol fom (f).
DNA c\.li:aIioa depcnds on DNA concentrction
The cyclizÂtion probabilily or j-factor represents in
fact the DNA concenEâtion such thrt cyclization ând
dimerization âreequallyprobable
oft]1efragment u3,
1.11.
The scheme of figule 3 tlinslates this situation inro
lerms of DNA looping for rhe ldc system. At low
concenûations of DNA and rcpressor, th€ inûa-
molecular .eaction for DNA, ie DNA looping. is
favored over the inlermolecuiar reactions and the
formâtion of 'sandwich'-type species.'Tandems' are
fomed in medium concenEations. All these slructurcs
have beenevidencedby gel retirdation ajld em assays,
on lhis basis 6l.
Remote control of transcriptiol
Distances
Originally noticed for their abiliiy to function at a
djst3l,]ce.enJr3ncersnow frequenliy appearto operate
bolh from a few ten to sevèral tirodsanasof bases.
Some examplesof such a peafoamance aûoûg proka-
ryotes are provided by rhe niùoge.l regulatory proteit
I merase tr would irclude an enhancer sequence located
Ftr
EI: E
tft
Fig J. Influen.e of D\A Jnd proreir concenrrrrons on
D\A loop iomrrion. wirn 1dcretre-or rs ùe proren.
NRI (also called NlrC or clflc, for reviews about rhis
ûansc.iptionÂi activâtor, see [46, 46a]), which
operrres from 30 to 1,100bp upsùeem rnd = 2000 bp
downsr.'rm [4], a7l. the ninosen firation proteii
NifA (= 100 to 2150 bp [48]), the DeoR fepressor(66
to 4600 bp t49l), lhe Hin eûhancer for site speciÂc
inversion and recombinelion(= 100 to 4000 bt [50])
and to a lesserextent,by the AraC protein involved in
repressiono[ ùe a/aBÂ]Doperon (32 to 500 bp [5LI).
The lolv affiniry of rhe ar3c prorein for one of ils sites
limits tIe ringe of operational distances [5 ] I.
When both shon and long disÈnce effects have
been obsewed, a featruealreadyconsisrentwith DNÀ
loop formation, there is geûerally some more dkect
evidencefor DNA looping. Loops could be visualized
by electron microscopy for i\RI [25], DeoR [25] and
Hin [211. They were detectedelectrophoreticÀUyin
case of arac [52]. NifA has not been purifed yer.
However, it is closely relared to NRI. Like NRI, il
functions with d5a{ontaining holoenzyme rarher tiun
oro-holoenzyme [53, 54]. The protein synrhesized i''
rriro is required for activation of a NifHlacz gene
fusion [53]. Chemical 'footprinting' shows ihat the
proposed distant sites are occupi€d i, ytvo [55] and
that like i\RI, irs presence is required for open
complex formarion airhe promoter in r}le cell [56. 5?].
Lite for some of ûre above-menlioned enharcels-
indirect evidence for D\A looping exisls t33l ùar
will be presenÈd laler-
Prokaryodc enhancers have generally lost the mejor
part 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, ring
closure probabilily is also very weak, just as low as
below 150 bp under lhe conditions deûned by Shore
etat [13]. An impoftant coûsequenceis !ha! prokaryo-
tic DNA is not sûikingly differenr from 'naked' lhear
DNA in solution.
Furthermore, rcgulation of gene exprcssion from
distânt sites is much more Éequentil1 eukaryotesth3n
m prokaryores[61]. Thus, in E co1i.rhe few ex$ples
of repressionor acdvalionst a distancewith a mrur3l
location of the distant sile from 90 to 900 bD. would
conslitute ex,cep(ions-On rhe condrry. in higher euka-
ryotes, a t)licai promoter transcribed by fu\A poly-
or funclioning 'ât least up to 10 kbp dom rhe RNA
start' [6, 62]. In eukaryotes, the chromatin is highly
organized inlo nucleosomes and higher orde! struc-
tures, while DNA packaging in bacteria is considercd
more labile, ùough not much is known [63]. The true
disùrnceof rction of aî eukârlotic enhaicer m3y then
be shoner rhen rhsi câlcuhred from naked DlçA.
Along these same lines, the distance effect should
rcflect the differences in DNA condensation between
 DNA loopinglor ùrs.riplion it prokaryotesud eukaryor.s
eukal-votcsand proka4otes and, for me, *ill consti-
tùtc a tool to detec! such differences.I! might be rel-
ated to this poin! that some mammâlian enharcers
which work over a few kb r'n viv, cannot work over
500 bp ln |ùro, in lhe absenceofchromâtin 1281.
Helical orientatian of the enhancerseqltence
Sdmulation sometimesdependson t}le corfecÎ helical
poçitioningof thc enh cer *ith re(pecito ùe proxi.
mal elements.This is reverled by itsenions (or de-
Ietions)ofsuccessivelyodd and even numbeGof heli-
cal tums between the sites of interest, at rcasonably
shon distances.as expected if DNA âcqukes some
torsional stiffness (see for example U'7,33, 42, 51,
6,1-661). An exEeme situatron is thtt of adjacent
proteinssuch as the I phagerepressorsu2l.
It is generally not stressedthat a periodic stimu-
latioû is ûot pel .re a prcof for a direct cooperative
inrera€tion belweer the proteins of interesl. Ivlediation
by another protein [2], 67], DNA conformational
changes a! a distûrce ol the 'sandwiches' of âgule 3
may exhibir the sarne property.
In some caes. on the contnry. tlere is some
evidence in favor of DNA looping while th€re is no or
poo. helical pe.iodicity for eniancer function. (Il is
assumed lhat all rhe length of the spacer belween 0
and l0 bp has been explored, since t}le interaclion
between tÏe t\lo proteins may not be optirnal in the
wild tlpe situation.) Thus, the DeoR reprcssor has
threc or four DNA binding sites and a loop might be
formed for different helical positions of the sites [25,
491. Only weal( periodicity was observed for lac
repressionin vivo, even arcund 80 bp between an
'ideal' /ac oDerÀtor and an ooeÉtor with a constitutive
muixtion (É Kr:imer, PhD thesis, 1988, Cologne:
Germany)- DNA is supercoiled in bacleda and the
situarion observedin virro u8l might be reprcduced
Similarly, a strict alignment is nol required i|t viv,
between the cal.t activator and the 'TATA box
elementsof the yeast gxlactokinasegene [68]. This is
also true for some other systems quoted in this work
(seealso [69] for a rccent example).According to th€
authors, an especially strong ùteraction between Gal4
protein and R-\A polymerase I], forcing DNA to
twist, might be responsiblefor this situation, as well
as the flexibilily of the interacting proteins (see also
t6sl).
The degree of periodic stimulation also depends on
the affrnity of the enhancer prctein for iis site- A
sûong inteûction in conjunction with the thermal
guctuationsof rhe rwisr likeiy stabilize DNA looping
at âny distance.Such a reducedeffect of phasingwas
obsewed ir vivo when a high NRI binding site repla-
ced a iow affinity one [,12]. The use of ideal' lac
1265
operalon instead of lhc wild type ones had probably
the sxme effecl, as snessedin uSl. The wl operalors
'idexl'
indeed forln less stableloops t}Ân the ones i?
rùr, under the same experimentùlcotditions t701. A
fortunate consequenceis that full inducibility of the
/dc operon can be ensured[18].
Orientation bith respectto îhe coding sequence
Sequenceinvelsion is genenlly perfomed at the same
lime as the site is moved away from its natural Io-
cation. It is found that inver:ion has nearly no effec!
on stimulaùon (numerous ex3mples are given in
reviews15,7.621r.bfluenceofsequenceinversionon
exprcssion over shon distancesis less docuûented-
The schemeof Êgurc 2 suggestslha! the bidirectional
stimulrtion is nor ûecessarily mainlained over shor!
distmces if the enhancersequenceis pârt of a loop.
This is in principle a way to recognizeDNA looping
from olher mechanisms r', riro (!t Amouyal md B
von wilcken-Bergmann, work in progrcss).
Intracellular concentrationsof enhancer DNA and
These concenùations classically vary for technical
reasons(eg multi-copy plasmidsor overproductionof
the protein) or in rcsponse to envircnmental chânges,
as in caseof ûitrogen depivation for NRI [46,46a]. A
giadient of concentration of rcgllatory prcteins can
also be produced naturally as repofted fû DrcsophiLt
developmen! [71]. These concentalions can also, to
some extent, be us€d as a physicochemicalpa.ameter
fo. the cell.
High amounls of DeoR reprcssor have the sa.rne
effect as distant sites for rcpressionin the deo operot
[72]. Thus, tie remole op€rators indirectly increîse
the local concentnlion of reDresso.. a function of the
loop sùessedby Record and aoliabolalors[15].
The cooperativily of repressionobserved between
rhe three /dc operrrors is lost wi!h a dimeric repressor
unlble to aggregate into lhe tetlameric foam and to
induce DNA loop fomation in the wild-q.-'pesiruation
[?3]. This result defrnitively stoppedthe conroversy
about the involvement of the dislaot operatorsitcs in
repression of the /d. operon (/n,rodrcuor). lt could be
obtained becausethe dimeric rcpressorwas deliber-
ately used in combinâtion with rhe low chromosomal
concentmtions of rcpressor and DNA, cotû?ry to
previous irl vilo works. \\lth higher concenEctionsof
repressor (iqr strains), ùe cooperalivily between the
threeoperalorsites was gready reduced,&1dno differ-
ence in the lelel of repressionberseen the teL'3meric
and the dimeric repressor wals observed. Therefore,
the high amountsof repressorhÀdinduced the predic-
ted i/! viva trânsirion lrom DNÀ looping to aîoûer
t266
modè of reprcssion (pfevious srction and 116l) and
lhe three ldc operirlorswcre now sepffately occupied
('Èndem' formation).
Concentrationsare âlso critical for the c!s/,/dr?r
effect ol enhancer sequences.In physicochemical
telms. this situirtion coven in(a- and ittednolecular
reactionsfor DNA. Some ûechanisûlssuch as sliding
imply that the enhancerfunctionsexciusivelyin a c,s-
posilion.On the conFary, DNA looping, normaily c,r-
acting, cxn be compensated in ,rdrJ. Up to ûow' the
arans-effect of EanscdpdonÂl enhance.s has been
exclusively observed when the two siles of interest
were mùintained in close proximity, r/I vi ro, with
catenanesI74, 751 or a non{ovalent bridge [28], and
in vi!o. in rhe natural ph€nomenaof tsansvectionin
Drosophila li6l. The question has been raised of
whethe. i! would be possible to detecr âllelic inter-
action of the tnisvection type in organisms where
therc is no apparent somatic paidng of chromosomes
[76]. A lrdns-interactioobetweentwo sitescarried by
two unlinked DNA moleculesshould be observedby
ra,.rs-complemenlationwith high amountsof DNA in
lhe cell, in order to form 's.ndwich'-t?e stluctues.
The NifA aperiodic activation of NdH promoter on
multi-copy plasmids is probably an exarnpleof such a
td,?s-effec! [33]. Along the same lines, acdvation of
the proka..yotic g/r,4 and NfFl genes is also possible
in lÏe absenceof enhâncer site aûd the presenceof
high concenùaliors of lhe conespondingprotehs NRI
[47,74] and NifA [7?]. The future will tell if equiv-
alenr siluationscan naturally occur.
Redun.lanct of infonnat io n
Enlnncer sequencesare generally repeated [5,62].
They may allow cooperative interactions between the
coresponding proteins and facilitate the contact
berweenthe enhancerprctein aîd the componentsof
lhe ùanscriotional machinery at the gromoter. This is
how rhe À èl repressorsstiniulate reiression ftom the
p@ prcmoEr ll2l and how NRI would also activate
expression [42]. The ennancer prot€ins rnay also
cooperûte no! by direcrly louching but by simul-
taneously touching some componenl of the tran-
scriptional machine.y [78, 79] or by aggegating ([5]
and refèrences therein). ReDeated sircs cxn also
simply increffe the probability of siûgle loop for-
malion at one site or favor the fomation of multipie
ioops, reinforcing â11the functions of single loops, as
observedfor the d"o opercn [25].
The other modesaf remotecantrol
Tr'rpping of the protcin by the distânt site and sliding
to the site of interest is ofter viewed âs ùe mair
altemative to DNA looping [5, 80]. Bur retenrior of
enhancerâctivity in trarr w|rs obseived in cxse of r
total or peftial disruption of the DNA Eack [23, 75].
Em did not reveal several(unspecific)locatioûsof /dc
and deo rcpressoron a frâgment (see all em picru.es
in [16] and [25]), though for 1dc repressor,the high
affinity 'ideal' Iac operalors were used. Such exper-
iments rule out sliding as exclusively acling undei the
prcEise in itro conditions und€r which they weae
performed. 1n vivo, the use of psordlen-modilied DNA
between the SV:10 enhancer ârd tlle humân B-slobin
gene iîhibited expressionof this gene l8l I. Bur F\orr-
len ahels DNA propenies([23] âJtdreferencesthe.ein).
On the cont âry, NifÀ activation was not inhibited by
lac repressor binding in the middle of DNA sequence
between upstream and downsteam elements in ,iva
[33]. Also, loss of cooperativity i'l vivo betvr'eer
distant a.ndproximrl sites with prcteins engineered so
rhar they becorre unable to aggregateF3,82,831
dirccdy suppons the existenceof DNA looping in the
cell. This is especially ûue when the loop irvolves a
unique and multimeric protein such as ldc rcpressor,
since otherwise, the qùestion of possible intemediate
factors mediating the p.olein-protein contacE is ûised
ir the absenceof an ,n vÈro proof for direct htenction.
Yer, sliding might ôssist DNA loopilg. Rece bio-
physical experimentssuggestit might be the case for
lac æpressor and wt operÀlors [8.+].
A distantly bound protein can also block Rr\A
elongation [66, 85, 861. The enhancer can also inter-
fere with other regulatory proteins and other processes
at the same location: the rcpressor hhders CRP ac-
tivator binding when located upsùe"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 looping
wirà this rcprcssor [37]. However, it presumably
exists in rivo. lndeed, a gal to tac opeûtor conversion
in vivo allows efficient repressionof the gdt opercn
[88], and this repressionis rclieved when a dimeric
/@crepressoris used [38]. Funhermorc,various rnodes
of rcmote conEol can be assumedby DNA looping, as
sEessedby Flashnerand GÉlla [85]. in caseofbiock-
age of RNA elongation.
Finally, in somes cirses, environmental chrnges
induce the rradsirion from DNA loopiîg to another
mechanisrn. Variations of concentations lead to sùch
a situarion n6, 731. Vadalions of DNA superhelicity,
due to an osmotic shock or anaerobiosis([89] and
references therein) or DNA trinscription [90-92],
rrlight also favor such ûrnsitions with low âf6nity
operirtorsto ensureloop breakagensl.
Acknowledgûent
DavidPenin is aclnowledgedfor cû€ful re.ding of thep.esen!
aniclein 1990.
 D\.\ loopingfor ûùscripllor i!1prôhrlores ù.t eùkrores t26'1

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