In vitro transcription from the Escherichia coli ilvIH promoter

Willins, D A; Calvo, Joseph M.

doi:10.1128/jb.174.23.7648-7655.1992

Cited by 22 publications

(17 citation statements)

References 19 publications

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“…Analyses of some of these sites by mutagenesis have provided evidence that they function in vivo and suggest that Lrp binding to promoter regions directly alters operon activity (9,25,30,31). This conclusion was strengthened by results which showed that the positive action of Lrp on ilvIH expression in vivo could be duplicated in a purified in vitro transcription system (35).…”

supporting

confidence: 61%

Lrp is a direct repressor of the dad operon in Escherichia coli

1996

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Expression of the degradative D-amino acid dehydrogenase (dad) operon is known to be increased when Escherichia coli is grown in the presence of D-or L-alanine. Alanine is thought to act as an inducer to block the action of a postulated repressor. This operon is also believed to be regulated by catabolite repression. We have used in vivo and in vitro experiments that show that the dad repressor is the leucine-responsive regulatory protein (Lrp). dad expression in a dad-lacZ operon fusion strain was increased four-to sevenfold when cells were grown in minimal medium containing alanine or leucine. A strain lacking Lrp had high-level constitutive dad expression. Gel retardation and footprinting studies revealed that Lrp binds in vitro to multiple sites over a large area in the dad promoter region. This binding was reduced by alanine or leucine. In vitro transcription assays, using a plasmid template and primer extension analysis, identified three major dad transcripts (Tr1, Tr2, and Tr3). The formation of these transcripts was differentially regulated by cyclic AMP-cyclic AMP receptor protein complex, and each was strongly repressed by Lrp. Alanine or leucine completely (for Tr1 and Tr2) or partially (for Tr3) reversed Lrp inhibition. Site-directed mutagenesis of an Lrp binding site strongly reduced Lrp binding and prevented Lrp repression of dad transcription in vivo and in vitro. Taken together, these results strongly suggest that Lrp and alanine or leucine act directly to repress and induce, respectively, transcription of the dad operon.The D-amino acid dehydrogenase (dad) operon is required by Escherichia coli for growth on L-or D-alanine as its sole carbon and energy source (33). This operon contains two structural genes, dadA, which codes for one subunit of D-amino acid dehydrogenase, and dadX, which encodes the catabolic alanine racemase (32). The racemase converts L-alanine to the D isomer, which is then oxidatively deaminated by the D-amino acid dehydrogenase to pyruvate and ammonia (6).Expression of dad is increased when either L-or D-alanine is present in the growth medium (34). However, L-alanine is thought to be the actual activator because D-alanine does not increase dad expression in strains that lack alanine racemase activity due to mutations in dadX (32). It has been suggested that L-alanine acts as an inducer by antagonizing a putative dad repressor (15). This operon has also been reported to be positively regulated by cyclic AMP-cyclic AMP receptor protein complex (cyclic AMP-CRP) (6). Consistent with this is a recent report showing that cyclic AMP-CRP binds in vitro to two areas in the dad promoter region (18).The leucine-responsive regulatory protein (Lrp) is a regulatory factor that controls, either positively or negatively, the expression of many genes, including numerous operons involved in amino acid metabolism (reviewed in references 3 and 21). In most cases, Lrp regulation of gene activity either requires or is antagonized by leucine. In some systems, alanine as well as leucine alters Lrp...

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supporting

confidence: 61%

Lrp is a direct repressor of the dad operon in Escherichia coli

1996

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“…lrp is in some cases a positive regulator and in others a negative regulator; in some cases leucine is required for action by Lrp, and in other cases leucine is not required. The best-characterized function of Lrp is as the positive transcriptional activator of the ilvIH operon of E. coli (32,39,40). In the absence of leucine in the medium, Lrp stimulates transcription of the ilvIH operon, whereas exogenous leucine results in repression of the ilvIH operon.…”

Section: Resultsmentioning

confidence: 99%

The bkdR gene of Pseudomonas putida is required for expression of the bkd operon and encodes a protein related to Lrp of Escherichia coli

1993

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Branched-chain keto acid dehydrogenase is a multienzyme complex which is required for the metabolism of the branched-chain amino acids in Pseudomonas putida. The Branched-chain keto acid dehydrogenase is an enzyme which is common to the metabolism of valine, leucine, and isoleucine in Pseudomonas putida (7) and eukaryotes (10,30). It catalyzes the oxidative decarboxylation of branchedchain keto acids formed by transamination of branchedchain amino acids. The reaction with 2-ketoisovalerate, formed from valine, is 2-ketoisovalerate + NAD+ + CoASH -> isobutyryl coenzyme A + CO2 + NADH + H'. The enzyme has been characterized from several sources, including P. putida (36), P. aeruginosa (24), bovine kidney (31), rabbit liver (29), rat kidney (27), and Bacillus subtilis (19). In the latter organism, it functions as a combined pyruvate and branched-chain keto acid dehydrogenase. Branched-chain keto acid dehydrogenase is a multienzyme complex which is composed of four proteins, Elao, E1l3, E2, and E3. The Elac4 component is the dehydrogenase and decarboxylase, the E2 component catalyzes transacylation between its lipoyl residue and coenzyme A, and the E3 component is lipoamide dehydrogenase, which catalyzes the oxidation of the lipoyl residue of E2. The structure of keto acid dehydrogenase complexes has been reviewed elsewhere (10,23,30

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“…(20) Primer extension analysis. Primer extension reactions employing reverse transcriptase were performed as described previously (31), using RNA isolated from strain CV1401 as described previously (2) (25).…”

Section: Methodsmentioning

confidence: 99%

“…In vitro, Lrp binds in a sequence-specific fashion to multiple sites upstream of the ilvIH promoters from E. coli and Salmonella typhimurium (28,30) and bends DNA upon binding (29). Binding of Lrp to these sites is both necessary and sufficient for transcriptional activation of the ilvIH operon (31). Although much has been learned recently about expression of operons regulated by Lrp, less is known about the regulation of the lrp operon itself.…”

mentioning

confidence: 99%

Regulation of the Escherichia coli lrp gene

Wang

Friedberg

et al. 1994

J Bacteriol

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Lrp (leucine-responsive regulatory protein) is a major Escherichia coli regulatory protein which regulates expression of a number of operons, some negatively and some positively. Operons that are affected by the presence or absence of Lrp includefanABC (3), gcv (10), ginA (5), gltBD (5), ilvIH (20), livJ and livK (7), lysU (6, 11), ompC and ompF (5), oppABCDF (1), papBA (3), sdaA (12), serA (12, 21), and tdh (12, 21). Some of these operons (e.g., ilvIH) are known to be directly controlled by Lrp, whereas others (e.g., ginA) are affected only indirectly by Lrp. Other operons that are affected by Lrp were identified by a plac Mu mutational analysis and by two-dimensional gel electrophoresis (5, 10). Many operons under control of Lrp are also subject to control by leucine. A striking feature of the Lrp regulon is the variety of ways that leucine and Lrp interact to regulate gene expression (16). Of the operons activated by Lrp, in some cases the activation requires leucine, in some cases the activation is negated by leucine, and in other cases the activation is independent of leucine. Similarly, for operons which are negatively regulated by Lrp, the same three subcategories have been observed: leucine negates the effect, leucine is required for the effect, and leucine has no effect. The molecular mechanisms underlying these six different patterns of regulation involving Lrp and leucine are only partially understood.Lrp, a dimer containing two identical subunits of molecular mass 18.8 kDa, is present in E. coli at a level of about 3,000 molecules per cell (32). Its amino acid sequence is evolutionarily related to that of AsnC, a regulatory protein that controls

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In vitro transcription from the Escherichia coli ilvIH promoter

Cited by 22 publications

References 19 publications

Lrp is a direct repressor of the dad operon in Escherichia coli

Lrp is a direct repressor of the dad operon in Escherichia coli

The bkdR gene of Pseudomonas putida is required for expression of the bkd operon and encodes a protein related to Lrp of Escherichia coli

Regulation of the Escherichia coli lrp gene

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