Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

When nutrients become limiting, many bacteria differentiate and become resistant to environmental stresses. For Escherichia coli, this process is mediated by the sigma s subunit of RNA polymerase. Expression of sigma s was induced by homoserine lactone, a metabolite synthesized from intermediates in threonine biosynthesis. Homoserine lactone-dependent synthesis of sigma s was prevented by overexpression of a newly identified protein, RspA. The function of homoserine lactone derivatives in many cell density-dependent phenomena and the similarity of RspA to a Streptomyces ambofaciens protein suggest that synthesis of homoserine lactone may be a general signal of starvation.
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PMID:Sensing starvation: a homoserine lactone--dependent signaling pathway in Escherichia coli. 754 40

Conjugation of the Agrobacterium Ti plasmid pTiC58 is regulated by a hierarchy involving induction by the opines agrocinopines A and B and a quorum-sensing system. Regulation by the opines is mediated by the repressor AccR, while quorum sensing is effected by the transcriptional activator TraR and its ligand, the acyl-homoserine lactone signal molecule Agrobacterium autoinducer (AAI). These last two elements combine to activate expression of the tra system at high population densities. Sequence analysis indicated that traR is the fourth gene of an operon, which we named arc, that is transcribed divergently from accR. Complementation analysis of mutations in the genes 5' to traR showed that the other members of the arc operon are not required for conjugation. Analysis of lacZ reporter fusions demonstrated that traR expression is regulated directly by AccR. Deletion analysis showed that AccR-regulated expression of traR initiates from a promoter located in the intergenic region between accR and orfA, the first gene of the arc operon. Reverse transcriptase-polymerase chain reaction (RT-PCR) and primer extension analyses indicated that the arc transcript initiates upstream of orfA and proceeds uninterrupted through traR. These results are consistent with a model in which quorum sensing is subordinate to the opine regulon because traR has become associated with an operon controlled by the opine-responsive transcriptional regulator.
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PMID:Hierarchical gene regulatory systems arising from fortuitous gene associations: controlling quorum sensing by the opine regulon in Agrobacterium. 1036 9

The Vibrio fischeri luminescence (lux) operon is regulated by a quorum-sensing system that involves the transcriptional activator (LuxR) and an acyl-homoserine lactone signal. Transcriptional activation requires the presence of a 20-base inverted repeat termed the lux box at a position centered 42.5 bases upstream of the transcriptional start of the lux operon. LuxR has proven difficult to study in vitro. A truncated form of LuxR has been purified, and together with sigma(70) RNA polymerase it can activate transcription of the lux operon. Both the truncated LuxR and RNA polymerase are required for binding to lux regulatory DNA in vitro. We have constructed an artificial lacZ promoter with the lux box positioned between and partially overlapping the consensus -35 and -10 hexamers of an RNA polymerase binding site. LuxR functioned as an acyl-homoserine lactone-dependent repressor at this promoter in recombinant Escherichia coli. Furthermore, multiple lux boxes on an independent replicon reduced the repressor activity of LuxR. Thus, it appears that LuxR can bind to lux boxes independently of RNA polymerase binding to the promoter region. A variety of LuxR mutant proteins were studied, and with one exception there was a correlation between function as a repressor of the artificial promoter and activation of a native lux operon. The exception was the truncated protein that had been purified and studied in vitro. This protein functioned as an activator but not as a repressor in E. coli. The data indicate that the mutual dependence of purified, truncated LuxR and RNA polymerase on each other for binding to the lux promoter is a feature specific to the truncated LuxR and that full-length LuxR by itself can bind to lux box-containing DNA.
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PMID:Conversion of the Vibrio fischeri transcriptional activator, LuxR, to a repressor. 1063 17

Luminescence in Vibrio fischeri is controlled by a population density-responsive regulatory mechanism called quorum sensing. Elements of the mechanism include: LuxI, an acyl-homoserine lactone (acyl-HSL) synthase that directs synthesis of the diffusible signal molecule, 3-oxo-hexanoyl-HSL (V. fischeri autoinducer-1, VAI-1); LuxR, a transcriptional activator protein necessary for response to VAI-1; GroEL, which is necessary for production of active LuxR; and AinS, an acyl-HSL synthase that catalyzes the synthesis of octanoyl-HSL (VAI-2). The population density-dependent accumulation of VAI-1 triggers induction of lux operon (luxICDABEG; genes for luminescence enzymes and for LuxI) transcription and luminescence by binding to LuxR, forming a complex that facilitates the association of RNA polymerase with the luxoperon promoter. VAI-2, which apparently interferes with VAI-1 binding to LuxR, operates to limit premature luxoperon induction. Hierarchical control is imposed on the system by 3':5'-cyclic AMP (cAMP) and cAMP receptor protein (CRP), which are necessary for activated expression of luxR. Several non-lux genes in V. fischeri are controlled by LuxR and VAI-1. Quorum regulation in V. fischeri serves as a model for LuxI/LuxR-type quorum sensing systems in other gram-negative bacteria.
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PMID:Quorum regulation of luminescence in Vibrio fischeri. 1094 79

The Escherichia coli mutant CWML2 was previously reported to exhibit improved physiological characteristics, including recombinant protein production. Here we investigate the molecular basis of this phenotype by comparing the cellular level of three RNA polymerase sigma subunits by immunoblot analysis. While the level of housekeeping sigma(D) was similar in parent and mutant, the levels of the flagella synthesis regulator sigma(F) and the stationary phase regulator sigma(S) were higher in the mutant strain, indicating a different motility and stationary phase phenotype. Evidence for this conclusion was provided by the significantly higher motility of CWML2, compared to its parent. Based on these results, we hypothesized that alterations in ppGpp regulation via a homoserine lactone-dependent mechanism may be relevant for the mutant phenotype. Indeed, transcription of the rspAB operon, which was previously described to be involved in the degradation of homoserine lactone, was found to be deregulated in CWML2 in a plasmid-based reporter protein assay. By overexpression of the E. coli rspAB operon, we could partly mimic the mutant phenotype and demonstrate that co-overexpression of RspAB is a pertinent metabolic engineering strategy to improve recombinant protein production.
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PMID:Co-overexpression of RspAB improves recombinant protein production in Escherichia coli. 1112 Jun 41

During quorum sensing in Vibrio fischeri, the luminescence, or lux, operon is regulated in a cell density-dependent manner by the activator LuxR in the presence of an acylated homoserine lactone autoinducer molecule [N-(3-oxohexanoyl) homoserine lactone]. LuxR, which binds to the lux operon promoter at a position centered at -42.5 relative to the transcription initiation site, is thought to function as an ambidextrous activator making multiple contacts with RNA polymerase (RNAP). The specific role of the alpha-subunit C-terminal domain (alphaCTD) of RNAP in LuxR-dependent transcriptional activation of the lux operon promoter has been investigated. The effects of 70 alanine substitution variants of the alpha subunit were determined in vivo by measuring the rate of transcription of the lux operon via luciferase assays in recombinant Escherichia coli. The mutant RNAPs from strains exhibiting at least twofold-increased or -decreased activity in comparison to the wild type were further examined by in vitro assays. Since full-length LuxR has not been purified, an autoinducer-independent N-terminally truncated form of LuxR, LuxRDeltaN, was used for in vitro studies. Single-round transcription assays were performed using reconstituted mutant RNAPs in the presence of LuxRDeltaN, and 14 alanine substitutions in the alphaCTD were identified as having negative effects on the rate of transcription from the lux operon promoter. Five of these 14 alpha variants were also involved in the mechanisms of both LuxR- and LuxRDeltaN-dependent activation in vivo. The positions of these residues lie roughly within the 265 and 287 determinants in alpha that have been identified through studies of the cyclic AMP receptor protein and its interactions with RNAP. This suggests a model where residues 262, 265, and 296 in alpha play roles in DNA recognition and residues 290 and 314 play roles in alpha-LuxR interactions at the lux operon promoter during quorum sensing.
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PMID:Role of the C-terminal domain of the alpha subunit of RNA polymerase in LuxR-dependent transcriptional activation of the lux operon during quorum sensing. 1214 22

A Tn5 mutant of Agrobacterium vitis F2/5 (M1154) differs from the wild-type strain in that it has lost its abilities to cause necrosis on grape and a hypersensitive-like response (HR) on tobacco. The Tn5 insertion occurred in an open reading frame (ORF) aviR that is homologous to genes encoding the LuxR family of transcriptional regulators, thereby suggesting that the HR and necrosis are regulated by a quorum-sensing system. Fewer N-acyl-homoserine lactone autoinducers were detected in extracts from M1154 compared with extracts from F2/5 and from aviR-complemented M1154. The complemented mutant regained full ability to cause grape necrosis and HR. Eighteen ORFs located on a 36.6-kb insert in cosmid clone CPB221, which includes aviR, were sequenced and aligned with homologous genes from A. tumefaciens C58 and Sinorhizobium meliloti Rm1021. The order of several clustered genes is conserved among the bacteria; however, rearrangements are also apparent. Reverse transcriptase-polymerase chain reaction analysis indicated that ORF2 and ORF14 may be regulated by an aviR-encoded transcriptional regulator. Single site-directed mutations in each of the ORFs, however, had no effect on expression of HR or necrosis as compared with the wild-type parent.
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PMID:A luxR homolog, aviR, in Agrobacterium vitis is associated with induction of necrosis on grape and a hypersensitive response on tobacco. 1284 31

Most LuxR homologues function as activators of transcription during the process of quorum sensing, but a few, including EsaR and ExpR(Ecc), negatively impact gene expression. The LuxR-activated luxI promoter and LuxR binding site, the lux box, were used in artificial contexts to assess the potential for transcriptional activation and DNA binding by EsaR and ExpR(Ecc). Although the acyl-homoserine lactone responsiveness of both proteins is the opposite of that shown by most LuxR family members, EsaR and ExpR(Ecc) have preserved the ability to interact with RNA polymerase and activate transcription despite their low affinity for the lux box DNA.
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PMID:The quorum sensing negative regulators EsaR and ExpR(Ecc), homologues within the LuxR family, retain the ability to function as activators of transcription. 1461 66

Quorum sensing-dependent activation of the luminescence (lux) genes of Vibrio fischeri relies on the formation of a complex between the autoinducer molecule, N-(3-oxohexanoyl)-L-homoserine lactone, and the autoinducer-dependent transcriptional activator LuxR. In its active conformation, LuxR binds to a site known as the lux box centered at position -42.5 relative to the luxI transcriptional start site and is thought to function as an ambidextrous activator capable of making multiple contacts with RNA polymerase (RNAP). The specific role of region 4 of the Escherichia coli sigma70 subunit of RNAP in LuxR-dependent activation of the luxI promoter has been investigated. Single-round transcription assays were performed in the presence of purified LuxRDeltaN, the autoinducer-independent C-terminal domain of LuxR, and a variant RNAP which contained a C-terminally truncated sigma70 subunit devoid of region 4. Results indicated that region 4 is essential for LuxRDeltaN-dependent luxI transcription, therefore 16 single and two triple alanine substitutions in region 4.2 of sigma70 between amino acid residues 590 and 613 were examined for their effects on LuxR- and LuxRDeltaN-dependent transcription at the luxI promoter. Taken together, the analyses performed on these variants of RpoD suggest that some individual residues in region 4.2 are important to the mechanism of activator-dependent transcription initiation under investigation.
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PMID:Involvement of region 4 of the sigma70 subunit of RNA polymerase in transcriptional activation of the lux operon during quorum sensing. 1463 24

The Vibrio fischeri LuxR protein is the founding member of a family of acyl-homoserine lactone-responsive quorum-sensing transcription factors. Previous genetic evidence indicates that in the presence of its quorum-sensing signal, N-(3-oxohexanoyl) homoserine lactone (3OC6-HSL), LuxR binds to lux box DNA within the promoter region of the luxI gene and activates transcription of the luxICDABEG luminescence operon. We have purified LuxR from recombinant Escherichia coli. Purified LuxR binds specifically and with high affinity to DNA containing a lux box. This binding requires addition of 3OC6-HSL to the assay reactions, presumably forming a LuxR-3OC6-HSL complex. When bound to the lux box at the luxI promoter in vitro, LuxR-3OC6-HSL enables E. coli RNA polymerase to initiate transcription from the luxI promoter. Unlike the well-characterized LuxR homolog TraR in complex with its signal (3-oxo-octanoyl-HSL), the LuxR-30C6-HSL complex can be reversibly inactivated by dilution, suggesting that 3OC6-HSL in the complex is not tightly bound and is in equilibrium with the bulk solvent. Thus, although LuxR and TraR both bind 3-oxoacyl-HSLs, the binding is qualitatively different. The differences have implications for the ways in which these proteins respond to decreases in signal concentrations or rapid drops in population density.
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PMID:Reversible acyl-homoserine lactone binding to purified Vibrio fischeri LuxR protein. 1472 87


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