Purification and Propertiesof a DNA-Binding Protein With
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Proc. Natl. Acad. Sci. USA Vol. 73, No. 7, pp. 2249-2253, July 1976 Biochemistry Purification and properties of a DNA-binding protein with characteristics expected for the Cro protein of bacteriophage X, a repressor essential for lytic growth (bacteriophage X cro gene/promoter-operator DNA) ATIS FOLKMANIS, YOSHINORI TAKEDA, JOSEF SIMUTH, GARY GUSSIN*, AND HARRISON ECHOLS Department of Molecular Biology, University of California, Berkeley, Calif. 94720, and * Department of Zoology, University of Iowa, Iowa City, Iowa 52242 Communicated by A. D. Kaiser, April 14, 1976 ABSTRACT The Cro protein specified by bacteriophage X viously (8), X DNA was labeled with P2p by growth of phage in is a repressor essential for normal lytic growth of the virus, thus low phosphate medium containing 5 ,Ci/ml of UP-labeled having a physiological role distinct from that of cI, the repressor that maintains lyso We have purified a X-specific DNA- inorganic phosphate. Phage were purified by precipitation with binding protein witheny.the requirements for synthesis and bio- polyethylene glycol and centrifugation to equilibrium in a CsCI chemical activities expected for Cro protein from studies in vivo. density gradient (2-3 times). DNA was extracted with redis- As isolated, the protein appears to be a dimer of molecular tilled phenol, the phenol was removed by extraction with ether, weight approximately 18,000 with DNA-binding properties that and the DNA was dialyzed into and stored in 10mM Tris-HCl, are very similar, but not identical, to those of the cI protein. We 0.2 mM EDTA, at pH 7.3. infer that bacteriophage X uses the same regulatory region of DNA for two different DNA-binding repressor proteins with DNA-Binding Assay. The DNA-binding assay used to seek subtle differences in binding activity specialized for different the activity of Cro protein has been described previously (7, 8). physiological roles. The assay measures retention of X [32P]DNA on a nitrocellulose filter (B-6, Schleicher and Schuell) by virtue of its tight binding The temperate bacteriophage X specifies two different repressor to a specific DNA-binding protein; an excess (100-fold) of un- proteins, cI and Cro, which play a major role in regulation of labeled "chicken blood" DNA (Calbiochem) is added to com- viral development. Each appears to have a specialized physi- pete for the binding of proteins that associate with DNA but ological role for one of the two possible life styles that the phage lack specificity for X DNA (22). The assay mixture contained: can pursue: lysogeny or lysis. The cI protein maintains lysogeny 0.2 ,ug of X [32PJDNA, 20 ,g of "chicken blood" DNA, 10mM through a repression of transcription of the genes essential for Tris'HCI at pH 7.3, 20 mM KCI, 10 mM MgCl2, 0.2 mM di- the earliest stage of lytic development; the Cro protein poten- thiothreitol, and 0.2 mM EDTA, in a total volume of 0.1 ml. tiates lytic growth through a repression of the early gene region One unit of DNA-binding activity is defined as the quantity during the late stage of lytic development (1-4) (A. Folkmanis, sufficient to retain 1 Mug of X DNA on the filter. The binding P. Mellon, H. Echols, and A. Skalka, manuscript in preparation). values are corrected for a "background" of DNA that is retained In spite of their different physiological functions, the bio- on the filter in the absence of binding proteins; this varies from chemical activities of the two proteins have been inferred from 5 to 15% with DNA preparation. in vivo experiments to be quite similar: inhibition of tran- Purification of Presumptive Cro Protein. Thirty liters of scription originating at the early promoters PL and PR (Fig. 1); C600 Su- cells were grown in a broth medium (1% Difco however, cI appears to stimulate transcription from the cI gene tryptone, 0.5% Difco yeast extract, 0.5% NaCI, 0.2% maltose) promoter PM during the maintenance of lysogeny, whereas Cro in three carboys. At a density of 4 X 108 cell per ml, cells were probably shuts off this transcription (see refs. 1-4 for a more infected with cIaml4Nam53vlvS phage at a multiplicity of detailed discussion). 10 phage per cell; 25 min after infection, crushed ice was added The cI protein has been purified and extensively character- and the cells were harvested by centrifugation in a Spinco 170 ized in vitro for both specific binding to X DNA and capacity continuous flow rotor. The cells were resuspended in 340 ml to repress RNA synthesis (5-9). The action of the Cro protein of 10% sucrose, 0.05 M Tris-HCI at pH 7.3, and were quick has previously been defined only through an analysis in vio. frozen in a dry ice-alcohol bath. The frozen cells were placed This report describes the purification of a DNA-binding protein in an ice bath for 20 min and then held at 200 until melted. At with properties expected for Cro protein and a partial charac- this time 34 ml of a solution of 0.25 M Tris.HCI at pH 7.3, 0.001 terization of its binding to the operator region that regulates M EDTA, 2 mg/ml of lysozyme and 18 ml of 2 M KC1 were early X genes. added and the mixture was kept at 00 for 40 min. The mixture was then made 0.01 M in MgCl2, the temperature was raised MATERIALS AND METHODS to 32', 0.5 mg of DNAse I was added, and after the addition of Bacteriophage and Bacteria. The Escherichia coli strains the DNAse had decreased the viscosity (about 5 min), the used and their relevant genetic characteristics were: W3350 Su- mixture was placed in an ice bath. Then 36 ml of 2 M KCI was (10), C600 Su+ (10), and C600 Su- (11). The X phage mutations added and the lysate was centrifuged at 25,000 rpm for 4 hr in used were: cI gene mutations cIts857 (12), cltsAt2 (13), clam 14 a Spinco 30 rotor. The supernatant fraction was then dialyzed (36); N gene mutations Nam53 and Nam7 (10); cro gene in Spectrapor no. 1 membrane tubing (Spectrum Medical In- mutations fedl (13) tof2 and tof6 (14); operator region point dustries) for 4 hr with one change against buffer A [10 mM mutations v2, vl, v3 (15, 16), vS326 (17), and prmll6 (18); and KPO4 at pH 6.35, 0.2 mM EDTA, 0.2 mM dithiothreitol, 5% operator region substitution mutations bio24-5 (19), imm434 (vol/vol) glycerol] with 0.1 M KC1. The dialyzed extract was (20), and imm2l (21). then applied to a 2.5 X 10 cm column of phosphocellulose Preparation of Phage DNA. Essentially as described pre- (Whatman P11) at a rate of 1.5 ml/min. The column was 2249 Downloaded by guest on September 26, 2021 2250 Biochemistry: Folkmanis et al. Proc. Natl. Acad. Sci. USA 73 (1976) 40 cro Recomb. genes clII N Ci cro cII Repin. genes -0. ci PM c 0 FIG. 1. The activity of cI and Cro inferred from experiments in co vivo. To maintain the repression essential for the B 20 lysogeny, cI protein z acts at OL and OR to repress leftward and rightward transcription in- 0 itiated at the early promoter sites PL and PR, respectively; the cI protein probably also activates leftward transcription of the cI gene 0N initiated at the maintenance promoter PM. The repression activity of cI keeps the entire phage genome repressed because the N gene product is required as a positive regulator of other genes. The Cro protein acts to turn off (or turn down) the expression of early genes during the late stage of lytic development, presumably through its capacity to act at OL and OR to repress early gene transcription; the Cro protein probably also represses transcription of the cI gene ini- 10 15 tiated at PM. After infection the initial transcription of the cI gene Tube Number occurs through activation by cII and c1II at a site to the right of the FIG. 2. cro gene. (See refs. 1-4.) Repin., replication; Recomb., recombination. Sedimentation properties of Cro activity. Material frac- tionated on phosphocellulose was sedimented in a 10-30% (vol/vol) glycerol gradient containing 0.4 M KCI at 50,000 rpm for 24 hr. washed with buffer A plus 0.1 M KCI and then eluted with a Fractions were collected and aliquots were assayed for DNA-binding 180 ml linear gradient (0.1 M to 1.0 M KCl) in buffer A. activity as described in Materials and Methods. Sedimentation was The X-specific DNA-binding activity eluted at from right to left. Arrows denote the position of marker proteins (B 0.35-0.45 M = bovine serum albumin, 0 = ovalbumin, C = chymotrypsin, and M KCI. These fractions were concentrated by precipitation with = myoglobin) ofmolecular weights 65,000,45,000,25,000, and 17,000, ammonium sulfate (80% saturation), and half of this sample was respectively. (0), DNA-binding activity for X DNA; (0), DNA-binding fractionated on a 2.5 X 81 cm column of Sephadex G-75 activity for Ximm434 DNA. (Pharmacia) in buffer A plus 0.4 M KCI (see Fig. 2 in Results). The Sephadex fractions with X-specific DNA-binding activity were diluted with buffer A to 0.1 M KCI (determined by con- would reflect Cro activity. We also included the operator region ductivity) and refractionated on a 2 X 3 cm phosphocellulose mutations vlv3 to try to reduce any self-repression by Cro column.