Transcript Analysis of the Citrate Synthase and Succinate Dehydrogenase Genes of Escherichia coli K12

Wilde, Robin J.; Guest, John R.

doi:10.1099/00221287-132-12-3239

Cited by 30 publications

(39 citation statements)

References 40 publications

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“…When the 263 bp DNA fragment that extended from position ¹331 to ¹68 bp relative to the start of sdhC transcription was used in the DNase I footprint assay Table 1). The locations of the two putative gltA promoters are indicated by P1 and P2 (Wilde and Guest, 1986).…”

Section: Resultsmentioning

confidence: 99%

Role of multiple ArcA recognition sites in anaerobic regulation of succinate dehydrogenase (sdhCDAB ) gene expression in Escherichia coli

Shen

Gunsalus

1997

Molecular Microbiology

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SummarySuccinate dehydrogenase (sdhCDAB ) gene expression in Escherichia coli is negatively regulated by the arcA and fnr gene products during anaerobic cell growth conditions. The controlled synthesis of this sole membrane-bound enzyme of the tricarboxylic acid cycle allows optimal participation in the aerobic electron transport pathway for the generation of energy via oxidative phosphorylation reactions. To understand how ArcA participates in the anaerobic repression of sdhCDAB expression, a family of sdhC-lacZ fusions was constructed and analysed in vivo. DNase I footprint and gel shift assays using purified ArcA protein revealed the location of four distinct and independent ArcA binding sites in the sdhC promoter region. ArcA sites, designated sites 1 and 2, are centred at ¹205 bp and ¹119 bp upstream of the sdhC promoter, respectively, whereas ArcA site 3 overlaps the ¹35 and ¹10 regions of the sdhC promoter. A fourth ArcA site is centred at þ 257 bp downstream of the sdhC promoter. They are bound with differing affinity by ArcA and ArcA phosphate. The in vivo studies, in combination with the in vitro studies, indicate that ArcA site 3 is necessary and sufficient for the ArcA-dependent repression of sdhC gene expression, while the DNA region containing ArcA site 2 contributes to maximal gene expression. The DNA-containing ArcA sites 1 and 4 provide minor roles in the ArcA regulation of sdhC expression. Lastly, the Fnr-dependent control of sdhCDAB gene expression was shown to occur independently of the ArcA and to require DNA sequences near the start of sdhC transcription.

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Section: Resultsmentioning

confidence: 99%

Role of multiple ArcA recognition sites in anaerobic regulation of succinate dehydrogenase (sdhCDAB ) gene expression in Escherichia coli

Shen

Gunsalus

1997

Molecular Microbiology

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“…However, a large 10-kb transcript including sdhCDABsucABCD has been detected. The transcript starting in front of sucA terminates at the 3Ј end of sucD (13,75). Expression from the sdhC promoter is depressed by anaerobiosis, inclusion of glucose in the growth medium, and inactivation of either the crp or the cya gene.…”

Section: Resultsmentioning

confidence: 99%

Functional Interactions between the Carbon and Iron Utilization Regulators, Crp and Fur, in Escherichia coli

et al. 2005

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In Escherichia coli, the ferric uptake regulator (Fur) controls expression of the iron regulon in response to iron availability while the cyclic AMP receptor protein (Crp) regulates expression of the carbon regulon in response to carbon availability. We here identify genes subject to significant changes in expression level in response to the loss of both Fur and Crp. Many iron transport genes and several carbon metabolic genes are subject to dual control, being repressed by the loss of Crp and activated by the loss of Fur. However, the sodB gene, encoding superoxide dismutase, and the aceBAK operon, encoding the glyoxalate shunt enzymes, show the opposite responses, being activated by the loss of Crp and repressed by the loss of Fur. Several other genes including the sdhA-D, sucA-D, and fumA genes, encoding key constituents of the Krebs cycle, proved to be repressed by the loss of both transcription factors. Finally, the loss of both Crp and Fur activated a heterogeneous group of genes under S control encoding, for example, the cyclopropane fatty acid synthase, Cfa, the glycogen synthesis protein, GlgS, the 30S ribosomal protein, S22, and the mechanosensitive channel protein, YggB. Many genes appeared to be regulated by the two transcription factors in an apparently additive fashion, but apparent positive or negative cooperativity characterized several putative Crp/Fur interactions. Relevant published data were evaluated, putative Crp and Fur binding sites were identified, and representative results were confirmed by real-time PCR. Molecular explanations for some, but not all, of these effects are provided.Processes that alter the rates of transcriptional initiation, elongation and termination, mRNA degradation, and mRNA translation control gene expression in all living organisms. Of these, regulation of transcriptional initiation is of greatest physiological importance in bacteria (34). About one-fourth of all Escherichia coli proteins interact functionally with nucleic acids, and about one-fourth of these are pleiotropic transcriptional regulators that control expression of multioperon regulons, primarily by influencing the transcriptional initiation step (57, 72). In fact, each regulon is defined by the activity of a transcription factor that coordinately controls expression of the operons included within that regulon (28). The global regulator, responsive to select cellular stimuli, binds to specific sites in the control regions of these operons. Examples of such pleiotropic transcription factors in E. coli include Crp, a primary sensor of carbon availability (8,15,23,65), NtrBC, a sensor of nitrogen availability (32, 63, 77), CysB, the sensor of sulfur availability (38,39,41,42), and Fur, a dominant sensor of iron availability (30,31,50,71). Crp when complexed with cyclic AMP (cAMP) binds to a consensus sequence in the DNA and activates transcription of many genes while repressing transcription of a few others. Some genes are subject to both positive and negative regulation by the cAMP-Crp complex due to th...

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“…1). Two of these operons, sdhCDAB and gltA, are constitutively expressed (24,25). Transcription of the remaining eight operons is activated by the following environmental conditions: umuDC, recN, and lexA-dinF by DNAdamage (26 -28); melAB by addition of isopropyl ␤-Dthiogalactoside and melibiose (29,30); lacZYA by isopropyl ␤-D-thiogalactoside (30); malEFG by maltose (31); and araBAD and araE by arabinose (32).…”

Section: Rnap Associates Preferentially With the Promoter-proximal Rementioning

confidence: 99%

Association of RNA polymerase with transcribed regions in Escherichia coli

Wade

Struhl

2004

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

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We examine the association of the ␤-, ␣-, and 70 -subunits of Escherichia coli RNA polymerase (RNAP) and the NusA elongation factor with transcribed regions in vivo by using chromatin immunoprecipitation. RNAP preferentially associates with the promoterproximal region of several operons, and this preference is particularly pronounced at the lexA-dinF promoter. When cells are grown in exponential phase, little or no 70 is associated with RNAP during early elongation. However, during stationary phase, 70 is retained in a fraction of elongating RNAP complexes throughout the melAB operon. In contrast, 70 is not observed in elongating RNAP complexes at the lacZYA operon during stationary phase. At both operons, NusA associates with RNAP during early elongation, and this association is greatly reduced during stationary phase. These observations suggest that in vivo association of 70 and NusA with elongating RNAP is regulated by growth conditions. transcription ͉ 70 ͉ NusA ͉ chromatin immunoprecipitation T he molecular understanding of transcriptional regulatory mechanisms in prokaryotes is well advanced, particularly in comparison to the understanding in eukaryotes. The association of RNA polymerase (RNAP) and associated proteins with transcribed regions of DNA has been studied extensively in vitro, and these biochemical experiments have provided great insight into the mechanisms of transcriptional initiation, elongation, and termination. In addition, there has been a great deal of genetic analysis, in which the transcriptional properties of mutant proteins and promoters have been assessed under various environmental conditions. Importantly, transcription in vitro is very efficient, occurring at rates comparable to those in living cells, and it faithfully mimics many aspects of transcription in vivo as defined by genetic analysis. However, there is very little work analyzing the association of RNAP and auxiliary proteins with transcribed regions in vivo.Escherichia coli RNA polymerase consists of five subunits: ␣ 2␤␤ Ј, but this core enzyme is unable to recognize promoters and accurately initiate transcription. RNAP associates with a number of accessory proteins during transcriptional initiation, elongation, and termination. During transcriptional initiation, core RNAP associates with a -subunit to form an RNAP holoenzyme. There are seven -subunits in E. coli, and each -subunit allows the RNAP holoenzyme to recognize a specific sequence at a subset of promoters (1). The predominant factor during exponential growth in rich media is 70 . For many years, it was widely believed that the transition to transcription elongation caused all 70 to be released from RNAP. A number of biochemical studies showed that ␤ and 70 could be purified from initiating but not from elongating RNAP complexes (2-5). These data are consistent with structural studies of RNAP that suggest would be displaced from RNAP holoenzyme by RNA products of 12-14 nt in length (6). However, more recent in vitro studies suggest that 70 can remain associated...

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Transcript Analysis of the Citrate Synthase and Succinate Dehydrogenase Genes of Escherichia coli K12

Cited by 30 publications

References 40 publications

Role of multiple ArcA recognition sites in anaerobic regulation of succinate dehydrogenase (sdhCDAB ) gene expression in Escherichia coli

Role of multiple ArcA recognition sites in anaerobic regulation of succinate dehydrogenase (sdhCDAB ) gene expression in Escherichia coli

Functional Interactions between the Carbon and Iron Utilization Regulators, Crp and Fur, in Escherichia coli

Association of RNA polymerase with transcribed regions in Escherichia coli

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