Characterization of a second MetR-binding site in the metE metR regulatory region of Salmonella typhimurium

Wu, Whei-Fen; Urbanowski, Mark L.; Stauffer, George V.

doi:10.1128/jb.177.7.1834-1839.1995

Cited by 16 publications

(22 citation statements)

References 24 publications

(29 reference statements)

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“…Interestingly, increased Hcy levels have been reported to stimulate metE transcription through the activation of MetR, a positive transcriptional regulator of metE. However, these studies were conducted using metJ mutant strains of E. coli (Byerly et al, 1990;Urbanowski & Stauffer, 1989;Wu et al, 1995), making it difficult to predict what impact the increase in methionine levels might have on metE transcription. It appears that under the growth conditions tested in the present study the repressive effect of MetJ-SAM on metE transcription outweighs the inducing effect of MetR-Hcy.…”

Section: Discussionmentioning

confidence: 96%

Global effects of homocysteine on transcription in Escherichia coli: induction of the gene for the major cold-shock protein, CspA

et al. 2006

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Homocysteine (Hcy) is a thiol-containing amino acid that is considered to be medically important because it is linked to the development of several life-threatening diseases in humans, including cardiovascular disease and stroke. It inhibits the growth of Escherichia coli when supplied in the growth medium. Growth inhibition is believed to arise as a result of partial starvation for isoleucine, which occurs because Hcy perturbs the biosynthesis of this amino acid. This study attempted to further elucidate the inhibitory mode of action of Hcy by examining the impact of exogenously supplied Hcy on the transcriptome. Using gene macroarrays the transcript levels corresponding to 68 genes were found to be reproducibly altered in the presence of 0.5 mM Hcy. Of these genes, the biggest functional groups affected were those involved in translation (25 genes) and in amino acid metabolism (19 genes). Genes involved in protection against oxidative stress were repressed in Hcy-treated cells and this correlated with a decrease in catalase activity. The gene showing the strongest induction by Hcy was cspA, which encodes the major cold-shock protein CspA. RT-PCR and reporter fusion experiments confirmed that cspA was induced by Hcy. Induction of cspA by Hcy was not caused by nutritional upshift, a stimulus known to induce CspA expression, nor was it dependent on the presence of a functional CspA protein. The induction of cspA by Hcy was suppressed when isoleucine was included in the growth medium. These data suggest that the induction of CspA expression in the presence of Hcy occurs because of a limitation for isoleucine. The possibility that Hcy-induced cspA expression is triggered by translational stalling that occurs when the cells are limited for isoleucine is discussed.

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Section: Discussionmentioning

confidence: 96%

Global effects of homocysteine on transcription in Escherichia coli: induction of the gene for the major cold-shock protein, CspA

et al. 2006

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“…Binding to these sites or extended binding sites stretching from Box I-like sequences towards the Ϫ35 regions of target genes was demonstrated by footprinting experiments for several LysR-type proteins (31). The functional role of a Box II-like sequence, called the activation site, has been demonstrated genetically for genes regulated by TrpI (13), CatR (27), and MetR (43). The extent of similarity between the two binding sites varies for different LysR-type proteins.…”

Section: Discussionmentioning

confidence: 99%

“…By mutational analysis similar to that described in this paper, the involvement of Box I-like sequences in activation of target genes has been demonstrated for certain other LysR-type proteins, such as NahR (32), MetR (6,39,43), NodD (11,15), and CatR (27). Several LysR-type proteins have been shown to bind to DNA regions that overlap the LysR consensus site (31), and this binding is decreased by the same mutations which impair activation of target genes (1,15,27,32).…”

Section: Discussionmentioning

confidence: 99%

Sites required for GltC-dependent regulation of Bacillus subtilis glutamate synthase expression

1995

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The Bacillus subtilis gltAB genes, coding for the two subunits of glutamate synthase, are transcribed divergently from the gltC gene, encoding a LysR-type transcriptional activator of gltAB. The predicted gltA and gltC transcription start sites are separated by 51 to 52 bp. A 15-bp, consensus binding site (Box I) for LysR-type proteins was found centered at position ؊64 with respect to the gltA transcription start. This site was shown by mutational analysis to be required both for GltC-mediated activation of gltA and for autorepression of gltC. Box II, which is similar to Box I, is centered 22 bp downstream of Box I and overlaps the ؊35 region of the gltA promoter. Box II was found to be essential for activation of gltA but not for gltC autoregulation. Introduction of approximately one additional helical turn of DNA between Box I and Box II enhanced gltA expression 7-to 40-fold under nonactivating conditions and about 2-fold under activating conditions. Expression of gltA was dramatically decreased when the distance between Box I and Box II was altered by a nonintegral number of helical turns of DNA. gltC autorepression was abolished by most of the inserts between Box I and Box II but was augmented by adding one helical turn.Glutamate synthase is an important enzyme of nitrogen metabolism in bacterial cells. In Bacillus subtilis it is the enzyme responsible for biosynthesis of glutamate, an essential component of the major pathway for ammonia assimilation and a direct nitrogen donor for the biosynthesis of amino acids and other nitrogen-containing compounds (34). The reaction catalyzed by glutamate synthase, transfer of an amide group from glutamine to 2-ketoglutarate, places the enzyme at the intersection of intracellular carbon and nitrogen metabolism.In some gram-negative bacteria, expression of glutamate synthase genes is regulated positively by the leucine-responsive protein, LRP (10), or negatively by the Nac protein (3); it may also be affected by the cyclic AMP (cAMP) receptor proteincAMP complex (26). In none of these cases is the complete pattern of glutamate synthase expression understood, however.In B. subtilis, transcription of the gltA and gltB genes, encoding the large and small subunits of glutamate synthase, respectively, is subject to nitrogen-source-dependent positive regulation by the product of the gltC gene (5). GltC protein, like the Nac protein of Klebsiella aerogenes (35), belongs to the LysR family of bacterial regulators of transcription. As is the case for many members of this family (with the notable exception of Nac), GltC is encoded just upstream of and in the opposite orientation to its target genes, gltA and gltB. In addition to stimulating transcription of gltAB, GltC represses transcription of its own gene (5). Such negative autoregulation is a feature shared by many proteins of the LysR family (31).The present study was undertaken to characterize the overlapping gltA and gltC promoter regions and to identify the DNA sequences involved in regulation of these genes. MATERIALS AN...

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“…For two of the genes, metE and metH, the MetR binding sites required for activation were defined genetically and biochemically and lie just upstream of the RNA polymerase binding site (3,22,38,42). It is possible that the MetR-mediated activation of the metE and metH genes involves a direct interaction of MetR with RNA polymerase, although there is no genetic or biochemical evidence to support this model.…”

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confidence: 99%

The glutamic acid residue at amino acid 261 of the alpha subunit is a determinant of the intrinsic efficiency of RNA polymerase at the metE core promoter in Escherichia coli

1996

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A mutation in the rpoA gene (which encodes the ␣ subunit of RNA polymerase) that changed the glutamic acid codon at position 261 to a lysine codon decreased the level of expression of a metE-lacZ fusion 10-fold; this decrease was independent of the MetR-mediated activation of metE-lacZ. Glutamine and alanine substitutions at this position are also metE-lacZ down mutations, suggesting that the glutamic acid residue at position 261 is essential for metE expression. In vitro transcription assays with RNA polymerase carrying the lysine residue at codon 261 indicated that the decreased level of metE-lacZ expression was not due to a failure of the mutant polymerase to respond to any other trans-acting factors, and a deletion analysis using a metE-lacZ gene fusion suggested that there is no specific cis-acting sequence upstream of the ؊35 region of the metE promoter that interacts with the ␣ subunit. Our data indicate that the glutamic acid at position 261 in the ␣ subunit of RNA polymerase influences the intrinsic ability of the enzyme to transcribe the metE core promoter.The efficiency with which transcription is initiated by RNA polymerase depends on the intrinsic strength of the promoter as well as on the influence of trans-acting regulatory factors. Positive control during transcription initiation is a common mechanism for the regulation of gene expression (23,28). Although a large number of trans-acting regulatory proteins and their binding sites on DNA have been identified (25, 28), relatively little is known about their mechanisms of action. The involvement of direct protein-protein contacts between RNA polymerase and transcription factors has been proposed to explain transcriptional activation (27, 29), though we have limited knowledge of the mechanism involved or the sites of contact, especially in RNA polymerase. Recently, several groups have reported that the sites of interaction of a number of activators with RNA polymerase are localized in its ␣ subunit, which is encoded by the rpoA gene, specifically in the carboxy-terminal region (13-16, 32, 34, 43). Studies indicate that the amino-terminal two-thirds of the ␣ subunit is sufficient for the formation of active enzyme molecules (9,11,12). More recently there have been reports that the ␣ subunit may also make contacts with DNA to activate transcription (1, 30). An AT-rich sequence located upstream of the Ϫ35 region of the Escherichia coli rRNA promoter rrnB P1 stimulates transcription in the absence of any accessory proteins, and mutations in the carboxy-terminal region of the ␣ subunit prevent this stimulation (30).The DNA-binding protein MetR belongs to the LysR family of bacterial activator proteins (10, 31) and is required for the activation of a number of methionine biosynthetic genes in E. coli and Salmonella typhimurium (4,8,21,22,39). For two of the genes, metE and metH, the MetR binding sites required for activation were defined genetically and biochemically and lie just upstream of the RNA polymerase binding site (3,22,38,42). It is possible that...

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Characterization of a second MetR-binding site in the metE metR regulatory region of Salmonella typhimurium

Cited by 16 publications

References 24 publications

Global effects of homocysteine on transcription in Escherichia coli: induction of the gene for the major cold-shock protein, CspA

Global effects of homocysteine on transcription in Escherichia coli: induction of the gene for the major cold-shock protein, CspA

Sites required for GltC-dependent regulation of Bacillus subtilis glutamate synthase expression

The glutamic acid residue at amino acid 261 of the alpha subunit is a determinant of the intrinsic efficiency of RNA polymerase at the metE core promoter in Escherichia coli

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