Detection and Isolation of the Repressor Protein for the Tryptophan Operon of Escherichia coli

^*Department of Biological Sciences, Columbia University, New York, N.Y. 10027
^†Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115
^‡Institut fur Genetik, Cologne, Germany

Abstract

DNA from a transducing bacteriophage carrying a fusion of the tryptophan and lactose operons of E. coli (λdtrp-lac) has been used to direct cell-free synthesis of β-galactosidase (EC 3.2.1.23). Whereas normal lac operon (λdlac) DNA requires adenosine-3′:5′-cyclic monophosphate (cAMP) for β-galactosidase synthesis, trp-lac DNA is unaffected by cAMP. This difference in cAMP dependence verifies the presence of a cAMP-requiring promoter in the lac operon that has been removed from the trp-lac DNA. Synthesis with trp-lac DNA is controlled by the protein product of the tryptophan repressor gene (trpR). Synthesis in extracts of trpR ^- (repressor-negative) cells is progressively reduced by increased additions of extract from trpR ⁺ cells. No trpR ^- product repression is seen when β-galactosidase synthesis is programmed by normal lac DNA. This highly sensitive and specific assay has facilitated quantitation and partial purification of the trp repressor.

Footnotes

↵ § Present address: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Mass. 02115.
↵ ¶ Present address: Department of Molecular Biology, University of Geneva, Geneva, Switzerland.