Proc. Nati. Acad. Sci. USA Vol. 85, pp. 9683-9687, December 1988 Genetics Interaction of spatially separated protein-DNA complexes for control of gene expression: Operator conversions (gal operon/repressor/distal sites/DNA loop) ROBERTA HABER AND SANKAR ADHYA* Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 Communicated by Jon Beckwith, September 19, 1988 (received for review July 18, 1988)

ABSTRACT Two operators, spatially separated from each that in the absence ofinducer, Gal repressor protein interacts other and from the promoters, repress the gal operon when with two operator regions (refs. 10 and 22; A. Majumdar and bound to Gal repressor. Conversion of either gal operator to a S.A., unpublished data). The external operator (OE) is lac operator results in derepression, although both Gal and Lac upstream from the promoters: the internal operator (Or) maps repressors are present, suggesting that mere occupation of within the galE structural gene. Both operators, whose operator sites is not sufficient to cause repression. Conversion centers ofsymmetry are separated by 114 base pairs, function ofboth operators to lac operators restores normal repression in in repression of both promoters. We discuss evidence that in the presence of Lac repressor protein. We propose that normal conjunction with Gal repressor protein, wild-type OE and 01 repression requires interaction between operator-bound like elements act in synergy, and we suggest that this synergy repressor molecules; this generates a DNA loop, which is part results in a higher order structure that is required for normal of a higher order structure. RNA polymerase and cyclic AMP repression. receptor protein are present in this complex but unable to initiate transcription because of the higher order structure. MATERIALS AND METHODS Such higher order DNA-multiprotein complexes could occur in a variety of genetic regulatory systems that are controlled from Bacterial Strains, Phage, Plasmids, and Repressor Proteins. distal sites by regulatory proteins. The following E. coli K-12 strains were used: RZ1032 (HfrKL16PO/45[1ysA(61-62)], duti, ungi, thil, relAl, Zbd- DNA control elements are sites within genes or operons that 279::TnlO, supE44) and JM 101 (Mlac-pro, thi, strA, supE, hsdR-jF' endA, sbcB, traD36, proAB, lacP, ZAM15) were interact with specific regulatory proteins to control gene from Jin Kim (National Cancer Institute); TG1 (Mlac-pro, expression. Environmental signals determine whether the supE, thi, hsd5/F'traD36, ProA+B+, lac", lacZAM15), DNA-binding proteins form effective complexes with their HB2151 (ara, Mlac-pro, thi/F' proA+B+, lacIq, lacZAMJS), corresponding sites (1-4). Such DNA sites, classically known and HB2154 (HB2151, mutL::TnlO) were from D. Chattoraj in prokaryotic systems, are now being found in eukaryotic (National Cancer Institute); SA2700 (F-, strr, relAl, systems. Positive-acting sites are called enhancers and up- gal::AOPE cMr) and C600 (F-, thil, thrl, leu106, lacYl, stream activators, and negative-acting sites are termed op- tonA21, supE44) were from the collection of this laboratory. erators or silencers (5-8). Although regulatory sites fre- Y2051 (AcI857, Aatt-int, gal::AOPEcmr), this laboratory; quently overlap or are contiguous with promoters, in both M13-gal, this study. prokaryotes and eukaryotes they have been found at distal Plasmids pUC19 and pBR322 were from the collection of regions on the DNA. In some systems, multiple regulatory this laboratory; plasmid pGR17 is a pBR322 derivative sites are observed, often spaced significant distances from carrying the gaiR gene (23); plasmid pDM1-1 is a pACYC each other and from promoters (5-16). derivative carrying the lacd gene (gift of H. Bujard, Univer- The mechanism(s) by which dispersed and multiple regu- sity of Heidelberg); p124, a pBR322 derivative containing the latory sites and their specific binding proteins control gene entire gal operon and p291, a pBR322 derivative containing expression is one of our considerations. Does each protein- the gal operators, promoters, and part of galE, are from this DNA complex contribute to regulation independently, or is laboratory. communication between protein-DNA complexes a part of Preparation of Gal repressor protein has been described regulation: If regulatory complexes do communicate with (23). Lac repressor was a gift of Kathleen Matthews (Rice each other, how does this occur? University). The gal operon of Escherichia coli is an excellent system Strain Construction. gal operators cloned into M13 phage with which to address these questions. The operon contains were converted to OL and OL' sequences by site-directed three structural genes-galE, galT, and galK (encoding mutagenesis, using published methods (24-26). The presence Uridine diphosphogalactose-4-epimerase, Galactose-1-phos- of the OL sequence on extrachromosomal elements was phate uridyltransferase, and Galactokinase, respectively)- followed by monitoring derepression of the single copy lac whose transcription is differentially regulated by two pro- operon on an LB plate containing 20 Ag of 5-bromo- moters, PI and P2 (Fig. 1). Cyclic AMP and Cyclic AMP 4-choro-3-indolyl f3-D-galactoside per ml. Conversion from receptor protein, acting as a complex, stimulate initiation of oL to 0L' was followed as loss of the ability to derepress the transcription at PI and inhibit initiation at P2 by site-specific chromosomal lac operon. After mutagenesis was verified by binding to DNA (17, 18). The operon is negatively regulated the dideoxy method of DNA sequencing (27), the replicative by Gal repressor protein, the product of the unlinked galR form DNA containing converted operators and flanking DNA gene(2, 19-21). Genetic analysis, as well as nuclease and was substituted in vitro for the homologous region of the alkyl protection and interference experiments, have shown wild-type gal operon in a pBR322 derivative. Two sets of

The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed at: National Insti- payment. This article must therefore be hereby marked "advertisement" tutes of Health, Laboratory of Molecular Biology, 9000 Rockville in accordance with 18 U.S.C. §1734 solely to indicate this fact. Pike, 37/4B06, Bethesda, MD 20892.

9683 Downloaded by guest on October 1, 2021 9684 Genetics: Haber and Adhya Proc. Natl. Acad. Sci. USA 85 (1988)

gaIR PG2 PG1 galE galT galK t I I ,,. I _ .4. ,T _ml +I OI Si_ 3? cAMP CRP

FIG. 1. The gal operon of E. coli and the unlinked gaIR gene. P1 and P2 are overlapping promoters, differentially regulated by cyclic AMP/cyclic AMP receptor protein (CRP). S1 and S2 represent start points of transcription for P1 and P2, respectively. Each transcript is polycistronic, encoding E, T, and K. Gal repressor protein, the gaiR gene product, interacts with two sites in the operon, OE and 0O, to inhibit gal expression. Both OE and 0O, whose centers of symmetry are 114 base pairs apart, function in repression of both promoters. plasmids resulted: the pI24 set, which contains the entire gal two operators into a lac operator would prevent repression of operon, and the p291 set, which carries only the gal opera- the operon because the heterologous nature of the two tors, promoters, and part of galE. A BstEII restriction protein-DNA complexes would hinder their interaction. To enzyme recognition site between the two operators was test this, we constructed a set of strains in which one or both exploited to construct plasmids with operator conversion at of the gal operators was modified such that it no longer both sites. interacted with Gal repressor protein but, instead, contained A phage Y2051 was used to transfer the converted opera- a synthetic lac operator sequence (32). tors from the plasmids to the gal operon of strain SA2700 as The conversion of gal operators, hereafter termed O¶ and described (28). Of, into lac operator sequences, termed OL, o0, and OF, was In Vitro Filter Binding. Cesium chloride-banded DNA was achieved by using synthetic oligonucleotides to mediate tritiated with S-adenosyl-L-(methyl-3H)methionine (Amer- site-directed mutagenesis of gal DNA that had been cloned sham) and Alu I methylase (New England Biolabs), according into an M13 phage vector (24-26). The DNA sequences used to the supplier's directions. Reaction mixtures (20 ul) con- in operator substitutions are shown in Fig. 2. The substituted taining 2 x 10-15 mol of plasmid DNA were incubated in sequences are identical in size and position to wild-type gal binding buffer (5 mM KCI/5 mM Tris, pH 7.6/5 mM sequences and should not change the overall geometry and MgSO4/0.05 mM EDTA/0.05 mM dithiothreitol/25 4g of angular orientation of the operators. bovine serum albumin per ml) with varying amounts of Gal Operator-Repressor Binding. The repressor binding abili- repressor and Lac repressor at room temperature for 30 min, ties of plasmids carrying various combinations ofgal and lac followed by filtration through nitrocellulose (Schleicher & operator sequences were tested in vitro under equilibrium Schuell). Filters were washed with 200 ,u1 of binding buffer binding conditions. The results, shown in Table 1, confirm and radioactivity bound to the filters was measured by that Gal repressor binds to DNA containing gal operators scintillation counting. (OG-OyI O- I, and OE-OF), but not to oE-o0 or OLEOi Determination of Relative Plasmid Copy Number. Cultures DNA. Lac repressor, on the other hand, binds only to DNA of strain C600 containing plasmids were grown to midloga- that contains the oL sequence (OL OL, OL-Of, O-01 and rithmic phase in LB broth containing ampicillin (50 ,4g/ml). OELk-O1). Lac repressor did not show detectable binding Equal numbers of cells from each transformed culture were to oE-of or &-oI'DNA. mixed with equal volumes ofC600 that had been transformed We also measured the relative affinities in vivo of the same with a smaller plasmid (pUC19). Plasmids were extracted gal and lac operator sequences for their cognate repressors, from each mixture, digested with EcoRI restriction endonu- using a previously described method (Table 2) (28, 33). C600, clease, electrophoresed on 1% agarose gels, and stained with a strain with single copy gal and lac operons as well as single ethidium bromide (1 ,ug/ml) (29). The pUC19 band in each copy Gal and Lac repressor genes, was transformed with lane of the gel was used as a standard against which the relative amount of each plasmid was determined. QG 5' GTGTAAACGATTCCAC Enzyme Assays. Cells were grown to midlogarithmic phase in minimal medium (see Table 2) or LB broth (see Tables 1 QG 5' GTGGTAGCGGTTACAT and 3). Cells containing plasmids were grown in the presence QI 5 TTGTGAGCGCTCACAA of the antibiotic. Galactokinase assays were appropriate Q 5' TTGTGAGCGCTCACAA performed as described (18). 6-Galactosidase assays were performed as described by Miller (30). QI 5' TTGGGAGCGGTCACAT RESULTS FIG. 2. Sequences used in operator conversions. Lines 1 and 2 show 16-base-pair OE and 0O sequences of gal, designated Og and We have investigated the role(s) of two nonoverlapping 0?. Line 3 (Ok) and line 4 (Of) show a 16-base-pair DNA sequence operators in repression of the gal operon. We reasoned that with perfect dyad symmetry. The latter sequence has been shown to if the formation of a repressor-operator complex at two bind Lac repressor 8-fold more efficiently than the natural lac operator sites is sufficient for repression, then substitution of operator sequence (32). Line 5 (Of') shows a 16-base-pair lac operator sequence with hyphenated dyad symmetry, which differs one of the gal operators with a different operator-e.g., a lac from the Ok/Of sequence by 3 base pairs. Note that neither the OL operator-would not affect its ability to evoke repression, nor the OL' sequence described here contains a central C&G base pair, and the operon would remain repressed if both Gal and Lac which is present in the wild-type lac operator. Seven base changes repressors were present. But if, as proposed previously, were required to convert O? to Ok, 6 base changes were needed to interaction between repressors bound at OE and 0 is re- convert O to Of, and 3 base changes were needed to convert 0? to quired for repression (10, 31), then conversion of one of the of'. Downloaded by guest on October 1, 2021 Genetics: Haber and Adhya Proc. Natl. Acad. Sci. USA 85 (1988) 9685 Table 1. In vitro binding of plasmid DNA to Gal and to bind Lac repressor demonstrates that the location of the Lac repressors operator within the operon affects the operator's ability to Plasmid retained on filter, fmol bind repressor in vivo (context effect). Since O¶ and OG appear to have equal affinities for Gal repressor in vitro Operator No Gal Lac (Table 1), our results suggest that gal operators also exhibit Plasmid genotype additions repressor repressor a context effect with respect to repressor binding. We suggest p124 Om-O& 0.3 1.1 0.2 that the lower affinity of repressor for an operator at the pH106 Os-tO 0.2 0.6 0.9 internal site is caused by RNA polymerase transcribing pH104 OLr-O 0.2 0.6 1.0 through the region. This lower repressor affinity of an pH107 oh-of 0.3 0.3 1.0 operator located at the 01 position can be enhanced by the pH110 os-OfL' 0.2 0.6 0.2 presence of a homologous operator sequence at OE. For pH109 op-f' 0.3 0.2 0.9 example, the combination GE-0I (line 2), which is the Equilibrium binding of operator DNA to repressor proteins. Filter situation in wild-type gal, caused greater derepression (7- binding reactions were performed as described. All reaction mixtures fold) of the chromosomal gal operon than the sum of the contained 2 fmol of plasmid DNA. The amount of protein added was derepression caused by plasmids carrying individual 0E none, 40 fmol of Gal repressor, or 40 fmol of Lac repressor. (3-fold) and O0 (2-fold) elements. Analogous results were obtained with the Off-Of plasmid (line 5), whose 330-fold multicopy plasmids that carry either wild-type or variously derepression of the chromosomal lac operon owing to the substituted operators, but not the structural genes being presence of oL-OL was higher than the sum of the derepres- assayed. Since no inducer is present, derepression of chro- OL mosomal gal and lac genes in the transformed strains is sion obtained with (150-fold) and OI (90-fold) individu- attributed to sequestering of cellular repressor proteins by ally. The helping effect of one operator sequence by another operators present on multicopy plasmids. The amount of present in cis was more dramatic when the lac operator derepression is a reflection of the of the sequence oI was used. On its own, of', which did not bind ability operators Lac repressor in vitro (Table 1), caused no derepression of present on multicopy plasmids to bind repressor molecules. OL Two different sets of plasmids were used for the experiments the lac operon (line 8), while on an isogenic plasmid described in Table 2. Within each set, the plasmid copy derepressed lac 20-fold (line 7). When OL and Of' were number is the same for the strains used in the corresponding present in cis, they caused a derepression of 130-fold (line 9). experiment (unpublished results). Therefore, strains trans- Thus, when OE and 0 contain homologous sequences, formed with isogenic plasmids could be compared. The binding of the corresponding repressor is more efficient. relative level of chromosomal derepression is solely a func- Cooperative binding of proteins to spatially separated DNA tion of the affinities of the plasmid-carried operators for sites has been observed in other systems (34, 35). repressors. From the titration experiments shown in Table 2, Effect of Operator Substitutions on gal Expression. After we made the following observations. transferring the converted operators from M13-gal to the The presence of the vector plasmid pBR322 did not affect chromosome, we examined whether repression of the gal repression of the chromosomal gal and lac operons, as operon with substituted lac operator(s) deviates from the reflected by the levels of Galactokinase and P-galactosidase, wild-type level of repression by assaying the level of Galac- respectively (line 1). The presence of OLko on the plasmid tokinase in strains in which the gal operon and the Gal and caused 2-fold derepression of the gal operon and 150-fold Lac repressor genes are present in single copy in the derepression of the lac operon (line 3). When the operators chromosome (Table 3). Normal repression is the degree to were in the reverse order (line 4), converse binding affinities which the wild-type gal operon, Of--O, is repressed in the were observed. A plasmid with genotype OG-of caused presence of Gal repressor. Lac repressor has no effect on the 3-fold derepression of gal and 90-fold derepression of lac. Galactokinase level in the wild-type strains (line 1). All When an individual operator was located at the internal site strains with heterologous operators (OL OG, oG-oL, and it consistently exhibited a lower affinity for repressor than OEO-'1) deviate from the normal level of repression, al- when the operator was at the external site. OLE and oL are though both Gal and Lac repressors are present (lines 2, 3, identical DNA sequences; therefore, the greater ability of and 4). This suggests that mere occupation of an operator by OLE a repressor molecule may be insufficient to cause normal Table 2. Measurement of repressor binding to operators in vivo Table 3. Levels of galactokinase in wild-type and converted gal Operator Galactokinase /3-Galactosidase operator strains of E. coli Plasmid genotype units units Repressor present pBR322 - 2.7 ± 0.1 10 p291 okoG 17.9 ± 1.2 10 Operator (A) (B) (C) (D) pHiO1 OL4-O? 4.2 ± 0.1 1500 Strain genotype Lac + Gal Gal Lac None pH103 Os-Of 7.2 ± 0.2 900 E125 Ok-O? 6 ± 2 6 ± 1 100 96 ± 11 pH108 OLE-oL 2.7 ± 0.1 3300 E69 0k-.O 49 ± 11 227 ± 3 87 ± 7 219 ± 24 p124 Om-O& 10 E104 Og-OI 19 ± 3 64 ± 3 42 ± 5 124 ± 21 pH104 OixoO 200 E189 Of-0f' 59 ± 10 55 ± 8 87 9 124 ± 24 pH110OOlO 10 E105 (14-OfL 1 ± 0 229 ± 28 1 0 144 ± 6 pH109 OE-OL1' 1300 E187 OJ-Of' 5 ± 1 233 ± 10 3 1 196 ± 12 Galactokinase and /3-galactosidase expression in E. coli strain Galactokinase expression in strains with wild-type and converted C600 transformed with multicopy plasmids carrying wild-type and operators. Gal repressor was inactivated by addition of D-fucose to converted operators. Units of galactokinase are nmol of product 2 mM. Strains with inactive Lac repressor were constructed by produced per min per ml of cells at A6w = 1.0. Values given are the transduction of a lac: :mini-TnJO marker into the appropriate strains mean ± SD of two experiments. Units of 83-galactosidase, a product using bacteriophage P1. Units of enzyme are nmol of product formed of the lac operon, are A.mol of product formed per mmn per ml of cells per min per ml of cells at A6N = 1.0 (18). Results are normalized at A600 = 1. p291, pH101, pH109, and pH108 are pBR322 derivatives relative to O-O, which is given a value of 100 when Gal repressor that contain a 664-base-pair fragment of the gal operon, which protein is inactivated; values given are the mean ± SD of five includes the operators and part of galE. p124, PH104, pH109, and experiments, except for lines 3 and 5, which are mean + SD of three pH11O are pBR322 derivatives that contain the entire gal operon. experiments. 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repression. However, there is partial repression in the het- inducer if pDM1.1 is present (unpublished results). We erologous cases. The operator sequence that contributes conclude that a high level of Lac repressor protein is made most to this partial repression (3-fold) is the tight binding when pDM1.1 is present. Under similar conditions of excess sequence, 0L (compare column C to column D in line 3). It Gal and Lac repressors, galactokinase synthesis in the three is known that Lac repressor bound to a lac operator located strains that contain heterologous operators, O-O?, OE-0I, several hundred bases downstream from a promoter attenu- and OE-01, never decreased to the normal repressed level ates transcription (36, 37). Whereas Lac repressor may of wild-type gal (Table 4). attenuate transcription in the o0-oj1 strain, Gal repressor The observation that saturating levels of repressor protein does not inhibit transcription in the Ok-o? strain (line 2, do not fully repress operons with heterologous operators was columns B and D). verified by using an additional more sensitive test for repres- We found that operons with both gal operators substituted sion of the gal operon. An 0L or OL' sequence at the internal by lac operators (0O and OEl-Oi ) were fully repressed: site changes the galE sequence and results in a nonfunctional the level of Galactokinase was very low in the presence of epimerase protein. If the galK gene is expressed in galE- Lac repressor, and Gal repressor had no effect (lines 5 and 6). cells, the addition of galactose causes toxicity because of the (We note that strains containing a substitution at the external accumulation ofgalactose phosphate intermediates (39). The site, OL, when fully derepressed, had 2-fold higher Galac- cell is sensitive to galactose and unable to grow on MacCo- tokinase levels than wild type. This finding does not have any nkey galactose plates ifthe operon is expressed. Ifthe operon bearing on our current question about the function of indi- is repressed, a galE- strain is galactose resistant. As shown vidual operator sites in the gal operon.) in Table 4, of-OG and oG-oL' strains, unlike -Of and OLE- We considered the possibility that the reason operons with of, are galactose sensitive, even in the presence of multi- heterologous operators were not repressed was because the copy repressor genes. This result confirms the observation DNA sites were not efficiently occupied. One way to increase that even in the presence of very high levels of repressor occupation is to increase the level of repressor proteins protein, oG-OI and or-oG are not fully repressed and (Table 4). The presence of multicopy plasmids bearing argues against a model that specifies that like operators are repressor genes greatly increases the intracellular level of required for repression solely to effect full occupation of the repressor- proteins and thus should saturate the operator regulatory sites. sequences with repressors. The two plasmids we used to in- crease repressor levels are pGR17, which carries the wild- DISCUSSION type gaiR gene, and pDM1.1, which carries lacd, the gene for Lac repressor (23, 36). The wild-type strain, oG-oG, re- The functional relationship between the two operators in the mained repressed after introduction of pGR17. When gal operon was studied after conversion of one or both oG-oG was exposed to 25 times more inducer (50 mM fucose) operators to lac operators. Although cooperative binding in than is required to derepress the same strain in the absence vitro was not observed under our conditions, in vivo binding of plasmid, the plasmid-bearing strain remained repressed assays indicated that the two gal operators, spatially sepa- (unpublished results). On the basis of this observation, we rated from each other and from the promoters, exhibit concluded that pGR17 directs the synthesis of a significantly unequal affinities for Gal repressor, because oftheir different higher amount of Gal repressor protein than is made by the locations in the operon. The operators function synergisti- single copy chromosomal gaiR gene. Similarly, OL-OIL and cally to inhibit gal operon expression. gal operons in which oL-01' remained repressed after introduction of the latIq only one of the operators was replaced with a lac operator plasmid. When pDM1.1 is present in oLEOj and OL-Olf, the were not fully repressed, even under conditions when both corresponding gal operon can be derepressed to only 10% of Lac and Gal repressor proteins were highly overproduced. the derepressed level attained in the absence of plasmid, This suggests that mere occupation of the two operator sites although exposed to 10-fold more inducer (2 mM isopropyl is not sufficient to effect normal repression. Our results are ,8-D-thiogalactopyranoside) than is required to inactivate the consistent with the model ofrepression that involves protein- amount of Lac repressor that is made from the single copy protein interaction between repressors bound to spatially lacI gene (unpublished results). In addition, the wild-type lac separated operators and consequent stabilization of a higher- operon remains repressed in the presence of 10-fold excess order structure containing a DNA loop (10, 31). It is con- Table 4. Effect of excess repressor on gal operon expression in wild-type and converted gal operator strains of E. coli Repressor gene on plasmid No plasmid lacP gaiR Operator Galactokinase Galactose Galactokinase Galactose Strain genotype units sensitivity units sensitivity E125 O-Of 2.3 ± 1.2 NA 1.0 ± 0 NA E105 Oj-0f 2.0 ± 1.7 - 0.5 ± 0.4 - E187 os-of 3.3 + 1.2 - 2.3 ± 0.6 - E69 Ok-O? 16.5 ± 4.9 NA 14.4 ± 3.5 NA E104 Ok-of 7.7 ± 1.5 + 4.7 ± 1.5 + E189 ok-of 22.7 ± 5.5 + 12.0 ± 4.5 + Effect of multicopy plasmids bearing repressor genes on gal operon expression in wild-type and operator-converted strains. The lacPs gene is on pDM1.1 (36). The lacp gene produces 10-fold more Lac repressor than lacI+ (38); gaiR is on pGR17 (23). pDM1.1 is compatible with pGR17 (36). Galactokinase assays and units were as described in the legend to Table 1. Values given are the mean ± SD of three assays, except for line 4, which is the mean ± SD of two assays. NA, not applicable; strains with the wild-type gal 0, sequence are galE+ and do not become galactose sensitive. +, Galactose sensitive; strains do not grow in the presence of galactose. -, Galactose resistant; strains grow in the presence and absence of galactose. Downloaded by guest on October 1, 2021 Genetics: Haber and Adhya Proc. Natl. Acad. Sci. USA 85 (1988) 9687

ceivable that Gal and Lac repressors can interact weakly, 15. Hall, M. N. & Johnson, A. D. (1987) Science 237, 1007-1012. thus contributing to the partial repression observed in strains 16. Maniatis, T., Goodbourn, S. & Fischer, J. A. (1987) Science with heterologous operators. The binding of Gal repressor 236, 1237-1244. protein to the gal operators does not impede RNA polymer- 17. Musso, R. E., Di Lauro, R., Adhya, S. & de Crombrugghe, B. (1977) Cell 12, 847-854. ase or cyclic AMP receptor protein from binding to their 18. Adhya, S. & Miller, W. (1979) Nature (London) 279, 492-494. respective regions of the DNA in vitro (ref. 40; A. Majumdar 19. Saedler, H., Gullon, A., Fiethen, L. & Starlinger, P. (1968) and S.A., unpublished data). We propose that RNA poly- Mol. Gen. Genet. 102, 79-88. merase and cyclic AMP receptor protein are present on the 20. Adhya, S. & Echols, H. (1966) J. Bacteriol. 92, 601-608. repressed operon in vivo; however, initiation of transcription 21. Nakanishi, S., Adhya, S., Gottesman, M. & Pastan, I. (1973) is blocked by the higher-order structure, which is maintained Proc. Natl. Acad. Sci. USA 70, 334-338. by protein-protein interaction between spatially separated 22. Majumdar, A. & Adhya, S. (1987) J. Biol. Chem. 262, 13258- operator-bound repressors. 13262. It is interesting to note that Lac repressor, which is 23. Majumdar, A., Rudikoff, S. & Adhya, S. (1987) J. Biol. Chem. believed to act at the lac operator by sterically hindering the 262, 2326-2331. binding of RNA polymerase to an overlapping promoter, can 24. Carter, P., Bedouelle, H. & Winter, G. (1985) Nucleic Acids distal Res. 13, 4431-4443. also act to repress operon expression from nonover- 25. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA 82, 488-492. lapping sites, presumably by the same mechanism as Gal 26. Kunkel, T. A., Roberts, J. D. & Zakour, R. A. Methods repressor. Previous reports that Lac repressor can bind to Enzymol. 154, 367-382. spatially separated lac operator constructs and form a DNA 27. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. loop are consistent with the observations of Lac repressor Acad. Sci. USA 74, 5463-5467. described here (41-43). 28. Irani, M., Orosz, L., Busby, S., Taniguchi, T. & Adhya, S. (1983) Proc. Natl. Acad. Sci. USA 80, 4775-4779. We thank Kathleen Matthews for a gift of Lac repressor protein 29. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular and Herman Bujard for pDM1.1. We also thank Alokes Majumdar, Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold Susan Garges, Jin Kim, and Roger Wartell for helpful discussions; Spring Harbor, NY). for R.H., this work is in partial fulfillment of Ph.D. requirements at 30. Miller, J. H. Experiments in Molecular Genetics (1972) (Cold The George Washington University, Washington, DC. Spring Harbor Lab., Cold Spring Harbor, NY). 31. 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