Control of metF gene expression in maxicell preparations of Escherichia coli K-12: reversible action of the metJ protein and effect of vitamin B12

Emmett, Mark R.; Johnson, James R.

doi:10.1128/jb.168.3.1491-1494.1986

Cited by 11 publications

(13 citation statements)

References 38 publications

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“…In the case of MetJ, it is capable of repressing or derepressing target genes within 30 min of a change of methionine concentration (35,36). Our NMR spectral data clearly indicate that MetJ's nonspecific interactions with genomic DNA are extensive, providing a mechanism for this efficiency.…”

Section: Discussionmentioning

confidence: 79%

MetJ repressor interactions with DNA probed by in-cell NMR

Augustus¹,

Reardon²,

Spicer

2009

Proc. Natl. Acad. Sci. U.S.A.

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Atomic level characterization of proteins and other macromolecules in the living cell is challenging. Recent advances in NMR instrumentation and methods, however, have enabled in-cell studies with prospects for multidimensional spectral characterization of individual macromolecular components. We present NMR data on the in-cell behavior of the MetJ repressor from Escherichia coli, a protein that regulates the expression of genes involved in methionine biosynthesis. NMR studies of whole cells along with corresponding studies in cell lysates and in vitro preparations of the pure protein give clear evidence for extensive nonspecific interactions with genomic DNA. These interactions can provide an efficient mechanism for searching out target sequences by reducing the dependence on 3-dimensional diffusion through the crowded cellular environment. DNA provides the track for MetJ to negotiate the obstacles inherent in cells and facilitates locating and binding specific repression sites, allowing for timely control of methionine biosynthesis.DNA-protein interactions ͉ gene regulation ͉ met repressor ͉ methionine regulon ͉ nonspecific DNA T he environment in which proteins and nucleic acids function within a cell is well known to be crowded and complex (1). Understanding the influence of the numerous large and small molecule components on the biophysical and functional properties of these macromolecules in cellular environments, however, is extremely difficult (2, 3). For example, the detailed mechanism of gene regulation is of great interest, but the study of these interactions within living cells has always been a challenge.The transcriptional repressor MetJ controls the expression in Escherichia coli of the Met regulon, which is composed of at least 12 genes scattered at 7 sites around the genome (Fig. 1). These genes code for proteins that are involved in methionine biosynthesis and transport (4, 5). The promoters of these genes contain from 2 to 5 tandem repeats of the MetJ binding site, which are called metboxes and have the consensus sequence AGACGTCT (6). When MetJ is activated by S-adenosylmethionine (SAM) it binds tightly to the metboxes and shuts down transcription (7,8). The different genes repressed by MetJ not only have a variable number of metboxes but also variable sequences. MetJ is thus capable of specific binding to several variations of the consensus sequence in vivo and fine-tuning its repression of each gene (4, 9). NMR spectroscopy has recently been shown to provide a valuable strategy for studying proteins in living cells (10-13). This in-cell spectroscopy gives atomic level fingerprints and even 3-dimensional spectral data that are used to assign resonances and characterize features of individual components in the cellular milieu (12). The method has been used to show ligand binding (14), protein-protein interactions (15) and in at least 1 case, a gain of structure presumably as a result of molecular crowding inside the cell (13). Here, we report NMR results on both in-cell and cell lysate studie...

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Section: Discussionmentioning

confidence: 79%

MetJ repressor interactions with DNA probed by in-cell NMR

Augustus¹,

Reardon²,

Spicer

2009

Proc. Natl. Acad. Sci. U.S.A.

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“…The electropherogram was stained with Coomassie blue, photographed, dried, and used to prepare an autoradiogram ( Fig. 1) (7,13,14). In maxicell samples of JJ122R(pEJ3-lB) (chromosomal metJ+ allele, metF+ allele carried on plasmid pEJ3-1B), expression of the 33,000-Mr (33K) MetF protein (22,31) was drastically reduced at 28 and 34°C by 200 F.M methionine (Fig.…”

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confidence: 99%

“…Plasmids pTJ77H and pEJ3-1B contain copies of the E. coli metF transcription unit inserted at the pBR322 HindlIl or BamHI restriction site, respectively (7,31). Strains GB793, GB793F, GB928, GB950.1, JJ117, JJ127B, and JJ127F resulted from bacteriophage P1 transductions (5 mid-borne inetF transcription unit resulted in elevation of the specific activity of the metF gene product at 28°C as well as at 34 and 42°C (Table 2).…”

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Characterization of two mutant metJ proteins with reduced, temperature-dependent capacity to regulate Escherichia coli K-12 met regulon elements

et al. 1989

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At 28 degrees C, but not at 34 or 42 degrees C, strains with the metJ193 allele repressed chromosomal met genes but not a plasmid-borne met promoter. Increasing the metJ193 gene dosage to two copies resulted in overrepression of chromosomal and plasmid-borne met promoters at 28 degrees C. Suppressing the metJ185 amber mutation with supF (tRNATyr) produced the MetJ185F protein. Strains producing MetJ185F repressed chromosomal met promoters but not a plasmid-borne met promoter at 42 degrees C. These are the first known defective MetJ proteins with documented temperature-dependent function.

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“…Maxicell samples were prepared from these cultures by UV irradiation as described by Emmett and Johnson (8). Before 14C-amino acids were used to label protein products, the exogenous methionine concentrations of these maxicell samples were manipulated as described in the Results (8 …”

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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

Collier

Johnson

1990

J Bacteriol

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The metJ193 allele encodes one of two identified temperature-sensitive Escherichia coli K-12 (34C). When 28°C cultures of strains bearing two metJl93 aDleles were transferred from methionine-containing medium to minimal medium, derepression of met regulon expression did not occur quickly enough to avoid a lag in growth due to the methionine deprivation. The inability of the MetJ193 protein to easily accomplish transition between apo-and active-repressor conformations was also demonstrated by using a maxicell system to study expression of a plasmid-borne copy of the E. coli metF transcription unit. These results confirm the importance of the leucine 56 residue for the structure and function in vivo of the wild-type MetJ protein.

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Control of metF gene expression in maxicell preparations of Escherichia coli K-12: reversible action of the metJ protein and effect of vitamin B12

Cited by 11 publications

References 38 publications

MetJ repressor interactions with DNA probed by in-cell NMR

MetJ repressor interactions with DNA probed by in-cell NMR

Characterization of two mutant metJ proteins with reduced, temperature-dependent capacity to regulate Escherichia coli K-12 met regulon elements

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

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