Communication. Vol. 262 .... from each time point and the rate was determined from a log plot of ... parent from these measurements that the secondary and/or.
THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 262, No. 11. Issue of April 15, pp. 4943-4946 1987 0 1987 by The American Society of Biologwal ChernisG Inc.
Communication
Printed in ~ s . A .
binding sites, andlower repressor-operator dissociation rates have been measured in the presence of both nonspecific and pseudooperatorsequences in the operator-containing DNA (4, 5). An intramolecular ternary complex with both DNA binding sitesof the repressor occupied, and consequentDNA (Received for publication, November 24, 1986) loop formation, has been postulated to explain the stabilizaPeggy A. Whitson$, Wang-Ting Hsiehs, tion of therepressor-operator complex in the presence of Robert D. Wellss, and Kathleen S . MatthewsSll intramolecular DNA sites(4-6, 38). Previous work has indicated significant effects on proteinFrom the $Department of Biochemistry, Rice University, Houston, Texas 77251 and the $Department of DNA interactions as a consequence of DNA topology (7-9). Biochemistry, Schools of Medicine and Dentistry, Wang and co-workers (10) reported a decrease in the dissoUniversity of Alabarnn at Birmingham, ciation rate constant of lac repressor from supercoiled covaBirmingham, Alabama 35294 lently closed Xplac DNA. We report here effects of negative supercoiling on the repressor-operator interaction in a variety The binding affinity of the Escherichia coli lactose of sequence contexts. Supercoiling of operator-containing repressor to operator-containing plasmids was increased by negative supercoiling of the DNA. The in- plasmid DNAs with and withoutpseudooperators resulted in creased affinities observed were dependent on the se- a decrease in the dissociation rate constant compared to the quence context of the DNA as well as the degree of rates observed withthe corresponding linear DNAs. The most supercoiling. Dissociation rate constants for plasmids dramatic effect was observed for pIQ, an operator and pseuto ternary containing a single operator site decreased as a func- dooperator-containing plasmid,presumablydue tion of the negative supercoil density. However, the complex formation of operator-repressor-pseudooperator. presence of pseudooperators in the plasmid DNA in These data extend previous in vitro studies (10-12) and sugaddition to the primary operator sequence resulted in gest critical roles in vivo for the DNA topology and sequence a significant decrease in the operator-plasmiddissocia- context in stabilizing the repressor-operator complex. tion rate at higher negative supercoil densities. ApEffects of Negative Supercoil Density on the Dissociation proximately eight ionic interactions were determined RateConstant-Thedissociation rateconstants for pIQ, for both the supercoiled plasmids and the linear DNAs anoperatorandpseudooperator-containing plasmid, and examined. These results suggest that the stabilization pLA322-8, a promoter-operator containingplasmid, as a funcprovided by the topology of supercoiled DNA affects tion of the negative supercoil density are illustratedin Fig. 1. the nonionic component of the protein-DNA interac- For both DNAs, the dissociation rate decreased as the negation. The ability to form a ternary complex of protein tive supercoil density increased. The dissociation rate for with two DNA segments is increased by the presence pLA322-8 decreased -4-fold as thenegative supercoil density of multiple operator-like sites on the DNA. Further- increased from 0 to 0.085, whereas an -230-fold decrease in more, supercoiling DNA with multiple operator-like sequences profoundly diminishes the dissociation rate the dissociation rate constant was observed for pIQ between and results in a remarkably stable ternary, presumably relaxed DNA and a negative supercoil density of 0.06. Labeled supercoiled pIQ which had been relaxed with tolooped complex (tM 28 h). These data suggest a critipoisomerase or linearized withPstI exhibited dissociation rate cal role in vivo for DNA topology and pseudooperaconstants comparable to those observed previously for linear tor(s) in transcriptional regulation of the lac operon. pIQ (4). The inset toFig. 1 illustrates dissociation rate determinations for relaxed and supercoiled pIQ. The plasmid pRW468, which harbors a 59-bp’ fragment containing only a portion of the promoter region and the entire lac operator, The lac repressor protein from Escherichia coli exerts negative transcriptional control over the structural genes of the exhibited behavior similar to pLA322-8, although to a lesser comlactose operon. Specific, tight bindingof the repressor protein degree.’ These data indicate that the repressor-operator plex is more stable as the negative supercoil density increases to theoperator region of the DNAphysically blockstranscription of DNA which codes for the lactose metabolic enzymes. and that the presence of the pseudooperators in addition to operator further enhances this apparent stability at higher Pseudooperators, which exhibit sequence homology to the primary operator, are located both in the lac Z and I genes negative supercoil densities. Since the dissociation rate constant forsupercoiled pIQ and display 10- and 100-fold lower affinities for the repressor, respectively (1).Operator binding affinity is increased when decreased dramatically compared to the linear form, we atpseudooperators are present in addition to the primary oper- tempted to determine the dissociation rate constant in the The reator on DNA (1-6). The repressor protein has two operator presence of isopropyl-1-thio-/3-D-galactopyranoside. pressor-operator dissociation was not measurable by the ni* This work was supported by National Institutes of Health Grant trocellulose filter-binding techniquewhen inducer was added < 5 s). Although the supercoiled structure significantly GM-22441 (to K. S. M.) and Grant GM-30822 (to R. D. W.), Robert A. Welch Foundation Grant C-576 (to K. S. M.),and National Science enhances repressor binding, dissociation of the protein-inFoundation Grant 83-08644 (to R. D. W.). The costs of publication ducer complex from the DNA remains quiterapid. Thus, the of this article were defrayed in part by the payment of page charges, This article must therefore be hereby marked “advertisement” in repressor-operator interactionwould be stabilized by the DNA
Supercoiling Facilitateslac Operator-RepressorPseudooperator Interactions*
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( h 2
accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. 11 To whom correspondence should be addressed Dept.of Biochemistry, P. 0. Box 1892, Rice University, Houston, T X 77251.
4943
‘ The abbreviation used is: bp, base pair.
* W.-T. Hsieh and R. D. Wells, manuscript in preparation.
D N A Topology Affects Operator Binding by lac Repressor
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100 50
F 1c a
v
*
$ x
5
Q
x
1
0.5 -0.02
-0.04 -0.06 SUPERCOIL DENSITY
-0.08
FIG. 1. Effects of negative supercoil density on the dissociation rate constants of the repressor-pIQ and the repressorpLA322-8 complexes. The plasmid pIQ (-6200 bp, gift from J. Betz, Universityof Colorado Health Sciences Center) contains both pseudooperator sequences, in addition to the promoter-operator region, and was produced by insertion of a 1.7-kilobase pair Him11 fragment (containingthe I gene, promoter, operator,and a portion of the Z gene) with EcoRI linkers into the Am1 site of pBR322 withthe EcoRI site deleted. The plasmid pLA322-8(-4100bp, gift from George Bennett, Rice University) contains a 95-bp Ah1 promoteroperator fragmentinserted into the linearizedpBR322 (cut at BarnHI and EcoRI). The DNAwaslabeledwith H h d methylase and Smethyl-[3H]adenosyl-~-methionine.2 The dissociation rate constants weremeasuredbyadding a 200- to 6000-foldexcessofunlabeled DNA (either pIQ or pLA322-8)to a pre-equilibrated solutioncontaining repressor and labeled pIQ or pLA322-8 (each at 5.0 X lo-" M) as described by Whitson and Matthews (4). The buffer for the assays was 0.01 M Tris-HC1, pH 7.5, 0.20 M KCl, 0.1 mM EDTA, 0.1 mM dithiothreitol,5% dimethyl sulfoxide. Time courses for pLA322-8 and pIQ were 3 and 40 h, respectively. Equilibrium was not attained for pIQ during this time periodat the higher negative supercoil densities (tH 28 h); therefore, the inducer-sensitive bindingwas subtracted from each time point and the rate was determined from a log plot of the curve as described previously (35). The DNA at various supercoil densities was prepared as described by Singleton and Wells (36). Each point represents two to four rate determinations from two separate preparations of supercoiled DNAs.Standard deviations are indicated by error bars, except where the deviations were less than the area of the symbol. 0,pLA322-8; A,pIQ. The inset illustrates the dissociation of pIQ in the presence (V)and absence (W) of calf thymus topoisomerase I (Bethesda Research Laboratories and gift of J. E. Larson, University of Alabama). The buffer for this procedure was the same as described above with the addition of 1 mM magnesium acetate. Repressor and pIQ (each at 5 X lo-" M) were incubated for 10 min before the addition of 40 units of topoisomerase I to one sample and further incubated for60 minat room temperature prior to the addition of a 200-fold excessof unlabeled pLA322-8.
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01 0.0:
-08
-06
I -0.4 -08 log (KCI)
-0.6
-0.4
FIG. 2. The dissociation rate constant for linear and supercoiled pLA322-8, pRW468, and pIQ as a function of the KC1 concentration. The dissociation rate constants were measured as described in Fig. 1 and Whitson and Matthews (4). Values for linear pLA322-8 and pIQ were determined previously (4). Each point represents the average of 2-4 rate determinations. Standard deviations are indicatedby error bars, except wherethe deviations are less than the area of the symbol. Initial repressor and operator concentrations were 5.0 X lo-" M, and a 200-fold excess of unlabeledoperator DNA was added at t = 0. The negative supercoil densities were as isolated from E. coli HBlOl (u 0.06) (36,37). Panel A, the dissociationrate constants forpLA322-8 and pRW468 at various[KCl]. 0, linear pLA322-8; 0, supercoiled pLA322-8; 0, supercoiled pRW468. Panel B,the dissociation rate constants for pIQ at various [KCl]. A,linear pIQ; A, supercoiled pIQ.
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rate constants for both DNAs at each salt concentration are decreased for the supercoiled DNA as compared to the linear DNA. However, the dissociation rate constant for supercoiled pIQ is less salt-sensitive than linear pIQ or either linear or supercoiled pLA322-8. The minimal salt sensitivity for the pIQ-repressor dissociationsuggests that negative supercoiling provides a secondary or tertiary DNA structurewhich further stabilizes the nonionic components of the intramolecular ternary complex of operator-repressor-pseudooperator. It is apparent from these measurements that the secondary and/or tertiary structure of the DNA plays a significant role in the structureintheabsence of inducer,butthepresence of binding process. The number of ionic contacts in a binding interaction can inducing sugar would elicit the rapid response necessary in be derived from the salt dependence of the equilibrium binding vivo to effectively utilize available lactose. constant. The slope of a plot of log K,, versus log[KCl] has Effects of [KCU on theRepressor-OperatorInteractionbeen shown theoretically to be equivalent to the number of The effects of saltonthedissociationrateconstantcan provide information about mechanisms of repressor-operator counterions released upon binding (14, 16-18). We were uninteraction (2, 4, 5 , 13-15). Previous data using linear DNAs able t o radiolabel pIQ witha sufficiently high specificactivity (
