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Interaction of tight binding repressors with lac operators
.I. Mol. Biol. (1984) 172, 405-416
Interaction of Tight Binding Repressors with lac Operators An Analysis by DNA-Footprinting ~'IAGNUS PFAHLt AN[) ~']ICHELI:E H ENDI¢ICKS
The Salk ln~titute for Biological Studies Regulatory Biology Laboratory p.o. Box 85800, San Diego, CA 92138, U.S.A. (Received 21 June 1983) 'ro increase our understanding of p r o t e i n - l ) N A interaction in general, and in particular t h a t of lac rel)ressor with lac operator, we have investigated the interaction of tight binding (ltb) repressors with wild type (WT) operator and 0 ¢ operators. Nine ()~ and a W T operator were cloned and sequeneetl. T h r e e different ()c and all O + were then ehosen for the fOotl)rint analysis of six pb repressors and W T repressor. I)istinet protection patterns for the various rel~ressor-operator pairs were observed at low repressor concentrations whereas, at high repressor (.(mcent rations, a stretch of 24 bases of the lower strand of the four different operators was protected in most eases. This protection pattern at high repressor etmeentration was ahnost eoml)letely r e d u n d a n t for all repressor-operator pairs. in spite tit" the fact that the attinities (if" the various pairsf ()+ and of some ()c o])erators at the 3' (right) en(I of the lower strand. I)imethylsulfoxide. which is known to increase the atlinity of () + |i)r rel)ressor, did not increase the n u m b e r of bases protected by W T repressor on the lower strand of 0 + The footprinting results 1)resented here clearly d e m o n s t r a t e t h a t luc rel)ressor can maximally protect a b o u t 24 bases o( the lower strand of the ol~erator anti t h a t the n u m b e r and kind of interaetitm.s ~)(.curring in this region (letermine the strength of the repressor-operat-,)w interact ion.
1. Introduction lac represst)r recognizes lac operator with very high specificity. The stability of the rolwessor-t~l)ora|or complex facilitates the biochemical analysis of the complex anti makes it a very suitable system to study the mechanism by whieh certain proleins recognize specific base sequences within double-stranded DNA. Moreover, from a Ifioehemical and genetic point of view, the lac repressor-lac operator system is the best-charaeterized example, since a large number of specific t Aut|mr tt~ wlmm e~wrespondence shouhl be addressed. 4O5 (11122-2836/8',~]040405-12 $03.00[0 ~) 1!t84 Academic Press l no. (London) lad,
1~,;
M. I ' F A I t l ,
AXI)
M. H E N I I I / I t ' K S
tnulations, ati't,t.ting Imth o p e r a t o r and represser, have heen isolated (for a review, see I~;.ur~t,ois & l+fahl. 197fi). We ln'eviously described the isolation and eharm.lt,rizatilex t h a n wihl t y p e (WT) rei>ressor wit h W T Oll<,rat<~r and also+ overer interaction a n d possibly prcd<'il~ + I)NA interat'tion ill ~t'nt'ral. In this ]ml~t,r. we have <'hmt,d and sequenced several 1)+ m u t a t i o n s and invosli;2alo
iw~+lvelim~ ~.Xl~erimenls. Very st r
2. E x p e r i m e n t a l
Procedures
(a) Bm't+,ri,d .~h'ai,.+ ~ml medin E.,+'k,.riehia col~ slr'ain.~ are ~,ssontiallv tlms~, des<'rihed (lq'ahl. 1!)7!)). HBII)I and 31312!)4 w~+n' +d~taim+
(1>) ('h+',+ir',l.,; ],+.lJrOl~yl-fl-|+-thi.,,.,ahu.t<,..dmI('X l'harma<,mttieals:' hvdrazixu, was f'l'1.ridin1+ from l,'isher Seientifi<.: li~rrniv a+,i
ZI~O dh~e <)+ - r ZI~XOdhu~ ()~ I]NA were. is, tared fi'um heat-inducible plut~es. The DNAs were, pt.rifivd and their +,on+-entration.~ wor(, rl~qermhl(.d as d(.,,~vrib(,d (]{iggs el at.. ]!)7()). ('all lhyrm,~ I)NA was ptH.t~hast.d I'r~tl~ ,'~i~111a and EroR[ linkvr |'t'a~nl~tlts ]mr,,'ha~{,t( J'rotzl l ] i i l , , l'la,~mid I)N..X. was purifi~'d ~n.~d~,seril~ed (Pfahl. 1982).
"I'I
4O7
(e) L~olatio. :tnd cloning of operolorfr~ment,~' F,'om 0.2 to 0.6 mg of 2h80 dluc 0 + or 0 ~ I)NA was tn.eeipitated with ethanol, washed. resuspended in Alul buffer, and digested with Atul. Tile digest was electrophoresed on a ,5% acrylamide gel (3 mm thick). The gel was then stained with eghidimn bromide. To identify the !)5 base-pair fi'agment eontaining the tac operator, a po,'tion of the ,4t,1 digest was incubated with lac represser and filtered through a (1.5 em nitrocellulose filter (pore size I).4/~m). The filter was washed and the bmmd I)NA was eluted by vortexing the filter in 501tl of e[eetvophoresis buffbr. Tlnis I)NA solution, of which 8()°~j was the 95 bp? ,4l, |'ragment containing the hie o~gj'ator, was run simultaneously with the non-filtered Alu digest on the aerylamide gel. The band eontaining the 95 bp operator fl'agment was cut out of the gel and the DNA was eleetroeluted from the gel slice. EeoNI linkers were then ligated to the 95 bp operator fi'agment; the I)NA was then digested with EcoRI and again run on a 5% polyacl3"hmtide gel The 1(17 hi+,,fi'agment was ehtted from the gel and ligated into the EeoRI site of plasmid pMB9. This I)NA was then used to transform strain MM2!14 or HBI01. Transformants were selected on minimal glucose-X-gal plates containing tetracycline. Dark blue colonies result when tile trust lae operon is induced by the molan' excess of WT operator t)inding lae repress
(f) DNA .~equencintt The 107 bp operator fi'agments eluted fi'om 5% polyaelTlamide gels were 5' labeled using [7-3~PJATP and T4 pelynoeleotide kinase. The labeled I)NA fragment, containing a single HlmIl site was digested with H p a l l , yielding a 61 bp fragment of which the Iowen' strand was 5' labeled. For DNA sequencing, the methods and tn'oeedures of 3laxam & Cilbert (I 980) were followed. (g) DNA foolpri+~linff in general, procedures of Galas & Schmitz (1978) were followed. The 5' lal)eled 61 hp fragments used for sequencing were ineubate{t in BP, buffer (0.01 ,m-KCI. 0.01 m-3lgC] 2. 0.01 m-Tris- HCI (pH 7.4), 0.0001 .~I-dithiothreitol), fen' 15 rain with various amoutlts (1)'5 #1. Itd. 5/tl, 25111) of purified represser (,-,fix 10 -~ .xl), l)Nase I was then added tbr 30 s. after which the I)Nase l reaction was stopped by the addition of EI)TA and calf t h y m u s 1)NA (final conch 6.25 x 10 -2 .~t and 200pg/ml, respectively). The I)NA was then precipitated twice using 2 volumes of 100°/o cold ethanol and a final salt concentration of 0.3 .m-sodium acetate. The precipitated I)NA was washed twice in 70°,~) ethanol, then resuspen(led in eleetrol)horesis buffer and loaded onto a Setlumleing gel.
3. Results
(e~) Characterizatfon, of operator fragments A set. of nine lac 0 c mutants, including most of the O c operators used previously to select and characterize l ub repressors in rive (Pfaht, 1979), and WT operator were cloned into plasmid pM B9 as deseribed in the methodology. To exclude any ambiguities concerning the sequence of the cloned operator fragments, all eioned operators including WT operator and known 0 ~ ~perators were s e q u e n c e d ( T h e
t Abbrevhdim~s rimed: hp. hase-pair; 1).MNO. dimethylsu|fi~xkte.
31. I ) F A H i .
ANI)
31. HI.;NI)RI(',KS
results are shown in Figure I. WT (~l)eralor and the lm<)wn ()~ ()l)erators ()R,VI, ORVI:~O and ()R\'116 (Smith & Na(ller, I.<171) revealed lhe previously rep()rte(] seqnences (Gill)erl ~ Maxam. 1.973: (;ilhert e! ~d.. 1975), T h e 0 ~ o p e r a t o r s nol previously isolated t u r n e d out to he identi(',al with either ORVI21) or O R \ ' l l 6 . This result was not e(>mpletely surprising, since m u l t i p l e ('~q)ies <)f s l r o n ~ (F m u l a n l s have heen isolated previously ((;ilhert et
Ihree O" operalor.~' Various (.on(.enlralions (>f W T repressor were i n e u h a t e d with lahele(t HpaII) ~;c,l~l 61 h l) f r a g m e n l . (.(mlaining eil.her W T Ol)erator (O +) or ,~ne of t h e t.hree (lifl'erent (F (~])erators. hef()re (ligesti()n wilh I)Nase I. T h e l)Xase l c()n(~entratio,i aml (ligesli()n time were kel)l ('(mslanl in the (lif['erent. e x p e r i m e n l s . Typical ti>ml)rinting resulls are s h o w n in Figures 2 and 3. 'ro o h t a i n the correlation helween the l)role(.h,(l I)NA re~ion and the <)i)erator sequence, sequenein~ v'eacli(ms were run in parallel wilh [lie DNase
+15
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AC AC AC CT TAAC AC TC GC CT ATTG T TAAAG T G TOT ORvl
>T
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AT
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Fro. I. l'.a,~" elmn~.s in I F mutali~m.~. Wild type Ol..r:lh)r I)NA and 9 IF InlltIlti(ms were elolled into phL~mid pMB9 as (h.serihed in Exl~'rim~.nhll Procedures and se(im,m,ed (Maxam & f;illu~rl. 1980). ,~'quel)(.e or l b . low,,r strand (,r the ~VT Ol)eralor i,~ ~ziv~'n nn lop w i l h symmetry r't.~d,,nx un(lerlined, Till. cl.nl~.r cJf ,~ymnlelry is at O. T I . ' ha.~. ehang,.s l'mmd in (F llllllatiollS are indieate(l. Seqller, ce hvlJhen.s haw~ l..en omille(l for elarily from Ibis and .~uh.se(luenl. Figures.
' [ ' I ( ; H T B I N I } I N ( I I~.I[~]PI{I~,',~,~OI{ F(}()TI~I:~IN'I'S
4119
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t;'1~:, :~. Fot~tprint ~)l" ~,~i"l" l'P|II'e,~tJl' Oil (J ÷ ~lui thrt.e 0 c oper.~t.tors, Puritled W T rept'es,~or (5 #1) wa.~ int.ubated w i t h G' hlheleO (lower stralld) Itp~+ll-EroRl ['r~LBment ecmt~ining either 0 + (lane B). ()RV, I (()1) (]ttl~.e AI, ()tl (laue (!). ~t + (,~, {[:ule D) ~])er+Itor. The rel)res.~ur-C)l~erat~r m i x t u r e s were then (ligt~sted w i t h I)Na.~e | llA'ld F'l.lll ()11 ~P(LtIel'l(-'iI~g gel.~ a~ deserihed in I'~,xl>erimel~tal Prt)(~edu:'es. A.~ a t:~mtt'(~l ()÷ I ) X A was digested in tl~.p ~ii)..~ell~P 11[ ][~.-[)l'd2.¢.si.].l " (little hi). (~t) }).l-itl (b)ThE. Sttl)'ie ['e21eticJIi tll~.XeJ.II'e~ but eleetrol)hore~ed [ur difl~re[tt [ength.~ o[' tinge, l)r¢~tecte<| areas ~ire in+|ieated f)y brackets.
(a)
A B
(b)
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D
A B
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rf,l~r,.s~.r w.r,, hn.uh~t.fl w i t h ()l (l~Ln,..\). ()* (l~zw. J{). ()ix (l~m~, ('). Jm~l (it, (l~m. I)) ~)l~'v'lHnt • nn~l
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hl~ ~,x~,han~o. ( ) ~ ,~hmv,~ n,dm'e.'l lWote(.tion in the middle and ~tt +6. All three ()~ ~q.,rators shiny ,~li~htly ,~t,n'~m~." prol.eetion at the right end (-1()). "1'~ .hl~dn m~,'~' int'~.'mation ~m the' I~mtertion Imtt~'rn~, we als~ ean'ri(,~l m~t f i . ~ l i ~ r i n t i n ~ : eXl,erim~mt.~ b e t w e e n W T r e p r e s s o r a n d t h e ['()ill" clifl'er~,nt ~q)c'rat~ws in l h u IWeSunr~ • ~t' l llq~ ( v / v ) dim~q hyl.~ult'~xide. I)~l,~() has I~e(,n sllowl~ t o i m ' r e a s u the, a l l i n i l y o f n,pre,~,~or f o r ~)peraioz'. A I()?{~ I)31N(-) e o n e ~ ' n l r a t i ~ m w ~ s l'~mnd t u st ~l~iliz~, t h e n , l ~ r e , ~ s o r - o p e r a ! o r ~ m ~ p l e × t e n | b l d ( P f a h l , I i)?~). 'rhc, M~ser.ved p m t e ~ q i o n patterns in .~°~.,,,. I),~IS() w e r e difl'erep.t l ' m m t h e imtler,m ~d~sel'ved befiu'e. I n Aene,'~tl, p r o t e c t i o n wa,~ s t r { m ~ e r , ,~o t h a t W T
rMw~,,~.~.' r~,,~,mhh~d an I ~ U'eln'r,~,~or under thu~' uondition,~. IAilt'~.r~.n~,~,.~ in iw~d~,{,ti~m lmtt~,rn,~ w~,r~, oh,~erved ~tt low rel.'e,~,~or eon~,entx'ati.n,~ ~mly. A .~uhemati~ rel~t'e.~entative (d' foml)rints is ~zivon in lggm'~' 4 (s~'t (b)). ()+ was l~r~doul(,d t~ Ih(' ,~ame e x t e n t a,~ b e f o r o . ()] rt, v e a l e d ]os,~ ditt'~,r~,m'~- t - ()+ a n d r{.dm,{,d pmt~.~,ti~m wa,~ only oh,~ex'ved ~t + 7 . (9~ and ()~1 at lmv n'el.'~,,~,~w r~m~'{.ntralion difl'~,'ed mor~. strongly from ()+ than in the ab.~enc~, of I)3IH(). 'rh~. ma.i~r d i l h ' r e n r e s i ~ ()+ w e n , a t + ], -- ] m-~d ~ : ~ f o r ()o ~.z1~] al +;3. + ]. - ] al}d --:l t()l' ( ) ! 1 .
(r) I " o . t p r i . l i . g ~.Mp.~is .J' !~' reln'e.~',sor,~ u'ilh 0 + ~ . d (Y W~emh.',~' 'l'hr d~,laih,d analv,%~ uf I ~b repre.~sors wit h var'im~,~ o p e r a t ~ r s rouhl yield results theft might allmv {.~mulusions on the n a t u r e of the ~lter'ed inter~wtion h~-twec,n the, T
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ACAC ACC TTA.AC AC TCGC CT ATTG T TAA_AGTG TG T +lO ~-5 0 -5 -lO O,
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the, .~,h~mmtir r~,l.'L,.~-ntati~m in (a). I"~tl.'int,~ w~,r. ~,~'ahmtud hy ~,ye and b.v del~.~il~.l~etry .~uanmer, The pruteeted area i~ the 24 Im,~es direutly ~m t~l~ of the" Imr. The I~r' ron.~i~t~ of 24 .~.b~znlen. one" for ~,~,.h Imst,, 'l'hc, n'mahfin,._, Im.~,~ ~t' |]H' hm'~,r strand of the ~pc,'al.r m'e Unl,'.h'et(,l. ,~]mxima] I~X'Otucthm (at :~5/d u'elw~,s.~w) i.~ n'lwe.~,nted in the. ()+ lane. I)it]'erenee.~ ob.~t,'ved in proteuti~m at Imv I'elwL,ssor (.(~l|c(,IHl'~Hi()ns al'l, indh.atecl hy dt,h'thm.~ in the Im:'.~ in the ()~. ()~, and ()~ lane.~. The deh'ti~m:; in the Imn'.~ are .~eah'd I to ,1, i.e. a very .~tx'.n~ hand sh~wh~,~ on t.h~, t'o-tl,'int tbr an ()~ with m~ Imnd shmvin~z fi~r the ()+ in the ~aum. lm.,4ti.n wmdd he l.'e.~ented a.~ ~ eOnnldete deh,ti~m ~d'the bar in the l) ~ I~me nt thr~ p~.~iti~m. I~ thi.~ Figure.. maxirmd ditt'erem,e.~ ~h.~m'ved were not ~zr'e~der titan "2. X()tM. tlmt ()~ i.~ m)! roml)h,teJy I.'~)h,rh,d ;d p.~ith)n --14) (jmlg~,d at the" Ifi~lm:4 repre~.~)r (.(.u,~,ntration ~s~,d). (h) ,~'rhemath. rel)r..~entati(m ()t" WT r('l)re.~.~or IW()tm'tion Imt tern.~ in the" I)r('.~em'(• ~)t' I()% I)M~().
.I1'2
51. I ' I , ' A H I , A N I )
M. H I , : N I ) R I ( ' K S
llltlltllll l'epv'ess(trs a n d ol)erators. I tb I n l l t a n t s h a v e l)e~ll s h o w n to over(+()ine ()+
inlllati()ns ill vivo. The i. vitro s(',reening system used hcrc will alh)w a mueh nlorc (h,taih,(l analysis, sivlce rel)V'essor t<) el)orator t'atios (Pan I)e greatly altere(l. Six ! tb repressors were investigated with tile |'our oi)erators used I)el'ore. In each (,ase, four different rel)ressor e o n e e n t r a t i o n s were used with one fixed operator (.(m(,(.ntrati()n. (:haraetcristie i)roteetion p a t t e r n s were el)served for ea('h rel)ressor-ol)erator pair. 'I'll(+, results are summarized in Figure 5. According to these results, the 1tb repressors ean he divided into two classes, one of" which distinguishes well I)etween WT anti O ¢ opev'ators. This class includes the R.7(i, R7Sa. R84 and R67 repressors. I+~xeept tbr the latter, all of these rel)ressors were shown previously to have only it small (3'5 to 9-fold) increase in affinity for' O + I)NA (i)fahl, 108lit). The other class ¢)t" I tb repressors contained tim two very tight bin(ling rel)ressors X86 anti R l I 3 . These two rel)ressors gave nearly identical prolet.ti(m Imtterns, for I)oth O + a n d ()c operators, at all represser concentrations. The I tit repressors also show an increased protection at the right end of some operators, position - 1 0 , with t h e R l 1 3 represser being the strongest in this r('gar¢l. In this (.ase (.omph,te proteeti()n was observed at - 1 0 tbr all four Ol)(,r'ators. WT rel)ressor In'otters this base only partially on 0 +. The I¢78a and the Illi7 r('prcssors were the only ones t h a t (lid not proteet l)osition - 1 0 on O + even at higher represser (.oneentrations. A similar effect was seen for O l with R7Sa. but not fi)r (-)6 and ()11, in whi('h ease the - I 0 position was proteeted to the same degree as in the %VT rel)ressor-O + pair. It should be emphasized t h a t all tilt, protection diagrams in Figure 5 are obtained from the analysis of several foot l)rints. (.Oral)rising several repress<)r to ()l)erator ratios. T h e (liagrams therefore give only (it,alitative results and do not express particular r e p r e s s e r - o p e r a t o r affinities. ()ur results are (letailed beh)w with the l)roteetion of 0 ¢ operators eOmlmred to the prote(,tion of" O + for each ltb rel)ressor" (i) R76 represser This represser was previously found to have a ninefohl iner'eased affinity tbr 0 ÷ (Pfahl. 1981a). Complete protection of the O + operator ineluding position - 1 0 was observed. In the case of O~, major decreases in protection were found in positions + 7 , + 5 , + 2 , + ! t h r o u g h - 1 , - 3 , - 5 , - 6 and - 1 0 . F o r 0 6 , major de(.reases in protection were observed at + 5 , + 2 , +1 and - 3 . For O~t major decreases in protection were found at + 5 , anti +1 through - 3 . (ii) R78a repres.~or This represser binds 3.5-fold tighter to O + (Pfahl, 1981a). Protection of 0 + was incomplete in that no protection was observed at+ - I 0 and decreased protection at - 9 . Major decreases in protection for O1 were found at position + 7 , + 5 , + 1, - ! anti - 3 . Increased protection for 0 6 was observed at - 9 and - 1 0 , and decreased protection at + 5 , + 2 , + ! , - 1 and - 3 . Increased protection was found |br ()11 at - 9 anti - 10, and decreased protection at + 11, + 6 , + 4 , + 1, - I anti - 3 .
TIGHT
#]INDING ICEPRI~SSOR FOOTPRINTS T
413
AT
AC ACAC C TTAACA CTCGC CTA TT,o TT~AGTGTGT +I0 R76 06 . 01
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AT
Fro. 5. Sci~ematic represe~ltation of footprints obtained with tight binding repressors (I'~). Various concentrations of each pb repressor were incubated with 0 ÷, 01, 06 ~nd 011 operator and digested with DNase I. The observed footprints were evaluated as described in the legend to Fig. 4. Marks t,nder the bar stand for it~ereased protection.
(iii) R84 repressor This repressor binds 3"5-fold tighter to O + (Pfah[, 198ta). With 01, decreased protection was observed at + 7 , + 5 , +2, + l , - - 1 a n d - 3 . With 06, the observed p a t t e r n was the s a m e as t h a t observed for O1 with only small differences in t h e intensities. With O l l , decreased protection was observed at + 5 , + l and - 3 . (iv) R I 1 3 repressor This repressor was f o u n d to bind almost 200 times tighter to 0 + (Pfahl, 1979). Complete protection was observed fbr O + including --10. For O1, increased
414
51. P F A H L A N D M. H E N 1 ) R I C K S
protection was observed at + 12 and - 1 0 , and decreased protection at + 7 . For O6, increased protection was observed at + 1 2 and - 1 0 . For O l t . increased protection was observed at positions + 1 2 , + 7 and --10, and no decrease was ol)served. (v) X 8 6 repressor This repressor was found to bind 50 times stronger to O + (3obe & Bourgeois, I972; Pfahl, 1976). With 01, slightly increased protection was observed at + 12 with O6 and O11 at + 12 and - 1 0 . F o r all three 0 ¢ operators, only very small decreases were observed in the protection. (vi) R67 repressor This repressor was shown to contain a m i x t u r e of weak and tight binding molecules (Pfahl, 1979). The t i g h t binding molecules have a 100-fold increased affinity for operator (Pfahl, 1981a). N~) protection of - 1 0 was observed with 0 + l)ecreased protection was observed for O~ and O 6 at. + 7 , + 5 , + 2 , + i , - 1 and - 3 . For O l~. decreased protection occurred at + 5 , + !, - ! and - 3 . For all three 0 ¢ operalnrs, increased protection was observed at. - 10. 4. D i s c u s s i o n
lq this s t u d y , we have investigated the interactions of W T repressor and six tight binding repressors with 0 + and three O ~"operators by 1)NA footprinting. A large variety of protection p a t t e r n s were observed at low repressor to operator ratios, whereas at [~igh repressor to operator ratios, a uniform protection profile was observed for a h n o s t all r e p r e s s o r - o p e r a t o r pairs. These latter results agree well with those obtained by others (Sehmitz & Galas, 1979), which showed t h a t the same 24 bp o p e r a t o r region ( - 1 3 to + i 0 in Fig. 4) is protected by W T repressor as well as by the very tight binding repressor ]12-x86, a double m u t a n t . The only exception to this rule are the R67 and the R78a repressors. In both cases, protection at the right end of the lower s t r a n d is a b s e n t at - 1 0 and also reduced at - 9 in the case of t.he R78a repressor. This reduced protection is observed mainly with W T o p e r a t o r and, to some extent, with O~. The other two 0 ~ operators, Os and O l t , show normal or enhanced protection at - 1 0 and - 9 . It is interesting to note here t h a t both the R67 and the R78a repressors carry m u t a t i o n s at a very different location (amino acids 255 to 205) t h a n other |tb repressors, whieh all carry a m i n o acid exchanges in the amino-terminal end (Pfahl, 1981b). The 255 to 295 region of t h e lac repressor has been postulated to contain infbrmation for repressor s u b u n i t interaction (Miller et al., 1979; Schmitz et al., 1976) or possibly be involved directly in D N A binding (Pfahl, 1981b). A change in the s u b u n i t interactions could allow the repressor s u b u n i t s to interact more tightly with certain regions of the o p e r a t o r I ) N A . Protection in the presence of DMSO seems to reduce t h e a m o u n t of repressor needed to obtain complete protection. However, DMSO does n o t a p p e a r to have the same effect, on all repressor o p e r a t o r pairs. For instance, in the eases of the 0 ~
TI(;HT BINDING REPI{ESSOR FOOTPRINTS
415
operators 06 and 011, tile 1)MSO effect appears to be reduced when compared to 0 +. Similar observations on the differential effect of DMSO on various 0 c operators }lave been made by direct binding studies (unpublished results). The main observation here, however, is that DMSO, in spite of increasing the affinity of WT repressor for 0 + tenfold, does not lead to the protection of additional bases on the lower strand. The data so far discussed reveal three points: (1) WT repressor and mutant repressors can protect only a limited number of bases against nuclease digestion (24 on the lower strand); (2) the number of bases protected at high repressor concentrations does not correlate to the strength of tile repressor-operator interaction; and (3) specific protection patterns can be observed only by careful titration experiments. Protection of O ~ operators differs more distinctly from 0 + for one class of Itb repressors (R67, R76, R78a and R84) than for the two very tight binding repressors x86 and R113. The decrease in the protection of O ¢ operators at low repressor concentrations occurs mostly in the middle of the protected region. It is somewhat difficult to visualize how DNaseI could act there (because of steric hindrance through repressor) while the outside regions are still protected. Possible explanations could be (1) the repressor is not constantly in close contact with the operator region, so that some "breathing" of the complex may be possible; (2) the weak protection patterns at low repressor to operator ratios could be an expression of two operator fragments binding one repressor molecule, in which case one of the interactions would be weaker than the other and result in only partial protection. (Tetrameric lac repressor has been assumed to contain two oper~rtor binding sites and some evidence has been obtained for this; Culard & Maurizot, 1981; O'Gorman et al., 1980); (3)the partial protection patterns ol)servcd could be the presence of partially inactive repressor molecules, due to (.heroical degradation or to special steric conformations of the repressor subunits. At this point none of the three above mechanisms can be ruled out; however, partially i11active repressor has been observed by many investigators, and usually all repressor preparations contain some repressor that is inactive or partially active in terms of its DNA binding. We have also previously shown that the R67 repressor is a mixture of weak and tight binding molecules (Pfahl, 1979). lac repressor tetramers containing some inactive or partially active subunits could easily yield partial footprinting patterns. At high repressor to operator ratios, the only partially active repressor molecules would be competed out by the fully active repressor molecules. The protected region of 23 to 24 bases of the lower strand is asymmetric with regard to the operator symmetry. The left half of the operator is better protected, which fits with the notion that in that region the stronger 0 ~ mutations occur (Gilbert et al., 1975). Schmitz & Galas (1980) have shown that protection of the upper strand is shifted two to three bases to the right protecting the operator somewhat asymmetrically in the right-hand direction; in this way a symmetric protection pattern is obtained when one looks at both upper and lower strand. The footprinting results are, in general, similar to results obtained by methylation protection (Ogata & Gilbert, 1979) and fit the models for the repressor-operat6r
41fi
M. PFAHL AND 51. HENI)RICKS
interaction recently suggested. In these models, protruding amino-terminal ends of the lac repressor sl,bunit, s are t h o u g h t to interact with the major groove of ol)erator B-I)NA (Adler et al.. 1!)72: Matthews et al., 1982; Sauer et al., 1982; Weber et al.. 1982). The repressor is t h o u g h t to interact with mostly _-,ne side of the I)NA helix, i.e. according to our results, each of the repressor subunits (2 per operator) would protect 1.2 turns of the 1)NA t;elix, probably the m a x i m u m the protruding amino-terminal end of a repressor subunit can protect or reach. The analysis of the amino acid residues exchanged in the pb repressor is now in progress and these results should yield f u r t h e r information on the molecular details of the lac repressor-operator interaction. We thank Allen Kool) for his hel l, in setting up the DNA technology, Suzanne Bourgeois f,)r diseu~sions and critical reading of the manuseril)t, and Karen Horosehak for excellent technical assistance. This work was supported by Public Health Service grant GM23671 from the National Institute of General Medical Seienees. REFERENCES Adler, K.. Beyreuther, K., Fanning, E., Geisler, N., Gronenborn, B., Klemn, A., Muller-Hill, B.. Pfahl, 51. & Schmitz, A. (1972). Nature (London), 237, 322-327. Bourgeois. S. & Pfahl. 5I. (1976). In Adt'ances in Protein Chemistry (Anfinsen, C. B.. Edsall, J. T. & Richards, F. 31.. eds). vol. 300. pp. !-99. Academic Press. New York. Ct, lard. F. & Maurizot. J. C. (1981). N~lcl. Acids Res. 9, 5175-5184. Galas. 1). & Schmitz. A. (1978). Nucl. Acids Res. 5, 3157-3170. Gilbert. W. & Maxam. A. (1973). Proc. Nat. Acad. Sci., U.S,A. 70, 3581-3584. Gilbert. W.. Gralla, J., Majors. J. & Maxam, A. (1975). In Symposium on Protein-Lifand l'nterttctions (Sund, H. & Blauer. (;., eds). pp. 193-206. Walter de Gruyter, Berlin. ,lobe. A. & Bourgeois. S. (1972). J. Mol. Biol. "72. 139-152. Matthews, B. W., OhlendorL I). H., Anderson. W. F. & Takeda. Y. (1982). Proc. Ntfl. Acad. 8ci.. U.S.A. 79, 1428-1432. Maxam, A. L. & Gilbert, W. (1980). Methods Enzymol. 65, 499-559. Miller. J. H., Coulondre. C.. Hofer, 5I.. Sehmeissner, X., Sommer, H.. Schmitz, A. & Lu, P. (1979). J. Mol. Biol. 131. 191-222. Ogata. T. R. & Gilbert. W. (1979). J. Mol. Biol. 132. 709-728. O'Gorman. R. B., l)unaway. 5I. & Matthews, K. S. (1980). J. Biol. Chem. 255, 1010010106. Pfahl, M. (1976). J. Mol. Biol. 106, 857-869. Pfahl, 5I. (1978). Biochim. Biopbys. Actor, 520, 385-290. Pfahl, 51. (1979). J. Bacteriol. 137, 137-145. Pfahl, M. (1981a). J. Mol. Biol. 14"/, 1-10. l'fid~l, 5I. (1981b). J. Mol. Biol. 147. 175-178. Pfahl, M. (1982). Cell. 31,475-483. Riggs, A. D., Suzuki, H. & Bourgeois, S. (1970). J. Mol. Biol. 48, 67-83. Sauer, R. T., Yocum, R. R., Doolittle, R. F., Lewis, 5I. & Pabo, C. O. (1982). Nature (London), 298, 447-451. Sehmitz, A. & Galas, D. J. (1979). Nztcl. Acids Res. 6, I I !-137. Sehmitz, A. & Galas, D. J. (1980). Nucl. Acids Res. 8, 487-505. Schmitz, A., Sehmeissner, U. & Lu, P. (1976). J. Biol. Chem. 251, 3359-3366. Smith, T. F. & Sadler, J. R. (1971). J. Mol. Biol. 59, 273-305. Weber, I. T., M,~Kay, !). B. & Steitz, T. A. (1982). Nucl. Acids Res. 10, 5085-5102.
Edited by J. H. Miller
Interaction of Tight Binding Repressors with lac Operators An Analysis by DNA-Footprinting ~'IAGNUS PFAHLt AN[) ~']ICHELI:E H ENDI¢ICKS
The Salk ln~titute for Biological Studies Regulatory Biology Laboratory p.o. Box 85800, San Diego, CA 92138, U.S.A. (Received 21 June 1983) 'ro increase our understanding of p r o t e i n - l ) N A interaction in general, and in particular t h a t of lac rel)ressor with lac operator, we have investigated the interaction of tight binding (ltb) repressors with wild type (WT) operator and 0 ¢ operators. Nine ()~ and a W T operator were cloned and sequeneetl. T h r e e different ()c and all O + were then ehosen for the fOotl)rint analysis of six pb repressors and W T repressor. I)istinet protection patterns for the various rel~ressor-operator pairs were observed at low repressor concentrations whereas, at high repressor (.(mcent rations, a stretch of 24 bases of the lower strand of the four different operators was protected in most eases. This protection pattern at high repressor etmeentration was ahnost eoml)letely r e d u n d a n t for all repressor-operator pairs. in spite tit" the fact that the attinities (if" the various pairs
1. Introduction lac represst)r recognizes lac operator with very high specificity. The stability of the rolwessor-t~l)ora|or complex facilitates the biochemical analysis of the complex anti makes it a very suitable system to study the mechanism by whieh certain proleins recognize specific base sequences within double-stranded DNA. Moreover, from a Ifioehemical and genetic point of view, the lac repressor-lac operator system is the best-charaeterized example, since a large number of specific t Aut|mr tt~ wlmm e~wrespondence shouhl be addressed. 4O5 (11122-2836/8',~]040405-12 $03.00[0 ~) 1!t84 Academic Press l no. (London) lad,
1~,;
M. I ' F A I t l ,
AXI)
M. H E N I I I / I t ' K S
tnulations, ati't,t.ting Imth o p e r a t o r and represser, have heen isolated (for a review, see I~;.ur~t,ois & l+fahl. 197fi). We ln'eviously described the isolation and eharm.lt,rizati
iw~+lvelim~ ~.Xl~erimenls. Very st r
2. E x p e r i m e n t a l
Procedures
(a) Bm't+,ri,d .~h'ai,.+ ~ml medin E.,+'k,.riehia col~ slr'ain.~ are ~,ssontiallv tlms~, des<'rihed (lq'ahl. 1!)7!)). HBII)I and 31312!)4 w~+n' +d~taim+
(1>) ('h+',+ir',l.,; ],+.lJrOl~yl-fl-|+-thi.,,.,ahu.t<,..d
ZI~O dh~e <)+ - r ZI~XOdhu~ ()~ I]NA were. is, tared fi'um heat-inducible plut~es. The DNAs were, pt.rifivd and their +,on+-entration.~ wor(, rl~qermhl(.d as d(.,,~vrib(,d (]{iggs el at.. ]!)7()). ('all lhyrm,~ I)NA was ptH.t~hast.d I'r~tl~ ,'~i~111a and EroR[ linkvr |'t'a~nl~tlts ]mr,,'ha~{,t( J'rotzl l ] i i l , , l'la,~mid I)N..X. was purifi~'d ~n.~d~,seril~ed (Pfahl. 1982).
"I'I
4O7
(e) L~olatio. :tnd cloning of operolorfr~ment,~' F,'om 0.2 to 0.6 mg of 2h80 dluc 0 + or 0 ~ I)NA was tn.eeipitated with ethanol, washed. resuspended in Alul buffer, and digested with Atul. Tile digest was electrophoresed on a ,5% acrylamide gel (3 mm thick). The gel was then stained with eghidimn bromide. To identify the !)5 base-pair fi'agment eontaining the tac operator, a po,'tion of the ,4t,1 digest was incubated with lac represser and filtered through a (1.5 em nitrocellulose filter (pore size I).4/~m). The filter was washed and the bmmd I)NA was eluted by vortexing the filter in 501tl of e[eetvophoresis buffbr. Tlnis I)NA solution, of which 8()°~j was the 95 bp? ,4l, |'ragment containing the hie o~gj'ator, was run simultaneously with the non-filtered Alu digest on the aerylamide gel. The band eontaining the 95 bp operator fl'agment was cut out of the gel and the DNA was eleetroeluted from the gel slice. EeoNI linkers were then ligated to the 95 bp operator fi'agment; the I)NA was then digested with EcoRI and again run on a 5% polyacl3"hmtide gel The 1(17 hi+,,fi'agment was ehtted from the gel and ligated into the EeoRI site of plasmid pMB9. This I)NA was then used to transform strain MM2!14 or HBI01. Transformants were selected on minimal glucose-X-gal plates containing tetracycline. Dark blue colonies result when tile trust lae operon is induced by the molan' excess of WT operator t)inding lae repress
(f) DNA .~equencintt The 107 bp operator fi'agments eluted fi'om 5% polyaelTlamide gels were 5' labeled using [7-3~PJATP and T4 pelynoeleotide kinase. The labeled I)NA fragment, containing a single HlmIl site was digested with H p a l l , yielding a 61 bp fragment of which the Iowen' strand was 5' labeled. For DNA sequencing, the methods and tn'oeedures of 3laxam & Cilbert (I 980) were followed. (g) DNA foolpri+~linff in general, procedures of Galas & Schmitz (1978) were followed. The 5' lal)eled 61 hp fragments used for sequencing were ineubate{t in BP, buffer (0.01 ,m-KCI. 0.01 m-3lgC] 2. 0.01 m-Tris- HCI (pH 7.4), 0.0001 .~I-dithiothreitol), fen' 15 rain with various amoutlts (1)'5 #1. Itd. 5/tl, 25111) of purified represser (,-,fix 10 -~ .xl), l)Nase I was then added tbr 30 s. after which the I)Nase l reaction was stopped by the addition of EI)TA and calf t h y m u s 1)NA (final conch 6.25 x 10 -2 .~t and 200pg/ml, respectively). The I)NA was then precipitated twice using 2 volumes of 100°/o cold ethanol and a final salt concentration of 0.3 .m-sodium acetate. The precipitated I)NA was washed twice in 70°,~) ethanol, then resuspen(led in eleetrol)horesis buffer and loaded onto a Setlumleing gel.
3. Results
(e~) Characterizatfon, of operator fragments A set. of nine lac 0 c mutants, including most of the O c operators used previously to select and characterize l ub repressors in rive (Pfaht, 1979), and WT operator were cloned into plasmid pM B9 as deseribed in the methodology. To exclude any ambiguities concerning the sequence of the cloned operator fragments, all eioned operators including WT operator and known 0 ~ ~perators were s e q u e n c e d ( T h e
t Abbrevhdim~s rimed: hp. hase-pair; 1).MNO. dimethylsu|fi~xkte.
31. I ) F A H i .
ANI)
31. HI.;NI)RI(',KS
results are shown in Figure I. WT (~l)eralor and the lm<)wn ()~ ()l)erators ()R,VI, ORVI:~O and ()R\'116 (Smith & Na(ller, I.<171) revealed lhe previously rep()rte(] seqnences (Gill)erl ~ Maxam. 1.973: (;ilhert e! ~d.. 1975), T h e 0 ~ o p e r a t o r s nol previously isolated t u r n e d out to he identi(',al with either ORVI21) or O R \ ' l l 6 . This result was not e(>mpletely surprising, since m u l t i p l e ('~q)ies <)f s l r o n ~ (F m u l a n l s have heen isolated previously ((;ilhert et
Ihree O" operalor.~' Various (.on(.enlralions (>f W T repressor were i n e u h a t e d with lahele(t HpaII) ~;c,l~l 61 h l) f r a g m e n l . (.(mlaining eil.her W T Ol)erator (O +) or ,~ne of t h e t.hree (lifl'erent (F (~])erators. hef()re (ligesti()n wilh I)Nase I. T h e l)Xase l c()n(~entratio,i aml (ligesli()n time were kel)l ('(mslanl in the (lif['erent. e x p e r i m e n l s . Typical ti>ml)rinting resulls are s h o w n in Figures 2 and 3. 'ro o h t a i n the correlation helween the l)role(.h,(l I)NA re~ion and the <)i)erator sequence, sequenein~ v'eacli(ms were run in parallel wilh [lie DNase
+15
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Fro. I. l'.a,~" elmn~.s in I F mutali~m.~. Wild type Ol..r:lh)r I)NA and 9 IF InlltIlti(ms were elolled into phL~mid pMB9 as (h.serihed in Exl~'rim~.nhll Procedures and se(im,m,ed (Maxam & f;illu~rl. 1980). ,~'quel)(.e or l b . low,,r strand (,r the ~VT Ol)eralor i,~ ~ziv~'n nn lop w i l h symmetry r't.~d,,nx un(lerlined, Till. cl.nl~.r cJf ,~ymnlelry is at O. T I . ' ha.~. ehang,.s l'mmd in (F llllllatiollS are indieate(l. Seqller, ce hvlJhen.s haw~ l..en omille(l for elarily from Ibis and .~uh.se(luenl. Figures.
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t;'1~:, :~. Fot~tprint ~)l" ~,~i"l" l'P|II'e,~tJl' Oil (J ÷ ~lui thrt.e 0 c oper.~t.tors, Puritled W T rept'es,~or (5 #1) wa.~ int.ubated w i t h G' hlheleO (lower stralld) Itp~+ll-EroRl ['r~LBment ecmt~ining either 0 + (lane B). ()RV, I (()1) (]ttl~.e AI, ()tl (laue (!). ~t + (,~, {[:ule D) ~])er+Itor. The rel)res.~ur-C)l~erat~r m i x t u r e s were then (ligt~sted w i t h I)Na.~e | llA'ld F'l.lll ()11 ~P(LtIel'l(-'iI~g gel.~ a~ deserihed in I'~,xl>erimel~tal Prt)(~edu:'es. A.~ a t:~mtt'(~l ()÷ I ) X A was digested in tl~.p ~ii)..~ell~P 11[ ][~.-[)l'd2.¢.si.].l " (little hi). (~t) }).l-itl (b)ThE. Sttl)'ie ['e21eticJIi tll~.XeJ.II'e~ but eleetrol)hore~ed [ur difl~re[tt [ength.~ o[' tinge, l)r¢~tecte<| areas ~ire in+|ieated f)y brackets.
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hl~ ~,x~,han~o. ( ) ~ ,~hmv,~ n,dm'e.'l lWote(.tion in the middle and ~tt +6. All three ()~ ~q.,rators shiny ,~li~htly ,~t,n'~m~." prol.eetion at the right end (-1()). "1'~ .hl~dn m~,'~' int'~.'mation ~m the' I~mtertion Imtt~'rn~, we als~ ean'ri(,~l m~t f i . ~ l i ~ r i n t i n ~ : eXl,erim~mt.~ b e t w e e n W T r e p r e s s o r a n d t h e ['()ill" clifl'er~,nt ~q)c'rat~ws in l h u IWeSunr~ • ~t' l llq~ ( v / v ) dim~q hyl.~ult'~xide. I)~l,~() has I~e(,n sllowl~ t o i m ' r e a s u the, a l l i n i l y o f n,pre,~,~or f o r ~)peraioz'. A I()?{~ I)31N(-) e o n e ~ ' n l r a t i ~ m w ~ s l'~mnd t u st ~l~iliz~, t h e n , l ~ r e , ~ s o r - o p e r a ! o r ~ m ~ p l e × t e n | b l d ( P f a h l , I i)?~). 'rhc, M~ser.ved p m t e ~ q i o n patterns in .~°~.,,,. I),~IS() w e r e difl'erep.t l ' m m t h e imtler,m ~d~sel'ved befiu'e. I n Aene,'~tl, p r o t e c t i o n wa,~ s t r { m ~ e r , ,~o t h a t W T
rMw~,,~.~.' r~,,~,mhh~d an I ~ U'eln'r,~,~or under thu~' uondition,~. IAilt'~.r~.n~,~,.~ in iw~d~,{,ti~m lmtt~,rn,~ w~,r~, oh,~erved ~tt low rel.'e,~,~or eon~,entx'ati.n,~ ~mly. A .~uhemati~ rel~t'e.~entative (d' foml)rints is ~zivon in lggm'~' 4 (s~'t (b)). ()+ was l~r~doul(,d t~ Ih(' ,~ame e x t e n t a,~ b e f o r o . ()] rt, v e a l e d ]os,~ ditt'~,r~,m'~- t - ()+ a n d r{.dm,{,d pmt~.~,ti~m wa,~ only oh,~ex'ved ~t + 7 . (9~ and ()~1 at lmv n'el.'~,,~,~w r~m~'{.ntralion difl'~,'ed mor~. strongly from ()+ than in the ab.~enc~, of I)3IH(). 'rh~. ma.i~r d i l h ' r e n r e s i ~ ()+ w e n , a t + ], -- ] m-~d ~ : ~ f o r ()o ~.z1~] al +;3. + ]. - ] al}d --:l t()l' ( ) ! 1 .
(r) I " o . t p r i . l i . g ~.Mp.~is .J' !~' reln'e.~',sor,~ u'ilh 0 + ~ . d (Y W~emh.',~' 'l'hr d~,laih,d analv,%~ uf I ~b repre.~sors wit h var'im~,~ o p e r a t ~ r s rouhl yield results theft might allmv {.~mulusions on the n a t u r e of the ~lter'ed inter~wtion h~-twec,n the, T
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auu(~Ulli
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the, .~,h~mmtir r~,l.'L,.~-ntati~m in (a). I"~tl.'int,~ w~,r. ~,~'ahmtud hy ~,ye and b.v del~.~il~.l~etry .~uanmer, The pruteeted area i~ the 24 Im,~es direutly ~m t~l~ of the" Imr. The I~r' ron.~i~t~ of 24 .~.b~znlen. one" for ~,~,.h Imst,, 'l'hc, n'mahfin,._, Im.~,~ ~t' |]H' hm'~,r strand of the ~pc,'al.r m'e Unl,'.h'et(,l. ,~]mxima] I~X'Otucthm (at :~5/d u'elw~,s.~w) i.~ n'lwe.~,nted in the. ()+ lane. I)it]'erenee.~ ob.~t,'ved in proteuti~m at Imv I'elwL,ssor (.(~l|c(,IHl'~Hi()ns al'l, indh.atecl hy dt,h'thm.~ in the Im:'.~ in the ()~. ()~, and ()~ lane.~. The deh'ti~m:; in the Imn'.~ are .~eah'd I to ,1, i.e. a very .~tx'.n~ hand sh~wh~,~ on t.h~, t'o-tl,'int tbr an ()~ with m~ Imnd shmvin~z fi~r the ()+ in the ~aum. lm.,4ti.n wmdd he l.'e.~ented a.~ ~ eOnnldete deh,ti~m ~d'the bar in the l) ~ I~me nt thr~ p~.~iti~m. I~ thi.~ Figure.. maxirmd ditt'erem,e.~ ~h.~m'ved were not ~zr'e~der titan "2. X()tM. tlmt ()~ i.~ m)! roml)h,teJy I.'~)h,rh,d ;d p.~ith)n --14) (jmlg~,d at the" Ifi~lm:4 repre~.~)r (.(.u,~,ntration ~s~,d). (h) ,~'rhemath. rel)r..~entati(m ()t" WT r('l)re.~.~or IW()tm'tion Imt tern.~ in the" I)r('.~em'(• ~)t' I()% I)M~().
.I1'2
51. I ' I , ' A H I , A N I )
M. H I , : N I ) R I ( ' K S
llltlltllll l'epv'ess(trs a n d ol)erators. I tb I n l l t a n t s h a v e l)e~ll s h o w n to over(+()ine ()+
inlllati()ns ill vivo. The i. vitro s(',reening system used hcrc will alh)w a mueh nlorc (h,taih,(l analysis, sivlce rel)V'essor t<) el)orator t'atios (Pan I)e greatly altere(l. Six ! tb repressors were investigated with tile |'our oi)erators used I)el'ore. In each (,ase, four different rel)ressor e o n e e n t r a t i o n s were used with one fixed operator (.(m(,(.ntrati()n. (:haraetcristie i)roteetion p a t t e r n s were el)served for ea('h rel)ressor-ol)erator pair. 'I'll(+, results are summarized in Figure 5. According to these results, the 1tb repressors ean he divided into two classes, one of" which distinguishes well I)etween WT anti O ¢ opev'ators. This class includes the R.7(i, R7Sa. R84 and R67 repressors. I+~xeept tbr the latter, all of these rel)ressors were shown previously to have only it small (3'5 to 9-fold) increase in affinity for' O + I)NA (i)fahl, 108lit). The other class ¢)t" I tb repressors contained tim two very tight bin(ling rel)ressors X86 anti R l I 3 . These two rel)ressors gave nearly identical prolet.ti(m Imtterns, for I)oth O + a n d ()c operators, at all represser concentrations. The I tit repressors also show an increased protection at the right end of some operators, position - 1 0 , with t h e R l 1 3 represser being the strongest in this r('gar¢l. In this (.ase (.omph,te proteeti()n was observed at - 1 0 tbr all four Ol)(,r'ators. WT rel)ressor In'otters this base only partially on 0 +. The I¢78a and the Illi7 r('prcssors were the only ones t h a t (lid not proteet l)osition - 1 0 on O + even at higher represser (.oneentrations. A similar effect was seen for O l with R7Sa. but not fi)r (-)6 and ()11, in whi('h ease the - I 0 position was proteeted to the same degree as in the %VT rel)ressor-O + pair. It should be emphasized t h a t all tilt, protection diagrams in Figure 5 are obtained from the analysis of several foot l)rints. (.Oral)rising several repress<)r to ()l)erator ratios. T h e (liagrams therefore give only (it,alitative results and do not express particular r e p r e s s e r - o p e r a t o r affinities. ()ur results are (letailed beh)w with the l)roteetion of 0 ¢ operators eOmlmred to the prote(,tion of" O + for each ltb rel)ressor" (i) R76 represser This represser was previously found to have a ninefohl iner'eased affinity tbr 0 ÷ (Pfahl. 1981a). Complete protection of the O + operator ineluding position - 1 0 was observed. In the case of O~, major decreases in protection were found in positions + 7 , + 5 , + 2 , + ! t h r o u g h - 1 , - 3 , - 5 , - 6 and - 1 0 . F o r 0 6 , major de(.reases in protection were observed at + 5 , + 2 , +1 and - 3 . For O~t major decreases in protection were found at + 5 , anti +1 through - 3 . (ii) R78a repres.~or This represser binds 3.5-fold tighter to O + (Pfahl, 1981a). Protection of 0 + was incomplete in that no protection was observed at+ - I 0 and decreased protection at - 9 . Major decreases in protection for O1 were found at position + 7 , + 5 , + 1, - ! anti - 3 . Increased protection for 0 6 was observed at - 9 and - 1 0 , and decreased protection at + 5 , + 2 , + ! , - 1 and - 3 . Increased protection was found |br ()11 at - 9 anti - 10, and decreased protection at + 11, + 6 , + 4 , + 1, - I anti - 3 .
TIGHT
#]INDING ICEPRI~SSOR FOOTPRINTS T
413
AT
AC ACAC C TTAACA CTCGC CTA TT,o TT~AGTGTGT +I0 R76 06 . 01
.
_
~
+5
.
-- ~
,-- _,___,
minim....~
~I
L
m '~
~
~
~
~
-
01 ~
mmmsm~Umml~ I
,
i
×06
m'=
~
0~
RI~
-5
.
-- I
---
,
'
--J,'
m
-I0
0
'
I
MF I
I~
m
R67 06 - ~ - - ~ - - L ~ . . . . ff
'
'1
+10 +5 0 -5 -I0 ACAC ACC TTAACAC TC GC CTAT TG TTAAAGTGGT T
AT
Fro. 5. Sci~ematic represe~ltation of footprints obtained with tight binding repressors (I'~). Various concentrations of each pb repressor were incubated with 0 ÷, 01, 06 ~nd 011 operator and digested with DNase I. The observed footprints were evaluated as described in the legend to Fig. 4. Marks t,nder the bar stand for it~ereased protection.
(iii) R84 repressor This repressor binds 3"5-fold tighter to O + (Pfah[, 198ta). With 01, decreased protection was observed at + 7 , + 5 , +2, + l , - - 1 a n d - 3 . With 06, the observed p a t t e r n was the s a m e as t h a t observed for O1 with only small differences in t h e intensities. With O l l , decreased protection was observed at + 5 , + l and - 3 . (iv) R I 1 3 repressor This repressor was f o u n d to bind almost 200 times tighter to 0 + (Pfahl, 1979). Complete protection was observed fbr O + including --10. For O1, increased
414
51. P F A H L A N D M. H E N 1 ) R I C K S
protection was observed at + 12 and - 1 0 , and decreased protection at + 7 . For O6, increased protection was observed at + 1 2 and - 1 0 . For O l t . increased protection was observed at positions + 1 2 , + 7 and --10, and no decrease was ol)served. (v) X 8 6 repressor This repressor was found to bind 50 times stronger to O + (3obe & Bourgeois, I972; Pfahl, 1976). With 01, slightly increased protection was observed at + 12 with O6 and O11 at + 12 and - 1 0 . F o r all three 0 ¢ operators, only very small decreases were observed in the protection. (vi) R67 repressor This repressor was shown to contain a m i x t u r e of weak and tight binding molecules (Pfahl, 1979). The t i g h t binding molecules have a 100-fold increased affinity for operator (Pfahl, 1981a). N~) protection of - 1 0 was observed with 0 + l)ecreased protection was observed for O~ and O 6 at. + 7 , + 5 , + 2 , + i , - 1 and - 3 . For O l~. decreased protection occurred at + 5 , + !, - ! and - 3 . For all three 0 ¢ operalnrs, increased protection was observed at. - 10. 4. D i s c u s s i o n
lq this s t u d y , we have investigated the interactions of W T repressor and six tight binding repressors with 0 + and three O ~"operators by 1)NA footprinting. A large variety of protection p a t t e r n s were observed at low repressor to operator ratios, whereas at [~igh repressor to operator ratios, a uniform protection profile was observed for a h n o s t all r e p r e s s o r - o p e r a t o r pairs. These latter results agree well with those obtained by others (Sehmitz & Galas, 1979), which showed t h a t the same 24 bp o p e r a t o r region ( - 1 3 to + i 0 in Fig. 4) is protected by W T repressor as well as by the very tight binding repressor ]12-x86, a double m u t a n t . The only exception to this rule are the R67 and the R78a repressors. In both cases, protection at the right end of the lower s t r a n d is a b s e n t at - 1 0 and also reduced at - 9 in the case of t.he R78a repressor. This reduced protection is observed mainly with W T o p e r a t o r and, to some extent, with O~. The other two 0 ~ operators, Os and O l t , show normal or enhanced protection at - 1 0 and - 9 . It is interesting to note here t h a t both the R67 and the R78a repressors carry m u t a t i o n s at a very different location (amino acids 255 to 205) t h a n other |tb repressors, whieh all carry a m i n o acid exchanges in the amino-terminal end (Pfahl, 1981b). The 255 to 295 region of t h e lac repressor has been postulated to contain infbrmation for repressor s u b u n i t interaction (Miller et al., 1979; Schmitz et al., 1976) or possibly be involved directly in D N A binding (Pfahl, 1981b). A change in the s u b u n i t interactions could allow the repressor s u b u n i t s to interact more tightly with certain regions of the o p e r a t o r I ) N A . Protection in the presence of DMSO seems to reduce t h e a m o u n t of repressor needed to obtain complete protection. However, DMSO does n o t a p p e a r to have the same effect, on all repressor o p e r a t o r pairs. For instance, in the eases of the 0 ~
TI(;HT BINDING REPI{ESSOR FOOTPRINTS
415
operators 06 and 011, tile 1)MSO effect appears to be reduced when compared to 0 +. Similar observations on the differential effect of DMSO on various 0 c operators }lave been made by direct binding studies (unpublished results). The main observation here, however, is that DMSO, in spite of increasing the affinity of WT repressor for 0 + tenfold, does not lead to the protection of additional bases on the lower strand. The data so far discussed reveal three points: (1) WT repressor and mutant repressors can protect only a limited number of bases against nuclease digestion (24 on the lower strand); (2) the number of bases protected at high repressor concentrations does not correlate to the strength of tile repressor-operator interaction; and (3) specific protection patterns can be observed only by careful titration experiments. Protection of O ~ operators differs more distinctly from 0 + for one class of Itb repressors (R67, R76, R78a and R84) than for the two very tight binding repressors x86 and R113. The decrease in the protection of O ¢ operators at low repressor concentrations occurs mostly in the middle of the protected region. It is somewhat difficult to visualize how DNaseI could act there (because of steric hindrance through repressor) while the outside regions are still protected. Possible explanations could be (1) the repressor is not constantly in close contact with the operator region, so that some "breathing" of the complex may be possible; (2) the weak protection patterns at low repressor to operator ratios could be an expression of two operator fragments binding one repressor molecule, in which case one of the interactions would be weaker than the other and result in only partial protection. (Tetrameric lac repressor has been assumed to contain two oper~rtor binding sites and some evidence has been obtained for this; Culard & Maurizot, 1981; O'Gorman et al., 1980); (3)the partial protection patterns ol)servcd could be the presence of partially inactive repressor molecules, due to (.heroical degradation or to special steric conformations of the repressor subunits. At this point none of the three above mechanisms can be ruled out; however, partially i11active repressor has been observed by many investigators, and usually all repressor preparations contain some repressor that is inactive or partially active in terms of its DNA binding. We have also previously shown that the R67 repressor is a mixture of weak and tight binding molecules (Pfahl, 1979). lac repressor tetramers containing some inactive or partially active subunits could easily yield partial footprinting patterns. At high repressor to operator ratios, the only partially active repressor molecules would be competed out by the fully active repressor molecules. The protected region of 23 to 24 bases of the lower strand is asymmetric with regard to the operator symmetry. The left half of the operator is better protected, which fits with the notion that in that region the stronger 0 ~ mutations occur (Gilbert et al., 1975). Schmitz & Galas (1980) have shown that protection of the upper strand is shifted two to three bases to the right protecting the operator somewhat asymmetrically in the right-hand direction; in this way a symmetric protection pattern is obtained when one looks at both upper and lower strand. The footprinting results are, in general, similar to results obtained by methylation protection (Ogata & Gilbert, 1979) and fit the models for the repressor-operat6r
41fi
M. PFAHL AND 51. HENI)RICKS
interaction recently suggested. In these models, protruding amino-terminal ends of the lac repressor sl,bunit, s are t h o u g h t to interact with the major groove of ol)erator B-I)NA (Adler et al.. 1!)72: Matthews et al., 1982; Sauer et al., 1982; Weber et al.. 1982). The repressor is t h o u g h t to interact with mostly _-,ne side of the I)NA helix, i.e. according to our results, each of the repressor subunits (2 per operator) would protect 1.2 turns of the 1)NA t;elix, probably the m a x i m u m the protruding amino-terminal end of a repressor subunit can protect or reach. The analysis of the amino acid residues exchanged in the pb repressor is now in progress and these results should yield f u r t h e r information on the molecular details of the lac repressor-operator interaction. We thank Allen Kool) for his hel l, in setting up the DNA technology, Suzanne Bourgeois f,)r diseu~sions and critical reading of the manuseril)t, and Karen Horosehak for excellent technical assistance. This work was supported by Public Health Service grant GM23671 from the National Institute of General Medical Seienees. REFERENCES Adler, K.. Beyreuther, K., Fanning, E., Geisler, N., Gronenborn, B., Klemn, A., Muller-Hill, B.. Pfahl, 51. & Schmitz, A. (1972). Nature (London), 237, 322-327. Bourgeois. S. & Pfahl. 5I. (1976). In Adt'ances in Protein Chemistry (Anfinsen, C. B.. Edsall, J. T. & Richards, F. 31.. eds). vol. 300. pp. !-99. Academic Press. New York. Ct, lard. F. & Maurizot. J. C. (1981). N~lcl. Acids Res. 9, 5175-5184. Galas. 1). & Schmitz. A. (1978). Nucl. Acids Res. 5, 3157-3170. Gilbert. W. & Maxam. A. (1973). Proc. Nat. Acad. Sci., U.S,A. 70, 3581-3584. Gilbert. W.. Gralla, J., Majors. J. & Maxam, A. (1975). In Symposium on Protein-Lifand l'nterttctions (Sund, H. & Blauer. (;., eds). pp. 193-206. Walter de Gruyter, Berlin. ,lobe. A. & Bourgeois. S. (1972). J. Mol. Biol. "72. 139-152. Matthews, B. W., OhlendorL I). H., Anderson. W. F. & Takeda. Y. (1982). Proc. Ntfl. Acad. 8ci.. U.S.A. 79, 1428-1432. Maxam, A. L. & Gilbert, W. (1980). Methods Enzymol. 65, 499-559. Miller. J. H., Coulondre. C.. Hofer, 5I.. Sehmeissner, X., Sommer, H.. Schmitz, A. & Lu, P. (1979). J. Mol. Biol. 131. 191-222. Ogata. T. R. & Gilbert. W. (1979). J. Mol. Biol. 132. 709-728. O'Gorman. R. B., l)unaway. 5I. & Matthews, K. S. (1980). J. Biol. Chem. 255, 1010010106. Pfahl, M. (1976). J. Mol. Biol. 106, 857-869. Pfahl, 5I. (1978). Biochim. Biopbys. Actor, 520, 385-290. Pfahl, 51. (1979). J. Bacteriol. 137, 137-145. Pfahl, M. (1981a). J. Mol. Biol. 14"/, 1-10. l'fid~l, 5I. (1981b). J. Mol. Biol. 147. 175-178. Pfahl, M. (1982). Cell. 31,475-483. Riggs, A. D., Suzuki, H. & Bourgeois, S. (1970). J. Mol. Biol. 48, 67-83. Sauer, R. T., Yocum, R. R., Doolittle, R. F., Lewis, 5I. & Pabo, C. O. (1982). Nature (London), 298, 447-451. Sehmitz, A. & Galas, D. J. (1979). Nztcl. Acids Res. 6, I I !-137. Sehmitz, A. & Galas, D. J. (1980). Nucl. Acids Res. 8, 487-505. Schmitz, A., Sehmeissner, U. & Lu, P. (1976). J. Biol. Chem. 251, 3359-3366. Smith, T. F. & Sadler, J. R. (1971). J. Mol. Biol. 59, 273-305. Weber, I. T., M,~Kay, !). B. & Steitz, T. A. (1982). Nucl. Acids Res. 10, 5085-5102.
Edited by J. H. Miller