Dual regulation of the mtr and aroL genes by the TyrR protein and Trp repressor of Escherichia coli

Virginia Mary Heatwole, Purdue University

Abstract

The mtr gene of Escherichia coli, which encodes a tryptophan-specific permease, was cloned using the mini-Mu system. The amino acid sequence of Mtr (414 amino acids), deduced by DNA sequence analysis, was found to be 33% and 53% identical to two other single-component, integral membrane proteins, the tyrosine-specific permease (404 amino acids) and the low affinity tryptophan permease (415 amino acids), respectively. Hydropathy plots, which can be used to predict membrane spanning regions of a protein, suggest these three permeases have a similar structure. The regulation of transcription of the mtr gene was studied using a single copy $mtr\sp{\prime}$-$\sp{\prime}lacZ$ transcriptional-translational fusion on a lambda phage, $\lambda$SLW20. Expression of $\beta$-galactosidase from this fusion was assayed in isogenic strains, lysogenic for $\lambda$SLW20, but lacking the TyrR protein or TrpR repressor or both regulatory proteins. Expression from the mtr promoter was repressed 97-fold by the Trp repressor via tryptophan and induced approximately 10-fold by the TyrR protein via phenylalanine or tyrosine. Primer extension analysis identified one major and one minor transcript from the mtr promoter. The major transcript was induced by the TyrR protein. The tryptophan-dependent interaction of the Trp repressor with the proposed operator, which overlaps the $-$10 of the promoter, was demonstrated by a restriction endonuclease protection assay. By computer analysis a potential Trp repressor operator was identified 56 nucleotides downstream of the transcriptional start site of aroL, the gene that encodes shikimate kinase II. Tryptophan-dependent interaction of the Trp repressor with this operator was demonstrated by a restriction endonuclease protection assay. An aroL$\sp\prime$-$\sp\prime$lacZ transcriptional-translational fusion was constructed on a lambda phage, $\lambda$SLW22. Expression from this fusion was assayed in a manner identical to $\lambda$SLW20 described above. This work demonstrated that expression from the aroL promoter was also repressed by the Trp repressor via tryptophan. Repression by the Trp repressor could not be observed in the absence of TyrR protein. A possible model for the mechanism of Tryptophan-mediated repression at this promoter, which involves the potential existence of a second promoter is discussed.

Degree

Ph.D.

Advisors

Somerville, Purdue University.

Subject Area

Molecular biology|Genetics|Biochemistry

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