Novel aspects of the regulation of L-tryptophan biosynthesis in Escherichia coli

Weiping Yang, Purdue University

Abstract

This thesis is concerned with three interrelated aspects of how L-tryptophan biosynthesis is regulated in Escherichia coli. Highly purified preparations of Trp repressor (TrpR) protein derived from Escherichia coli strains that were engineered to overexpress this material were found to contain another protein, of 21 kDa. The second protein, designated WrbA (for tryptophan (W) repressor-binding protein), was cloned and sequenced. The WrbA protein was found by several criteria to enhance the formation and/or stability of noncovalent complexes between TrpR holorepressor and its primary operator targets. The WrbA protein alone does not interact with the trp operator. During the stationary phase, cells deficient in the WrbA protein were less efficient than wild type in their ability to repress the trp promoter. It is proposed that the WrbA protein functions as an accessory element in blocking TrpR-specific transcriptional processes. A divergent operon, of opposite orientation to the trp EDCBA operon of E. coli, has been identified and characterized. This operon, designated the trpH operon, encodes a polypeptide of 114 residues. A functional target for TrpR was identified within the trpH promoter by gel mobility-shift analysis and by DNase I footprinting. TrpR has been shown to stimulate the transcription of the trpH promoter independent of L-Trp. The inability to make the trpH gene product confers a phenotype of enhanced sensitivity to 5-methyltryptophan. The gene product of the trpH operon appears to affect the process of attenuation in the trp operon. It is proposed that TrpH may be an RNA binding protein and whose role is to counteract the attenuation process in the trp operon of E. coli. Anti-idiotype antibodies have been made from an anti-TrpR antibody preparation. These anti-idiotype antibodies were able to interact in a specific manner with the trp operator DNA, as shown by gel mobility-shift analysis. In a competitive ELISA assay, the binding of the anti-idiotype antibody to the trp operator was reversed by TrpR protein. It is proposed that a subset of the anti-idiotype antibodies may structurally mimic the DNA binding motif of TrpR, thereby enabling these antibodies to interact with the trp operator in a sequence specific manner.

Degree

Ph.D.

Advisors

Somerville, Purdue University.

Subject Area

Biochemistry

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