Proc. Natl. Acad. Sci. USA Vol. 81, pp. 6100-6104, October 1984 Genetics Demonstration of two operator elements in gal: In vitro repressor binding studies (operon/gaLR gene/electrophoretic mobility/DNA-protcin complex) ALOKES MAJUMDAR AND SANKAR ADHYA Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20205 Communicated by Allan Campbell, June 18, 1984 ABSTRACT Genetic and DNA base sequence analyses of F-hsdS20 recA13 aral4 proA2 lacY1 gal2 rpoL20 xyl5 su'II, cis-dominant mutations that derepress the gal operon ofEsch- used for large-scale preparation of gal plasmids (a gift of G. erichia coli suggested the existence of two operator loci needed Gaitanaris); SA1984 = F-strR galRA XcIts857 (Xint- for gal repression. One (OE) is located immediately upstream XcIII)AXN' (Xcro-chlA)A, made during this study and used to the two overlapping gal promoters and the other (01) is in- to maintain a PL-galR fusion plasmid. side the first structural gene. We have investigated the ability Fig. 1 Upper shows the genetic structure of the E. coli gal of wild-type and mutant OE and 01 DNA sequences to bind to operon and its restriction endonuclease sites relevant to the gal repressor. The repressor has been purified from cells con- construction of the following plasmids (i-v), which were the taining a multicopy plasmid in which the repressor gene is source ofgal DNA fragments used in protein binding assays. brought under the control of phage A PL promoter. The DNA- (i) pMI3 was generated by cloning the 260-bp gal DNA repressor interactions are detected by the change in electro- spanning sites b-e between the EcoRI and HindIII sites of phoretic mobility of labeled DNA that accompanies its com- pBR322 (7). The gal DNA (fragment A) contains a wild-type plex formation with repressor protein. The purified repressor OE (O) allele. shows concentration-dependent binding to both O and Ot (il) pAM230 was generated by restriction of gal DNA at but not to cEand 0( sequences. These results authenticate the site b, digestion with DNase exoIII and nuclease S1 to the proposed operator role of the two homologous gal DNA control position marked c, and addition of an EcoRI linker. The gal elements and thereby establish that the negative control of the DNA between sites c and f (fragment B) was subsequently gal operon requires repressor binding at both OE and 0, cloned between EcoRI and HindIII sites in pBR322. which are separated by >90 base pairs. pAM230 contains a wild-type 01 allele (Ot). Fragment B is 500 bp long. Three structural genes, E, T, and K, constitute the gal oper- (iii) p291 contains the wild-type alleles of both OE and 01 on of Escherichia coli (1). They are transcribed from either gal DNA (O Ot). p291 was generated by cloning the 664-bp of two partially overlapping promoters PG1 and PG2, which segment between sites b and f in gal (fragment C) between are modulated by cAMP and its receptor protein (CRP) in the EcoRI and HindIll sites of pBR322 (6). The distance be- opposite ways (2, 3). cAMP'CRP complex activates PG1 and tween OE and 0° is 97 bp. inhibits PG2 by binding to a single site, cat (refs. 4 and 5; see (iv) pBdC6 contains a 1100-bp gal DNA piece (fragment Fig. 1 Upper). The gal promoters are also regulated nega- D) spanning the segment a-f, between the EcoRI and Hin- tively by a gal repressor protein, the product of an unlinked dIII sites of PBR322 (11). Fragment D carries a mutation in galR gene (8-10). We have isolated and characterized cis- the OE allele (OE81 O'). dominant constitutive mutations in the gal operon and locat- (v) pAM401 was generated by cloning the gal DNA of an ed them by DNA base sequence analysis (3, 6, 11). These 01 mutant between sites d and f into the BstEII and HindIII studies have revealed the existence of two homologous 17- sites of pMI3. The resulting 664-bp-long gal DNA (fragment base-pair (bp) sequences in gal DNA, mutation in either one E) in pAM401 contains a mutant 01 allele (O 0i'6). of which causes derepression of PG, and PG2 in the presence (vi) pBR322 was obtained from Bethesda Research Labo- of gal repressor (Fig. 1). One of the sites (OE) is located im- ratories and digested with HindIII and BamHI to generate mediately upstream to the promoter region-i.e., around the 346-bp fragment F. -60 bp from the start site of PGl-promoted transcription. (vii) pAM2 and pAM3. pAM2 contains a complete gal re- The other (0r) is located downstream to the promoter and pressor gene (galR') within a HincII and EcoRI DNA frag- inside the first structural gene, galE. Because of the cis-act- ment (12) fused to the PL promoter of bacteriophage X in the ing nature of the two sites and of the sequence homology plasmid pKC30 of R. N. Rao (26). The HincII-EcoRI gaiR between them, we have proposed that the gal operon is neg- DNA fragment was generated by restriction digestion of a atively regulated by two operators, OE and 0° (6). Both of XgalR-transducing phage (13). The HincII site is upstream of them are required for repression ofthe two gal promoters. In the NH2 terminus and the EcoRI site is downstream of the this paper, we report on the binding of purified gal repressor COOH terminus of the gaIR gene. The PL-galR fusion was to DNA fragments that contain either wild-type or mutant made by the gaiR HincII site and the X Hpa I site located OE and 01 base sequences. downstream to PL. pAM3 contains the COOH-terminal seg- ment of the gaIR gene cloned in a similar fashion but by fus- MATERIALS AND METHODS ing a different HincII site, located within the gaiR structural The E. coli strains used in this paper are SA1796 = F-strR gene (12) to the X Hpa I site in pKC30. These two plasmids galRs8, used to test the presence of a gal operator allele (7); are maintained in the host strain SA1984 lysogenic for X. The RW1401 = C600 galA, used for cloning wild-type and mutant details of construction of pAM2 and pAM3 will be published gal DNA fragments (a gift of R. Weisberg); HB101 = elsewhere. Microbiological Technics. Standard microbiological media, agar plates, and methods, including transformation of cells The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: CRP, cAMP receptor protein; bp, base pair(s). 6100 Downloaded by guest on October 2, 2021 Genetics: Majumdar and Adhya Proc. NatL Acad Sci. USA 81 (1984) 6101 PG1 PG2 OE cat EN I-60 t -35 + 1 +27 det f 0+ A 260 I I B 500 c OI C I- 664 b C D t 1100 a 0c E I 664 t f F 346 I I FIG. 1. (Upper) Genetic map of the initial part of the gal operon. O' and O. are the two control elements (6) shown to be operators in this paper; PG1 is the cAMP-dependent promoter and PG2 is the cAMP-independent promoter; cat is the cAMP CRP complex binding site; EN is the start site of translation of the first structural gal gene. The lettered arrows pointing upward indicate the following natural or engineered restriction nuclease sites in gal DNA: a, a natural EcoRI site present in bacterial DNA brought closer to gal by a deletion (A320); b, an engineered EcoRI site (7); c, an engineered EcoRI site (see text); d, a natural BstEII site; e, a natural Hpa II site engineered into a HindIII site (7); f, a natural HindIII site. The numbers indicate the corresponding distance in base pairs from the start site of transcription initiation from PG1. (Lower) DNA fragments A-F generated by restriction nucleases from their corresponding plasmids and used in repressor binding assays of Fig. 3. The following lettered arrows indicate restriction sites of pBR322: g, a natural HindIII site; h, a natural BamHI site. The size of each fragment is indicated in base pairs at its right. with plasmid DNA, were used (14, 15). sponding plasmids and labeled at their 5' ends with [y- 32P Labeling of DNA. DNA fragments generated by restric- 32P]ATP by polynucleotide kinase. The size and the relevant tion nucleases was labeled at the 5' end with [y32P]ATP as genetic makeup of the fragments are diagramed in Fig. 1 described by Maxam and Gilbert (16). The labeled fragments (Lower). These fragments were tested for their ability to bind were then purified by elution from gels (16). to gal repressor, as shown later. DNA-Protein Binding Assays. DNA complexed with pro- Purified gal Repressor. The gaiR gene encodes the repres- tein characteristically shows slower electrophoretic mobility sor of gal operon and maps between lysA and thyA genes at on acrylamide gels than free DNA. This property of DNA 60 min on E. coli chromosome (8, 9). We have fused the has been utilized successfully by Garner and Revzin (17) and wild-type gaiR gene to a strong promoter of bacteriophage X by Fried and Crothers (18) to demonstrate specific binding of in a hybrid plasmid vector pKC30, creating the plasmid proteins to DNA. We used the same principle to study the pAM2 (Materials and Methods). Because high-level tran- binding of gal repressor to 5'-end-labeled [32P]DNA frag- scription of the PL promoter in pAM2 is deleterious to its ments.