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The Nucleotide Sequence of the Lac Operator

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The Nucleotide Sequence of the Lac Operator Proc. Nat. Acad. Sci. USA Vol. 70, No. 12, Part I, pp. 3581-3584, December 1973 The Nucleotide Sequence of the lac Operator (regulation/protein-nucleic acid interaction/DNA-RNA sequencing/oligonucleotide priming) WALTER GILBERT AND ALLAN MAXAM Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138 Communicated by J. D. Watson, Augut 9, 1973 ABSTRACT The lac repressor protects the lac operator bind again to the repressor, and is about 27 base-pairs long. against digestion with deoxyribonuclease. The protected Here we shall describe its sequence. fragment is double-stranded and about 27 base-pairs long. We determined the sequence of RNA transcription METHODS copies of this fragment and present a sequence for 24 base pairs. It is: Sonicated DNA Fragments. Sonicated [82P]DNA fragments 5'--T GG AATT GT GA GC GG AT AAC AATT 3' were made by growing a temperature-inducible lysogen of 3'--AC C TT AACA C TC GC C T ATT GTT AA5' Xcl857plac5S7 at 340 in a glucose-50 mM Tris HCl or TES The sequence has 2-fold symmetry regions; the two longest (pH 7.4) medium in 3 mM phosphate, heating at 420 for 15 are separate4 by one turn ofthe DNA double helix. min at a cell density of 4 X 108/mI, then washing and resus- pending the cells at a density of 8 X 108/ml in the same me- The lactose repressor selects one out of six million nucleotide dium with 0.1 mM phosphate. 100 mCi of neutralized H3s2PO4 sequences in the Escherichia coli genome and binds to it to was added to 10 ml of cells, and the incorporation was con- prevent the expression of the genes for lactose metabolism. tinued for 2 hr at 34°. The cells were washed, suspended in 2 How does this protein, a 150,000-dalton tetramer of identical ml of TE buffer [10 mM Tris *HCl (pH 7.5)-i mM EDTA], subunits, recognize its target? To answer this question we have sonicated with six 15-sec bursts, and extracted with phenol. determined the sequence of the repressor-binding site: the The aqueous phase was extracted with ether, and the residual operator. ether was removed with a stream of N2. The mixture of radio- Genetically the operator is the locus of operator constitutive active DNA and RNA was then made up to about 6 ml with (o0) mutations, cis-dominant changes that render the adjacent binding 10 mM MgCl2, 10 mM KCl, 10mM structural genes less sensitive to repressor control (1). The buffer [containing 0O Tris HCl (pH 7.5), 0.1 mM EDTA, 0.1 mM dithiothreitol, isolation of repressors (2, 3) and the demonstration that 50 of bovine-serum albu- proteins to 5% dimethylsulfoxide, and jtg/ml mutations interfere with the binding of these min]. 20 ,g of lac repressor was added and 1-ml aliquots of this DNA (4, 5) showed that the operator is the DNA binding site six 25-mm Schleicher and for the mixture were filtered on separate repressor protein. Schuell B-6 filters. The filters were presoaked in binding buffer We have isolated the lactose operator as a double-stranded without bovine-serum albumin; the filtration was fast. Each fragment of DNA that has been protected by the lactose ml of then DNase The first filter was rinsed twice with 0.3. binding buffer, repressor against digestion by pancreatic (6). transferred to a second filtration apparatus and eluted twice step in this purification isolated DNA fragments, about 1000 mM in lac promoter-operator region. with 0.25 ml of 0.1 IPTG binding buffer. The eluate base-pairs long, that carry the was extracted with phenol and dialyzed against TE buffer. The lac repressor, when mixed with sonicated fragments of sonic the lac DNA from a phage that carries the lac genes, binds to a cel- This one-step process yields fragments containing operator, which repressor will bind again to filters with about lulose nitrate filter only those fragments containing the efficiency. The overall yield is about 0.2% of the input operator. IPTG (isopropyl-#,u-thiogalactoside, a synthetic 50-70% inducer of the lac operon) specifically elutes from the filter label. those fragments that are found to the repressor (7). The lac Unlabeled sonicated fragments were made by purifying repressor binds to these DNA fragments, as it does to whole 100-300-mg amounts of phage. A 150-liter fermentor culture phage DNA, with a dissociation constant of 10-13 M in 0.01 M of a XcI857S7plac5 lysogen was heat-induced at A550 = 0.6 in salt and 0.01 M Mg++ (8). [The repressor binds IPTG with a M63 minimal medium supplemented with glucose and constant of 1.3 X 10- M (2).] Since the repressor dissociates casamino acids. After 3 hr the bacteria were harvested and from the operator with a half-life (under our conditions) of frozen. 300-700 g of bacteria were thawed, lysed by suspension 15 min, we treat a mixture of repressor and sonic fragments in phage buffer [0.1 M NaCl-10 mM Tris -HCl (pH 7.4)-10 with DNase for a very short time, 1 min, and isolate any mM MgSO4] containing 0.1 ug/ml of DNase, and centrifuged protected region before the repressor can release the DNA. at 17,000 X g for 30 min. The phages were precipitated with The protected fragment we obtain is double-stranded, can 2.1% NaCl and 6.5% polyethylene glycol. The precipitated phages were dissolved in phage buffer, run on a block CsCl gradient, and then on an equilibrium CsCl gradient. After brief dialysis to remove CsCl, purified phages were made up to Abbreviations: TES, N-tris(hydroxymethyl)methyl-2-amino- 20 mM EDTA and sonicated with eight 15-sec bursts at full ethanesulfonic acid; CMCT, 1-cyclohexyl-3-(2-morpholinoethyl)- power, and extracted with phenol. The DNA was dialyzed carbodiimide metho-p-toluenesulfonate; IPTG, isopropyl-,3, D- against TE buffer. thioga-lactoside. The DNA solution was made up to binding buffer, and 5-ml 3581 Downloaded by guest on October 3, 2021 3582 Biochemistry: Gilbert and Maxam Proc. Nat. Acad. Sci. USA 70 (1978) TABLE 1. Pyrimidine tracts from the lac operator Tract Moles Yields 60~ Denatured pCp 4-5 (4.6) 15% Gel En pTp 7 (7.0) 40 pTpTp 4 (3.8) - / pTpCpCp 1 30 pCpTpCp 1 (2.0) -J pTpTpCpCp 1 (0.7) 20 - Native 12% Gel Pyrimidine tracts were isolated and fingerprinted. The se- quences were determined by partial digestion of phosphatase- * I I I treated material by spleen and by venom phosphodiesterase. 6 9 12 The relative molar yields are the averages of three experiments, Mobility (cm) taking the TCC and CTC isostichs together as 2 mol/mol of operator. FIG. 1. Gel electrophoresis of operator fragments. 32P-Labeled operator fragments were subjected to electrophoresis: native fragments (0) on a 12% polyacrylamide gel in 90 mM Tris. aliquots of DNA with repressor were filtered on a 47-mm B-6 borate (pH 8.3)-5 mM MgCl2 with double-stranded synthetic 66/60, 50/45, and 22/17, or filter. The filter capacity is 25 mg of DNA with 0.25 mg of DNA markers of length (0) 77/77, after denaturation (U) on a 15%' polyacrylamide gel in 90 M active repressor (a 2-fold excess). The filter was washed with 3 Tris-borate (pH 8.3)-2.5 mM EDTA-7 M urea with DNA ml of binding buffer and then eluted with 1 mM IPTG. strands of length (0) 50, 45, 22, and 17. This experiment was Operator Fragments. 32P-Labeled operator was made by done by Dr. T. Maniatis (14); the synthetic DNAs were the generous H. H. G. Khorana. mixing sonic 32P-labeled fragments with 2 Ag of pure unlabeled gift of Drs. Van de Sande and lac fragments and 1 /Ag of repressor in 0.5 ml of binding buffer, digesting with 0.05-0.1 mg of DNase for 1 min, filtering enzyme or core). For labeling with a single NTP, the mixtures rapidly on a B-6 filter, washing with 0.5 ml of 00 binding buffer also contained one [a-'2P]NTP (5-50 MM) at specific activities without Mg++ and containing 20 mM EDTA, and shaking the of 20-150 mCi/Mmol (New England Nuclear Corp.), and three filter on a Vortex mixer in 0.2 ml of 50 mM NH4HCO3 with other cold NTPs at 300 MuM. Reactions using these NTP 0.1% Na dodecyl sulfate. concentrations were quite efficient, incorporating 20-70% of Unlabeled operator was prepared by mixing 300 Mg of sonic the label, about a 30-fold net synthesis. For primed synthesis, lac fr 16s,1 cpm of 32P-labeled sonic lac fragments (as the mixtures contained either all four labeled NTPs at 5 MM or tracer), and 150 ,ug of active repressor (300-600 Mug of repres- only GTP at 5 AMM, the others at 20 MM, and dinucleotide sor) in 3 ml of binding buffer, and treating with 1 mg of primers at 100-500 MM or oligonucleotide primers at 50-100 DNase for 1 min at room temperature. The mixture was MuM. Reactions using these lower NTP concentrations with filtered on a 47-mm Schleicher and Schuell B-6 filter, washed primers were less efficient, incorporating about 10% of the with 3 ml of 20 mM EDTA, and shaken on a Vortex mixer in 1 label, but synthesized better-defined species of RNA. ml of 50 mM NH4HCO3 with 1% Na dodecyl sulfate.
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