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Interaction of Spatially Separated Protein-DNA Complexes For 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 Conversion of either gal operator to a S.A., unpublished data).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).
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