PMID:6338493
| Citation |
Joachimiak, A, Kelley, RL, Gunsalus, RP, Yanofsky, C and Sigler, PB (1983) Purification and characterization of trp aporepressor. Proc. Natl. Acad. Sci. U.S.A. 80:668-72 |
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| Abstract |
We have isolated homogeneous trp aporepressor from an overproducing strain of Escherichia coli carrying a plasmid containing trpR preceded by tandem trp operon promoters. Dye-affinity and ion-exchange chromatography were used in conjunction with a gel electrophoresis assay in which the repressor, when bound to the trp operator, protects an Rsa I restriction site from endonuclease cleavage. Crystals suitable for x-ray diffraction studies were grown from a variety of concentrated salt solutions. Hydrodynamic properties and electrophoretic analysis of unmodified and covalently crosslinked aporepressor show that the free aporepressor has an isoelectric point of 5.9 and is a dimer containing two identical 12.5-kilodalton subunits in the presence or absence of L-tryptophan. The repressor . operator complex binds poorly to nitrocellulose filters, but restriction-site protection studies indicate that, in the presence of tryptophan, one dimer is bound to the operator site with an apparent dissociation constant less than 2 X 10(-9) M. Preliminary equilibrium dialysis experiments suggest that tryptophan binds to the aporepressor with a dissociation constant of 1.6 X 10(-5) M. |
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| Keywords |
Amino Acid Sequence; Apoproteins/isolation & purification; Crystallography; Escherichia coli/genetics; Gene Expression Regulation; Isoelectric Point; Molecular Weight; Operon; Protein Binding; Repressor Proteins/isolation & purification; Repressor Proteins/metabolism; Spectrum Analysis; Transcription Factors/isolation & purification; Tryptophan/genetics; Tryptophan/metabolism |
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