IN VITRO CONSTRUCTION OF OPERON FUSIONS AND THEIR APPLICATION TO THE STUDY OF TRYPTOPHAN REGULATORY MECHANISMS IN ESCHERICHIA COLI

GREGG BOGOSIAN, Purdue University

Abstract

An investigation of repression in the trp system of Escherichia coli was undertaken using operon fusions and plasmids constructed using recombinant DNA technology. The promoters of the trp operon and the trpR gene were fused to lacZ, enabling the activity of these promoters to be evaluated through measurements of (beta)-galactosidase production. Plasmids were constructed which facilitated the manipulation of intracellular levels of Trp repressor. The trpR gene was found to be regulated autogenously. Intracellular Trp repressor levels were essentially invariant under most conditions studied. This feature of the trp system plays a significant role in imparting a broad range of expression to the trp operon. Strains with artificially elevated levels of Trp repressor displayed complete growth inhibition on minimal media which contained high levels of tryptophan. The inhibition was attributable to the acquisition of a compound nutritional requirement satisfiable by a combination of isoleucine, leucine, valine, threonine, serine, phenylalanine and tyrosine. Trp repressor lowered the rate of transcription of genes which encode enzymes for the biosynthesis of some of these amino acids. The regulatory implications of this finding are discussed. In the absence of repression and attenuation, the trp promoter was found to be subject to a third system of control. This newly discovered regulatory system has several components, including the products of trpB and trpS. The activity of the trp promoter was regulated over a 2500-fold range by the effectors of this third control system. The available experimental data are consistent with a model wherein trpB product may activate transcription at the primary trp promoter while trpS protein may inhibit transcription at the same site.

Degree

Ph.D.

Subject Area

Genetics

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