Abstract
The galR gene, which encodes the Gal repressor protein in Escherichia coli, has been fused to the strong pL promoter of bacteriophage lambda in plasmid pKC31. The pL promoter is kept repressed by a thermolabilie lambda repressor, CIts857, to prevent cell killing. Heat induction of the pL-galR fusion plasmid synthesizes large amounts of active Gal repressor. The protein has been purified to homogeneity in three steps. The purification is greatly aided by the reversible insolubility of active repressor in crude extract at salt concentrations of less than 200 mM. The amino-terminal amino acid sequence determined by automated Edman degradation is: N-Ala-Thr-Ile-Lys-Asp-Val-Ala-Arg-Leu-Ala-Gly-Val-Ser-Val-Ala-Thr-Val-. Comparison of this sequence with that deduced from the DNA sequence of the galR gene showed that the formyl methionine residue preceding alanine at position 1 is cleaved off. The repressor is present in solution as a dimer of a 37-kDa subunit. The protein binds to gal DNA containing wild type and not mutant operator sequences. As predicted, this sequence-specific binding is inhibited by the presence of D-galactose or D-fucose, both of which are in vivo inducers of the gal operon. Gal repressor inhibits the expresison of gal operon by binding to two spatially separated operators which flank, but do not overlap, the gal promoter segment. Experiments to study the mechanism of repressor action are discussed.
Highlights
From the Laboratory of Molecular Biology and the $Laboratoryof Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892
The galR gene showed that the formyl methionine residue preceding alanine at position 1 is cleaved off.The repressor is present in solution as a dimer ofa 37-kDa Strains-The following E. coli strains used in this study are all subunit
C600, F- supE44 thrl leuB6 thi lacy1 tonA2; type and not mutant operator sequences. This sequence-specific binding is inhibited by the presence of D-galaCtOSe or D-fucose, both of which are in vivo inducers of the gal operon
Summary
Repressor is present in solution as a dimer ofa 37-kDa Strains-The following E. coli strains used in this study are all subunit. C600, F- supE44 thrl leuB6 thi lacy tonA2; type and not mutant operator sequences As predicted, this sequence-specific binding is inhibited by the presence of D-galaCtOSe or D-fucose, both of which are in vivo inducers of the gal operon. The bacteriophage A strain, AcZts857Sam7lysA+galR+s,ubsequently designated XgalR168 (catalog No y168), carries the Gal repressor gene and was used to subclone the latter into plasmid vectors. Two promoters,P1 and P2, control the transcriptionof the cation of the Gal repressor, cells carrying the pL-galR fusion plasmid gal operon of Escherichia coli (1, 2; see Fig. 1).Cyclic AMP and its receptor protein, acting as a complex, stimulate P1 and inhibit P2. Replacement of the PstI-EcoRI DNA segment wild type allele, i.e. galR+/galR“ diploid cells do not make Gal enzymes even in the presenceof D-galactose [5, 6]. NH2-Epimerase sure liquid chromatography as described by Zimmerman et al [22]
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