Operator-constitutive mutations of the Escherichia coli metF gene

Abstract

The Escherichia coli metF gene codes for 5,10-methylene-tetrahydrofolate reductase, the enzyme that leads to the formation of N-methyltetrahydrofolate, supplying the methyl group of methionine. Transcription of metF, as well as of most of the methionine genes, is repressed by the metj gene product complexed with S-adenosylmethionine. A metF'-'lacZ gene fusion was used to isolate mutants that have altered expression from the metF promoter. The nuicleotide sequences of the metF regulatory region from five such mutants were determined. The mutations were located in the region previously defined as the potential target of the methionine repressor by its similarit to other binding sites. The mutationally defined metF operator thus consists of a 40-base-pair-long region, with five 8-base-pair imperfect palindromes spanning the metF transcription start. The altered operators do not recognize the purified repressor in an in vitro transcriptiontranslation system, although the repressor binds efficiently to the metF wild-type operator.

Regulation of methionine synthesis in Escherichia coli: effect of the MetR protein on the expression of the metE and metR genes.

“…The first four amino acids of the MetE protein from E. coli and S. typhimurium are identical (18,29), and the present results show that 16 of the first 18 amino acids of the MetR protein in both organisms are also identical (18). Seven potential regulatory met boxes (3)(4)(5)(6)(7) (that contain at least 50% identity with the met box consensus sequence, 5'-AGACGTCT-3') are overlined in the sequence shown in Fig. 2.…”

“…Considerable knowledge has been obtained at the molecular level on the mechanism of this regulation. Studies on the expression of several of the met genes have shown that a repressor protein, the product of the metJ gene, can bind to a specific region on the DNA (met box) that is present in all met genes thus far examined (3)(4)(5)(6)(7). This interaction of the MetJ protein with the met operator region is markedly enhanced by the presence of S-adenosylmethionine (AdoMet), a primary metabolic product of methionine (5,8,9).…”

A novel regulatory factor affecting the transcription of methionine biosynthesis genes in Escherichia coli experiencing sustained nitrogen starvation

“…7a), (ii) is highly de-repressed in our RNA-seq analysis in DyeaG bacteria ( Fig. 4c and Table S4), and (iii) is widely used for in vitro analyses of MetJ-DNA interactions [24,25]. As shown in Fig.…”

The Escherichia coli K-12 metJ193 allele contains a point mutation which alters the hydrophobic pocket responsible for in vitro binding of S-adenosylmethionine: effects on cell growth and induction of met regulon expression

“…The pertinent DNA sequences of the metA, metC, metF, and metJ/metBL promoter regions contain from two to five imperfect, tandem repeats of the Met-box consensus sequence (3,4,7,18,24,25). The homology of individual Met-box sequences varies significantly within and between met regulon elements (3, 4).…”