Nucleotide sequence of the leader region of the phenylalanine operon of Escherichia coli

Abstract

The isolation of X transducing phages carrying the phenylalanine (phe) operon of Escherichia coli (1) has facilitated the cloning of this operon onto plasmid vectors. We have mapped the phe operon by cleavage with restriction endonucleases and by genetic analysis. In this paper we report that there is a transcribed leader region of about 170 base pairs (bp) immediately preceding pheA, the sole structural gene of the phe operon. The function of this region in the regulation of phe operon expression is discussed. MATERIALS AND METHODSConstruction and Preparation of Plasmids. The methods used for ligation, transformation, and preparation of plasmid DNAs were essentially those described in Selker et al. (2).Restriction Mapping. The methods used for restriction endonuclease digestion and analysis of DNA fragments by agarose and polyacrylamide gel electrophoresis have been described (3). A detailed restriction analysis of the phe operon will appear elsewhere.In Vitro Transcription. Plasmids and restriction fragments were transcribed and the 32P-labeled RNA products were analyzed by electrophoresis on 10% polyacrylamide/Tris borate/EDTA gels containing 7M urea as described (4).

Specificity of attenuation control in the ilvGMEDA operon of Escherichia coli K-12

“…1) (4,32,38). In principle, the attenuation mechanism controlling the ilv operon appears to be quite similar to that found to control operons involved in tryptophan (35), leucine (19), phenylalanine (61), histidine (3), and threonine (17) biosynthesis: the leader region specifies a short peptide containing a disproportionate frequency of the amino acids that are found to regulate the operon. Thus, the rate at which the leader can be translated serves to sense whether the regulatory amino acid is in short or ample supply.…”

“…This apparently greater sensitivity of the ilv leader may contribute to the fact that the relatively short peptide it specifies (32 amino acid residues) can have sensing sites for three different amino acids. Recall that the leader of the histidine operon specifies a peptide containing a run of seven histidine residues (3); that of the pheA gene specifies seven phenylalanine residues, but they are arranged in two runs of three and a single residue separated by single nonregulatory amino acids (61). In contrast, the peptide specified by the ilv…”

Mapping of chorismate mutase and prephenate dehydrogenase domains in the Escherichia coli T‐protein

“…The tyrA gene, which codes for the T-protein, was subcloned from plasmid pKB45, a derivative of pMB9 that contains a 6-kb segment of E. coli chromosomal DNA [13]. Several primers (Table 1) were used to amplify specific fragments from pKB45.…”

“…Expression levels for all fragments lacking the native N-terminal sequence (plasmids PSQC2, 3,4,5,6,7,8,13) were low. Good levels of expression were observed with all other fragments.…”

Isolation of the gene (miaE) encoding the hydroxylase involved in the synthesis of 2-methylthio-cis-ribozeatin in tRNA of Salmonella typhimurium and characterization of mutants

“…Interestingly, the absence of this modified nucleoside has several dramatic effects on gene regulation and cell physiology. A miaA mutant is affected in growth rate, attenuation regulation, and translational step time and has increased uptake and synthesis of aromatic amino acids (11,21,22,30,72,74). These effects are most likely caused by a decreased efficiency and an increased codon context sensitivity of those tRNAs that normally contain ms2i6A37 or ms2io6A37 (5,22,48,67).…”