The regulation of transcription of the gene for the tryptophan-specffic permease, mtr, was evaluated in several genetically marked Escherichia coli strains through the use of a single-copy lacZ reporter system. The expression of mtr was repressed 97-fold by tryptophan via the Trp repressor and induced 10-fold by phenylalanine or tyrosine via the Tyr repressor. By primer extension analysis two distinct mtr transcripts and their corresponding promoters were identified. One transcript was induced by the Tyr repressor. The tryptophan-dependent interaction of Trp repressor with an operator target within the mtr promoter was demonstrated by means of a restriction endonuclease protection assay.

The mtr gene of Escherichia coli K-12 encodes an L-tryptophan-specific permease. This gene was originally identified through the isolation of mutations in the 69-min region of the chromosome, closely linked to argG.Cells with lesions in mtr display a phenotype of 5-methyltryptophan resistance. The mtr gene was cloned by using the mini-Mu system. The amino acid sequence of Mtr (414 codons), deduced by DNA sequence analysis, was found to be 33% identical to that of another single-component transport protein, the tyrosine-specific permease, TyrP. The hydropathy plots of the two permeases were similar. Possible operator sites for the tyrosine and tryptophan repressors are situated within the region of DNA that is likely to be the mtr promoter.In Escherichia coli, the aromatic amino acids are concentrated in the cytoplasm mainly via four operationally distinguishable transport systems. These systems are a general aromatic amino acid permease, encoded by aroP, and three other transport systems specific for phenylalanine, tyrosine, and tryptophan (6). The general aromatic amino acid permease has strong affinity for the aromatic amino acids (Km for all three amino acids, 10' M), whereas the individual transport systems have a lower affinity but a higher specificity for their respective aromatic amino acids (Km for each, ca. 10-6 M). Mutations in the structural genes for each specific permease have been found (6,19,39). The genes for the general aromatic amino acid permease, aroP, and the tyrosine-specific permease, tyrP, have been cloned (10,14) and sequenced (20,43). The regulation of transcription from the tyrP and aroP promoters has been thoroughly investigated (11,23,24). AroP and TyrP are hydrophobic proteins that are associated with the cytoplasmic membrane (10, 44).The tryptophan-specific transport system is encoded by a single gene designated mtr. This gene is situated near the 69-min region of the bacterial chromosome (19), and the locus was first identified through the isolation of mutations that impart resistance to the antimetabolite 5-methyltryptophan. By P1 transduction, mtr is 60 to 80% linked to argG (19,25). Mutations in mtr result in a loss of tryptophanspecific uptake (C. Yanofsky, cited by Oxender [34]). In addition to the aroP and mtr systems for L-tryptophan uptake, a gene for a low-affinity tryptophan permease (K, 10-5 M) is situated within the tna operon (14).This report describes the cloning and structural characterization of the mtr+ gene. The cloned DNA has structural and genetic properties predicted for a segment from the 69-min region of the chromosome. Several lines of evidence support the notion that the region cloned and sequenced in this study encodes the tryptophan-specific transport protein Mtr. Structural comparisons of Mtr with the two other wellcharacterized aromatic amino acid permeases of E. coli are also presented. Preparation of mini-Mu lysates. Mini-Mu lysates were prepared as described previously (18). MC1040-2 carrying plasmid pEG5005 was used to prepare a mini-Mu lysate. Lys...

(Fig. 1) by using the Consensus program (9). The Fitconsensus program (9) was modified so that the consensus table could be used to scan all the E. coli sequences in the GenBank and EMBL data bases. This search revealed a potentially strong half operator in the aroL promoter region. Visual inspection of the aroL nucleotide sequence identified a potential full Trp repressor operator site that included the computer-predicted half operator (Fig. 2).The possible contradiction between the in vitro binding studies and the in vivo analysis of regulation cited above, coupled with our identification of a potential Trp repressor operator site, suggested that the Trp repressor, not the Tyr repressor, may be responsible for the tryptophan-mediated repression.Tryptophan holorepressor binds to an operator downstream of the aroL promoter. Within the putative Trp repressoroperator site is an RsaI restriction endonuclease cleavage site (Fig. 2). Purified Trp repressor was shown to specifically protect this RsaI site from cleavage in a tryptophan-dependent manner (Fig. 3) Construction of an aroL-lacZ transcription-translation fusion on a X phage. To test whether the Trp repressor affects expression from the promoter in vivo, a transcriptionaltranslational fusion was constructed on a X phage by the