Escherichia coli heterogenotes, which produce hybrid molecules between the chimaeric protein repressor-galactosidase and the enzyme 8-galactosidase, were constructed. Repressor-galactosidase. in which fully active lac repressor is covalently linked to active P-galactosidase, is an aggregate with a core structure of four P-galactosidase parts and two peripheral lac repressor dimers. The lac repressor dimers, which are separated by tetrameric 1-galactosidase, retain all the biological activities of tetrameric lac repressor. Substitution of repressor-galactosidase subunits with P-galactosidase subunits leads to hybrid molecules with y P-galactosidase subunits aggregated with (4-y) repressor-galactosidase subunits (where y = 1, 2 or 3). A 2: 2 hybrid, i. e. a tetrameric P-galactosidase core with one lac repressor dimer grafted to it, binds at least 100 times less strongly to 3zP-labelled Aplac DNA than pure lac repressor or repressor-galactosidase.The data suggest a model in which lac repressor binds with two subunits to lac operator and with the other two subunits elsewhere on the DNA, possibly on sequences like the lac operator.Heteromultimeric enzymes which are often found in heterozygotes are formed by random association of enzyme subunits coded by different alleles [ l ] . We have prepared hybrid aggregates between subunits of the chimaeric protein repressor-galactosidase and the enzyme P-galactosidase. Chimaeric repressor-galactosidases have proved useful in probing the relationship between structure and function of lac repressor. Chimaeras, in which 60 -80 amino acids of the NHZ terminus of lac repressor are grafted to functioning P-galactosidase, are capable of binding to the phosphate backbone of DNA, thus proving that the NHzterminal domain of lac repressor contains the DNAbinding site [2-41. Using a chimaera, in which fully active lac repressor is covalently linked to the NHz terminus of active P-galactosidase, we were able to investigate the kind of interaction of tetrameric lac repressor with lac operator DNA [5,6]. Applying diimidates and iodine as crosslinking agents, and electron microscopy, we propose that this particular chimaera is a tetramer aggregated via the P-galactosidase parts in a DZ symmetry, as in the wild-type molecule, carrying two lac repressor dimers at the periphery of the fl-galactosidase core (see Fig. 5). Such a symmetry implies that only two lac repressor subAbbreviation. iPrSGal, isopropyl-thio-8-D-galactoside; 1 : 3, 2 : 2 and 3 : 1 hybrids, complexes with 1, 2 or 3 repressor-gdlactosidase polypeptides aggregated with 3,2 or 1 /J-galactosidase polypeptides, respectively.Enzyme. p-Galactosidase (EC 3.2.1.23).units, properly aggregated, are sufficient to bind specifically to lac operator. In order to test this hypothesis we constructed hybrid aggregates which carry only one lac repressor dimer per P-galactosidase tetramer (Fig. 5 D). A similar attempt has been made by Geisler and Weber [7], who constructed hybrid lac repressors consisting of native and protease-treated subunits....
A protein possessing both lac repressor and f-galactosidase activities in a single polypeptide of about 155,000 daltons was purified from a deletion mutant of Escherichia coli in which the lacI and Zgenes are fused. A 77-residue cyanogen bromide peptide containing the fusion joint was isolated. A radioimmunoassay with an antibody prepared against CNBr2 (residues 3-92) of ,i-galactosidase was used to monitor its purification. The sequence of the joining peptide was determined by analysis of tryptic peptides and by automatic sequencer analysis. The site of joining is from residue 355 of lac repressor to residue 24 of P-galactosidase (or 356 to 25), indicating that the last 4 residues at the carboxyl terminus of lac repressor and the first 23 residues at the amino terminus of ftgalactosidase are not essential for the activities of these two proteins. The exact site of the fusion is not known because lac repressor residue 356 and P-galactosidase residue 24 are both leucine residues. Examination of the nucleotide sequences around the two end points of the deletion revealed a homology of 9 identities in a stretch of 11 base pairs.Strains of Escherichia coli with fusions of the lad and Z genes, coding for lac repressor and fl-galactosidase, respectively, have been isolated and shown to produce proteins containing a portion of lac repressor covalently attached near the amino terminus of 03-galactosidase (1). These deletion mutants were found among lac+ revertants of strains carrying the lacZ U118 nonsense mutation. Because the site of this mutation in the gene corresponds to amino acid residue 17 (2), at least 17 amino acid residues at the amino terminus of fl-galactosidase are absent in the repressor-galactosidase hybrids. Several of these strains produce chimeric proteins possessing both lac repressor and f3-galactosidase activities (1). One of these was purified from strain 71-56-14 and was found to be composed of monomers of about 155,000 daltons containing an amino-terminal sequence identical to that of lac repressor (1). AF'pro+iqli+Z+] (1) was grown in minimal medium with 1% glycerol at 35°. The initial steps in the isolation of repressor-galactosidase from 650 g of cells were identical through the ammonium sulfate precipitation to those used for f3-galactosidase (8). The 0-35% ammonium sulfate precipitate was then dialyzed against 0.08 M Tris/1 mM MgCl2/1 mM dithiothreitol/0. 1 mM EDTA, pH 7.5 and applied to a 2.5 X 40 cm column of DEAE-Bio-Gel A (Bio-Rad) equilibrated with the same buffer. After washing, a linear gradient of 0.01-0.15 M NaCl in 1200 ml of buffer was applied. The main peak of f-galactosidase activity was pooled and the protein was chromatographed again on a 1.5 X 20 cm column of DEAE-Bio-Gel under the same conditions but with a gradient of 500 ml. Enzyme (9) and a-complementation assays (10) were carried out as before.Radioimmunoassay. The procedure used was similar to that described (7) with several modifications. Magnesium was omitted from all buffers. The tryptic peptide T8 (residu...
The chimaeric protein repressor-galactosidase, in which fully active Jac repressor is covalently linked to the active enzyme #-galactosidase, was used as a system for probing the quaternary structure of Jac repressor. Electron micrographs revealed repressor-galactosidase to be a tetrameric aggregate.When Jac repressor, alone, was crosslinked with dimethyl suberimidate, dimers, trimers, tetramers, and oligomers of the protein subunit were produced, whereas crosslinking of the tetrameric repressor-galactosidase resulted in the production of only dimers of the chimaera. Treatment lac repressor, a tetramer of identical subunits, specifically interacts with its DNA target, the lac operator, thus preventing the expression of the structural genes of the lac operon. Genetic and biochemical analysis point to the NH2-terminus of lac repressor as the region which interacts specifically with DNA [see review by Muller-Hill (1)]. The primary structure of the lac repressor protein (2) and the lac operator (3) have been determined. Little, however, is known about the tertiary and quaternary structure of wac repressor since crystals large enough for single crystal x-ray diffraction analysis have not yet been obtained. Electron micrographs of the protein in solution exist (4, 5). The most detailed structural analysis was carried out by Steitz et al. (6), who proposed a model of quaternary structure from electron micrographs and powder x-ray diffraction analysis of microcrystals. For this model, lac repressor subunits are arranged in an exact or quasi D2 symmetry. As a conclusion to their investigations Steitz et al. proposed a model for repressor-operator interaction, where it was suggested that two operator binding sites exist per tetramer, in the case of D2 symmetry. This model, if correct, might imply that lac repressor dimers are capable of recognizing the lac operator sequence, if somehow the repressor tetramer could be dissociated into the appropriate stable dimers. Repressor-galactosidase* is a fusion-protein, in which fully active lac repressor is covalently attached by its COOH-terminus to the NH2-terminus of active Abbreviation: NaDodSO4, sodium dodecyl sulfate. * We will use the term "repressor-galactosidase" for the protein chimaera purified from Escherichia coil strain iql (71-56-14), as described (7). The terms "repressor-part" and "galactosidase-part" are used for those amino acid sequences in repressor-galactosidase, which are coded by the lac repressor gene and by the fl-galactosidase gene, MATERIALS AND METHODS Purification of Repressor-Galactosidase. Repressor-galactosidase protein was purified as described previously (7), with the modification that chromatography on DEAE-cellulose (Biorad) was performed after the phosphocellulose step. DEAE-cellulose was equilibrated with buffer D [0.01 M Tris at pH 7.27 (at 22°), 0.01 M MgAc, 0.01 M 2-mercaptoethanol, and 0.1 M NaCI]; the pure protein eluted at 0.21 M NaCl, when a linear gradient of 0.1 M NaCI to 0.5 M NaCl in buffer applied to the column. Accordin...
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