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Communication Vol. 262 No. 11. Issue of April 15, pp. 4943-49 46 1987THE OURNAL F BIOLOGICALHEMISTRY0 1987 by The American Society of Biologwal ChernisG Inc.Printed in ~ s . A .

Supercoiling FacilitatesacOperator-Repressor-Pseudooperator Interactions*(Received for publication, November 24, 1986)

Peggy A. Whitson , Wang-Ting Hsiehs,RobertD. Wellss, and Kathleen S . MatthewsSllFrom the Department o Biochemistry, Rice University,Houston, Texas 77251 and the DepartmentofBiochemistry, Schools of Medicine and Dentistry,University o Alabarnn at Birm ingha m,Birming ham, Alabama 35294

The binding affinity of the Escherichia coli lactoserepressor to operator-containing plasmids was in-creased by negative supercoiling of the DNA. The in-creased affinities observed were dependent on the sequence context of the DNA as well as the degree ofsupercoiling. Dissociation rate constants for plasmidscontaining a single operator site decreased as a func-tion of the negative supercoil density. However, thepresence of pseudooperators in the plasmid DNA inaddition to the primary operator sequence resulted ina significant decrease in the operator-plasmiddissocia-tion rate at higher negative supercoil densities. Ap-proximately eight ionic interactions were determinedfor both the supercoiled plasmids and the linear DNAsexamined. These results suggest that the stabilizationprovided by the topology of supercoiled DNA affectsthe nonionic component of the protein-DNA interac-tion. The ability to form a ternary complex of proteinwith two DNA segments is increased by the presenceof multiple operator-like sites on the DNA. Further-more, supercoiling DNA with multiple operator-likesequences profoundly diminishes the dissociation rateand results in a remarkably stable ternary, presumablylooped complex tM- 28 h). These data suggest a criti-cal role in vivo for DNA topology and pseudoopera-tor(s) in transcriptional regulation of the lac operon.T h e lac repressor protein from Escherichia coli exerts neg-at ive transcript ional controlover the structu ral genes of thelactose operon. Specific, tigh t bind ingof the repressor proteinto th e perator region of the DN A hysical ly blocks ranscrip-tion of D NA w hich codes for the lactose metabolic enzymes.

Pseudo operators, which exh ibit sequence homology to heprimary operator, are located both in the lac Z a n d I genesan d display 10- an d 100-fold lower affinities for the repressor,respectively (1).Operato r binding affin ity is increased whenpseudooperators are present in addit ion to the primary oper-a tor on DNA (1-6). Th e repressor protein has two operatorGM-22441 (to K. S. M.) and Grant GM-30822 (to R. D. W.), Robert* This work was supported by National Institutes of Health GrantA. Welch Foundation Grant C-576 (to K. S. M . ) ,and National ScienceFoundation Grant 83-08644 (to R. D. W.). The costs of publicationof this article were defrayed in part by the payment of page charges,This article must therefore be hereby marked advertisement inaccordance with 18U.S.C. Section 1734 solely o indicate this fact.To whom correspondence should be addressed Dept.of Biochem-istry, P. 0. Box 1892, Rice University, Houston, TX 77251.

bind ing sites, and ower repressor-opera tor dissociation rateshave been measured in the prese nce of both nonspecific a ndpseudooperator sequences in he operator-containing DN A4, 5). An intram olecular ternary complex with both DN Abinding sitesof th e repressor occupied, and cons eque ntDNAloop form ation, has been pos tulated to explain the stabiliz a-tion of therepressor- operator complex in he pres ence ofintramolecular DNA sites 4-6, 38).Previous work has indica ted significant effects on prote in-DNA interact ions as a consequence of DNA topology 7-9).Wan g and co-workers (10) reported a decrease in the disso-ciation rate cons tant of lac repressor from supercoiled cova-lently closed Xplac DNA. W e re por t here effects of negativesupercoil ing on the repressor-operator interact ionn a varietyof sequence contexts. Supercoiling of operator-containingplasmid DNA s with and without seudooperators resulted ina decrease in t he dissociation r ate co nstan t compared to th erates observed with h e corresponding linear DNAs. Th e mostdram atic effect was observed for pIQ, an oper ator and pseu-dooperator-containing plasmid,presumablydue to ernarycomplex formation of operator-repressor-pseudooperator.These data extendprevious in vitro studies 10-12) and sug-gest critical roles in vivo for the DN A topology an d sequencecontext in stabilizing the repressor- operator complex.Effects o Negative Supercoil Density o n the DissociationRateConstant-The issociation rate onsta nts for pIQ,anoperator ndpseudo operator-co ntaining plasmid, andpLA322-8, a prom oter-operato r containing lasmid, as a func-t ion of th e negative supercoil density are illustrated n Fig. 1.For both DNAs, th e dissociation rate decreased as the nega-tive supercoil density increased. The dissociation rate forpLA322-8 decreased -4-fold as th e egative supercoil densityincreased from 0 to 0.085, whereas a n -230-fold decrease inthe dissociation r ate c ons tant was observed for pIQ betweenrelaxed D NA and a negative supercoil den sity of 0.06.Labeled supercoiled pIQ w hich had been relaxed w ith to-poisomerase or linearized withPstI exhibited dissociation ratecons tants comparable to tho se observed previously for linearpIQ 4). The inset to ig. 1 llustrates dissociation rate deter-minations for relaxed and supercoiled pIQ. The plasmidpRW468, which harbors a 59-bp fragment containing only aportion of the prom oter region an d the enti re lac operator,exhibited behavior similar to pLA322-8, althou gh to a lesserdegree. These data ind icate that the repressor-operatorom-plex is more stab le as the egative supercoil density increasesan d th at th e presence of the pseudooperators in addition tooperator further enhance s this apparent stability at highernegative supercoil densities.Sinc e he dissociation ra te cons tant forsupercoiled pIQdecreased dramatically compared to the linear form, we at -tempted to determine the dissociation rate constant in thepresence of isopropyl-1-thio-/3-D-galactopyranoside.he re -pressor-operator dissociation was not measurable by the ni-trocellulose filter-binding technique when inducer was added( h 2 < 5 s). Although the supercoiled structure significantlyenh ance s repressor binding, dissociation of the prote in- in-ducer complex from the DN A remains quite apid. Thus, therepressor- operator interaction ould be stabilized by th e DNAThe abbreviation used is: bp, base pair.W.-T. Hsieh and R. D. Wells, manuscript in preparation.

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4944 D N A Topology Affects OperatorBinding by lac Repressor

0.02 0.04 0.06 0.08SUPERCOIL DENSITY

FIG. 1. Effects of negative supercoil densityon he dissocia-tion rate constants of the repressor-pIQ and the repressor-pLA322-8 complexes. The plasmid pIQ (-6200 bp, gift from J.Betz, Universityof Colorado Health Sciences Center) contains bothpseudooperator sequences, in addition to the promoter-operator re-gion, and was produced by insertion of a 1.7-kilobase pair Him11fragment (containing he I gene, promoter, operator, nd a portion ofthe Z gene) with EcoRI linkersnto the A m 1 site of pBR322 with theEcoRI site deleted. The plasmid pLA322-8 -4100bp, gift fromGeorge Bennett, Rice University) contains a 95-bp Ah1 promoter-operator fragmentnserted into the linearized pBR322 (cut at BarnHIand EcoRI). The DNAwas abeledwith Hhd methylase and S -methyl-[3H]adenosyl-~-methionine.2he dissociation rate constantswere measured by adding a 200- to 6000-fold excess of unlabeledDNA (either pIQ or pLA322-8)o a pre-equilibrated solution ontain-ing repressor and labeled pIQ or pLA322-8 (eacht 5.0 X lo- M) sdescribed by Whitson and Matthews (4). The buffer for the assayswas 0.01 M Tris-HC1, pH 7.5, 0.20 M KCl, 0.1 mM EDTA, 0.1 mMdithiothreitol,5% dimethyl sulfoxide. Time courses for pLA322-8ndpIQ were 3 and 40 h, respectively. Equilibrium was not attained forpIQ during this time period at the higher negative supercoil densitiest H- 28 h); therefore, the inducer-sensitive bindingwas subtractedfrom each time point and the rate was determined from a log plot ofthe curve as described previously (35). he DNA at various supercoildensities was prepared as describedy Singleton and Wells (36). Eachpoint represents two to four rate determinations from two separatepreparations of supercoiled DNAs.Standard deviations are indicatedby error bars, except where the deviations were less than the area ofthe symbol.0 pLA322-8;A, IQ. The inset illustrates he dissociationof pIQ in the presence V) nd absence W) of calf thymus topoisom-erase I (Bethesda Research Laboratories and gift of J. E. Larson,University of Alabama). The buffer for this procedure was the sameas described above with the addition of 1 mM magnesium acetate.

Repressor and pIQ (each at 5 X lo- M)were incubated for 10 minbefore the addition of 40 units of topoisomerase I to one sample andfurther incubated for 0 minat room temperature priorto the additionof a 200-fold excessof unlabeled pLA322-8.structure n heabsence of inducer,but hepresence ofinducing sugar would elicit the rapid response necessary invivo to effectively utilize available lactose.Effects of [KCU on theRepressor-OperatorInteraction-The effects of salton hedissociation ateconstantcanprovide information about mechanisms of repressor-operatorinteract ion (2, 4, 5 , 13-15). Previous data using linear DNAsof various sizes and sequence contexts have suggested tha tthe presence f intramolecular sites affects the stabilityf therepressor-operator complex 2-5). In Fig. 2, the dissociationrate constants a t various KC1 concentrations are reported forlinear and supercoiled pLA322-8 and pIQ. The dissociation

10050

1ca*x 5Qx

1

0.5

010.0:

log KCI)

coiled pLA322-8, pRW468, and pIQ as a functionof the KC1FIG. 2. The dissociation rate constant for linear and super-concentration. The dissociation rate constants were measured asdescribed in Fig. 1 and Whitson and Matthews 4). Values for linearpLA322-8 and pIQ were determined previously 4). Each point rep-resents the average of 2-4 rate determinations. Standard deviationsare indicatedby error bars, except where he deviations are less thanthe area of the symbol. Initial repressor and operator concentrationswere 5.0 X lo- M , and a 200-fold excess of unlabeledoperator DNAwas added at t 0. The negative supercoil densities were as isolatedfrom E . coli HBlOl (u- 0.06) (36,37).Panel A, the dissociation rateconstants forpLA322-8 and pRW468 at various [KCl]. 0 linearpLA322-8; 0, supercoiled pLA322-8;0 supercoiled pRW468. PanelB, he dissociation rate constants for pIQ at various [KCl]. A, inearpIQ;A , supercoiled pIQ.

08 -06 0.4 08 -0.6 0.4

rate constants for both DNAs at each salt concentration aredecreased for th e supercoiled DNA as compared to the linearDNA. However, the dissociation rate constant forupercoiledpIQ is less salt-sens itive than linear pIQ or either linear orsupercoiled pLA322-8. The minimal salt sensitivity for thepIQ-repressor dissociation uggests th at negative supercoilingprovides a secondary or tertiary DNA structure hich furtherstabilizes the nonionic componentsf the intramolecular ter-nary complex of operator-repressor-pseudooperator. It is ap-parent from these measurements that the secondary and/ortertiary st ructure of the DNA plays a significant role in thebinding process.

The number of ionic contacts in a binding interaction canbe derived from the salt dependencef th e equilibrium bindingconstant. The slope of a plot of log K,, versus log[KCl] hasbeen shown theoretically to be equivalent to the numb er ofcounterions released upon binding (14, 16-18). We were un-able to radiolabel pIQ witha sufficiently high specificactivitytomeasure heequilibriumconstant (

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4946 D N A TopologyAffec ts Opera tor Binding by l cRepressorconstant for l inear pIQ k , / k , b 2 X 1014M-). K* alculatedusing this value for supercoiled pIQ is -2000 as compared to59 for inearpIQ Table I). Basedon he effective localconcentration of theseudooperator M ), K can beconverted to a pseudo operator-b inding constant to the re-pres sor- ope rator complex of 2 x 10 for supercoiledDNA. Thus, supercoil ing and th epresence of intramolecularpseudooperator-binding sites are vital n uiuo considerations.Th e rat io of equil ibrium constants for protein binding osupercoiled and linear DNA can e utilized to determine theDN A unwinding angle 10, 0, 21). Th e u nw indin g angle forpLA322-8 is -60. However, if the DNA forms a loop struc-ture , heunwinding anglecalculation or pIQ would notaccurately reflect the a ctual D NA unw indin gngle.3Conclusions-Examination of linear DN As has indic atedth at th e ize of the oper ator equence, as well as th e equencecontext, are impor tant factors in repressor b inding 2-5, 12,22). The plasm ids used in the p resent study dem onstrate theinfluence of the pseudooperators, the prom oter region, andDN A topology on repressor binding to operator-co ntainingDNA. Thestab ility of repressorcomplexeswith plasmidscontaining operator an d pseudoo perator equences has beendem onstr ated previously 2-6, 38). Th e dram atic increase inthe half-life of the represso r-D NA complex when these sec-ondary binding sequences are present in addit iono operatorin supercoiled DN A suggests tha t the y are significant factorin the egulatory mechanism for this system. An intramolec-ular ernar y complex of pseudo operator-rep ressor-op eratorstabilized by t he topological restrictions of supercoiled DNAis onsistentwi th he data obtained. A similar ernarylooped) DN A structure involving b oth the ara0 2 and araIregulatory regions in supercoiled DN A has been proposed fortranscriptional regulation of the araB AD genes 23) an d int he gal operon 24) in E . coli.Equilibrium a nd dissociation rate determin ations forre-pressor protein binding o various supercoiled DN As confirma functional role of the tertia ry stru ctu re of the DNA. Al-though the numberf ionic conta cts for linear and upercoiledDN A is similar, large differences in the dissociation rate andequilibrium con stants indica te a significant role of the DN Astructure in stabiliz ing the protein . DN A complex in uiuo.Unu sual DNA structures characterized o date include thefollowing: left-handed DN A, which is foundprincipally atalternating purine-py rimidine sequences 25-27); cruciforms,which occur at inverted repeats 28-30); and slipped struc-tures, which have been postu lated to occur at direct repeats31, 32). Negative supercoiling of the D NA at physiologicaldensities stabilizes these u nusual structures 28, 30, 33). Th enature of the structural change in the E. coli lac promoter-operator region induced by negative supercoiling is currentlyunder investigation. Supercoiling of the closed, circular DNAof the E . coli genome in the cell 34) may stabilize unusualstructures of the ypes ndicated; n addit ion, one or bothpseudo operator sequences are available or intramo lecularternary complex formation. Thus, once the repressor proteinis bound to the opera tor , the te r t ia ry s t ruc ture genera tedythe supercoiled DNA and the intramolecular ternaryomplexwith the pseudooperator region DN A loop) result nanexceptionally stable pr otein-DN A interaction which remainsinducer-sensitive.

T. S. Hsieh, personal communication.

1.2.3.4.5.6.7.8.9.10.

11.12.13.14.15.16.17.18.19.20.21.22.23.24.25.

26.27.

28.29.30.31.32.33.34.35.36.37.38.

REFERENCESPfahl, M., Gulde, V., and Bourgeois, S. 1979) J Mol. Biol. 1 2 7 ,Winter, R. B., and von Hippel. P. H. 1981) Biochemistry 2 0 ,339-3446948-6960Winter, R.~B., erg, O..G.,andvon Hippel,P. H. 1981)Biochem-Whitson, P. A,, and Matthews, K. S. 1986) Biochemistry 25,istry 20,6961-69773845-3852Whitson, P. A., Olson, J. S., and Matthews, K. S. 1986) Bio-Besse, M., von Wilcken-Bergmann, B., and Muller-Hill, B. 1986)Gellert, M. 1981)Annu. Reu. Biochem. 50,879-910Geider,K., and Hoffmann-Berling, H. 1981) Annu.Rev.Wang, J. C. 1985)Ann u. Reu. Biochem. 54,665-698Wang, J. C., Barkley, M. D., and Bourgeois, S . 1974) NatureChan, H. W., and Wells, R. D. 1974) Nature 252,205-209Chan, H. W., Dodgsen,J. B., and Wells, R.D. 1977)BiochemistryBarkley, M. D., Lewis, P. A., and Sullivan, G . E. 1981) Biochem-Lohman, T. M. 1985) CRC Crit. Reu. Biochem. 19 , 191-245Lohman, T. M., de Haseth, P. L., and Record, M. T., Jr. 1978)Record, M. T., Jr., de Haseth, P. L., and Lohman, T. M. 1977)Record, M. T., Jr., Anderson, C. F., and Lohman, T. M. 1978)de Haseth, P. L., Lohman, T. M., and Record, M. T., Jr. 1977)Mossing, M. C., and Record, M. T., Jr. 1985) J Mol. Biol. 186,Davidson, N. 1972) J Mol. Biol. 66,307-309Hsieh, T.-S.,and Wang, J. C. 1975) Biochemistry 14,527-535OGorman, R. B., Dunaway, M., and Matthews, K. S. 1980) JHahn, S., Hendrickson, W., and Schleif, R. 1986) J . Mol. Biol.Majumdar, A., and Adhya, S. 1984) Proc. Natl. Acad. Sci.U.S A .Wells, R. D., Bre nqn, R., Chapman, K. A., Goodman, T. C.,Hart, P. A., Hillen, W., Kellogg, D. R., Kilpatrick, M.W.,Klein, R. D., Klysik, J., Lambert, P. F., Larson, J. E., Miglietta,J. J., Neuendorf, S. K.,OConnor, T. R., Singleton, C. K.,Stirdivant, S. T., Veneziale, C. M., Wartell, R. M., and Zacha-rias, W. 1982) Cold Spring Harbor Symp. Qwlnt.Biol. 47,77-84Rich, A. 1982) Cold Spring Harbor Symp. Quant. Biol. 47 , 1-12Wells, R. D., Erlanger, B. F., Gray, H. B., Hanau, L. H., Jovin,T. M., Kilpatrick, M. W., Klysik, J., Larson, J. E., Martin, J.C., Miglietta, J. J., Singleton, C . K., Stirdivant, S. M., Venezi-ale, C.M., Wartell, R. M., Wei, C. F., Zacharias, W., andZarling, D. 1983) Gene Expression, ULCA Symposium on Mo-lecular and Cellular Biology (Hamer, D., and Rosenberg, M.,eds) Alan R. Liss, Inc., New York 8, 3-18

chemistry 25,3852-3858E M B O J . 5,1377-1381

Biochem. 50,233-260

251,247-249

16,2356-2366istry 20,3842-3851

Bwphys. Chem. 8, 281-294Biochemistry 22,4791-4796Q. Reu. Biophys. 111,103-178Biochemistry 16,4783-4791295-306

Bid . Chem. 255 , 10100-10106188,355-36781,6100-6104

Panayatatos, N., and Wells, R. D. 1981) Nature 289,466-470Lilley, D. M. 1980)Proc. Natl. Acad. Sci. U. s.A . 77,6468-6472Singleton, C.K., and Wells, R. D. 1982) J. Biol Chem. 258,Mace,H. A. F., Pelham, H. R.B., and Travers, A. A. 1983)McKeon, C., Schmidt, A., and de Crombrugghe, B. 1984)J Biol.Singleton, C. K., Klysik, J., Stirdivant, S. M., and Wells, R. D.Pettijohn, D. 1982) Cell 30,667-669Riggs, A. D., Bourgeois, S., and Cohn, M. 1970) J Mol. Biol. 5 3 ,Singleton, C. K., and Wells, R. D. 1982) Anal. Biochem. 122,Bauer, W. R. 1978) Ann u. Reu. Biophys. Bioeng. 7,287-313Mossing, M. C., and Record, M. T.,r. 1986) Science 233,889-

7661-7668Nature 30 4 , 555-557Chem. 259,6636-66401982) Nature 2 9 9, 312-316

401-417253-257

892