A mutation in the rpoA gene encoding the alpha subunit of RNA polymerase that affects metE-metR transcription in Escherichia coli

Jafri, Samina; Urbanowski, Mark L.; Stauffer, George V.

doi:10.1128/jb.177.3.524-529.1995

Cited by 27 publications

(22 citation statements)

References 46 publications

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“…We have, however, excluded a possible role of cyclic AMP receptor protein in mediating either a direct or indirect down regulation at the metE promoter (17). Both in vivo and in vitro experiments described in this paper demonstrate that the rpoA(E261K) mutation does not identify a site of contact with any other unidentified activator protein at the metE promoter, nor is it involved in an interaction with a possible DNA element upstream of the metE Ϫ35 promoter sequence.…”

mentioning

confidence: 82%

“…We previously reported that the rpoA(E261K) mutation results in at least six phenotypic changes from the wild-type phenotype (Table 2), including an inability to grow on L agar plates at 42ЊC when the NaCl concentration was reduced from 1 to 0.5% (17). In a search for mutants that could suppress the various phenotypes, a mutant that was no longer sensitive to low salt concentrations at high temperature was isolated.…”

Section: Resultsmentioning

confidence: 99%

“…We addressed this problem using an in vitro transcription assay employing purified wild-type RNA polymerase or the mutant polymerase with the ␣ E261K substitution. Two DNA fragments were used as templates, one carry- (17). Presumably this increase in the level of metE expression is sufficient to allow growth on GM without methionine.…”

Section: Resultsmentioning

confidence: 99%

“…Chromosomal DNA was isolated from E. coli cells, and the rpoA gene was amplified by PCR as described previously (17). The primers used were based on the sequence of the E. coli genes flanking rpoA.…”

Section: Methodsmentioning

confidence: 99%

“…Previously, in a search for mutants that might delineate an interaction of the MetR protein with the ␣ subunit of RNA polymerase, we isolated an rpoA mutant having a ninefold lower level of metE-lacZ expression than that of the wild type, even in a metR background (17). This mutation was shown to change a glutamic acid residue at amino acid 261 to lysine (E261K) in the C-terminal domain of the ␣ subunit of RNA polymerase.…”

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confidence: 99%

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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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confidence: 82%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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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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RNA polymerase alpha subunit binding site in positively controlled promoters: a new model for RNA polymerase-promoter interaction and transcriptional activation in the Escherichia coli ada and aidB genes.

Landini

Volkert

1995

The EMBO Journal

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The ada and aidB genes are part of the adaptive response to DNA methylation damage in Escherichia coli. Transcription of the ada and the aidB genes is triggered by binding of the methylated Ada protein (meAda) to a specific sequence located 40–60 base pairs upstream of the transcriptional start, which is internal to an A/T‐rich region. In this report we demonstrate that the Ada binding site is also a binding site for RNA polymerase. RNA polymerase is able to bind the −40 to −60 region of the ada and the aidB promoters in the absence of meAda, and its binding is mediated by the alpha subunit. This region resembles the UP element of the rrnB P1 promoter in location, sequence and mechanism of interaction with RNA polymerase. We discuss the function of UP‐like elements in positively controlled promoters and provide evidence that Ada does not act by enhancing RNA polymerase binding affinity to the promoter region. Instead, Ada stimulates transcription by modifying the nature of the RNA polymerase‐promoter interaction, allowing RNA polymerase to recognize the core promoter −35 and −10 elements in addition to the UP‐like element.

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Two-dimensional electrophoresis protein profile of the phytopathogenic fungus Botrytis cinerea

et al. 2006

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Botrytis cinerea is a phytopathogenic fungi causing disease in a number of important crops. It is considered a very complex species in which different populations seem to be adapted to different hosts. In order to characterize fungal virulence factors, a proteomic research was started. A protocol for protein extraction from mycelium tissue, with protein separation by 2-DE and MS analysis, was optimised as a first approach to defining the B. cinerea proteome. Around 400 spots were detected in 2-DE CBB-stained gels, covering the 5.4-7.7 pH and 14-85 kDa ranges. The averages of analytical and biological coefficients of variance for 64 independent spots were 16.1% and 37.5%, respectively. Twenty-two protein spots were identified by MALDI-TOF or ESI IT MS/MS, with some of them corresponding to forms of malate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Two more spots matched a cyclophilin and a protein with an unknown function.

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A mutation in the rpoA gene encoding the alpha subunit of RNA polymerase that affects metE-metR transcription in Escherichia coli

Cited by 27 publications

References 46 publications

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

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

RNA polymerase alpha subunit binding site in positively controlled promoters: a new model for RNA polymerase-promoter interaction and transcriptional activation in the Escherichia coli ada and aidB genes.

Two-dimensional electrophoresis protein profile of the phytopathogenic fungus Botrytis cinerea

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