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)

Based on the integral role that argininosuccinate synthase (AS) plays in the production of nitric oxide in vascular endothelial cells and urea in liver, an analysis was carried out to determine whether signals reside in the AS mRNA to account for tissue differences in AS function and location. Reverse transcriptase-PCR and sequence analysis showed that the AS mRNA coding region was the same for both endothelial cells and liver; however, 5'-RACE analysis (rapid amplification of cDNA ends) identified AS mRNA species in endothelial cells in addition to a major 43-nucleotide (nt) 5'-untranslated region (UTR) AS mRNA with overlapping extended 5'-UTRs of 66 and 92 nt. Comparison to the genomic sequence immediately upstream of the reported transcription start site for the human and mouse AS gene suggested that expression of all three species of bovine endothelial AS mRNA are driven by a common promoter and that 5'-UTR diversity in endothelial cells results from three transcriptional initiation sites within exon 1. RNase protection analysis and real-time reverse transcriptase-PCR verified and quantitated the differential expression of the extended 5'-UTR species relative to the major 43-nt 5'-UTR AS mRNA. In vitro translation studies showed a less pronounced but similar discordant expression. Sequential deletions starting from the 5' terminus of the 92-nt 5'-UTR construct resulted in a corresponding increase in translational efficiency, but the most pronounced effect resulted from mutation of an upstream open reading frame, which restored translational efficiency of the 92-nt 5'-UTR AS mRNA. When the different AS mRNA 5'-UTRs, cloned in front of a luciferase reporter gene, were transfected into endothelial cells, the pattern of luciferase expression was nearly identical to that observed for the different 5'-UTR AS mRNAs in endothelial cells. Given the different roles ascribed for argininosuccinate synthase, urea versus NO production, these results suggest that sequence in the AS gene represented by position -92 to -43 nt from the translation start site in the extended AS mRNA 5'-UTRs plays an important role in differential and tissue-specific expression.
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PMID:Endothelial argininosuccinate synthase mRNA 5'-untranslated region diversity. Infrastructure for tissue-specific expression. 1196 59

Attenuated Salmonella strains with defined gene deletions have been extensively evaluated as suitable live carriers of passenger antigens. A number of strategies for antigen delivery by these strains have been attempted, ranging from plasmid-based to chromosomal integration systems. We report here the chromosomal integration of the T7 RNA polymerase gene (T7pol) in the attenuated strain Salmonella enterica serovar Typhi (Salmonella typhi) CVD908 (aroC(-), aroD(-)). The T7pol gene was amplified by PCR from Escherichia coli BL21(DE3) and cloned in the pNir3 plasmid under the control of the anaerobically inducible nirB promoter. Then it was subcloned in a pKTN701 derivative, suicide plasmid with the R6K ori, and flanked by the aroC gene. After evaluation of its functionality in E. coli SY327, the aroC-T7pol-aroC cassette was integrated into the aroC locus of S. typhi CVD908 by homologous recombination. The resulting strain, S. typhi CVD908-T7pol, was able to transcomplement two plasmids bearing the luc or the lacZ reporter genes controlled by the T7 promoter and produce luciferase and beta-galactosidase under anaerobic culture conditions. Therefore, an inducible system for recombinant antigen production in attenuated S. typhi was achieved.
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PMID:Attenuated Salmonella enterica serovar typhi live vector with inducible chromosomal expression of the T7 RNA polymerase and its evaluation with reporter genes. 1198 32

Eukaryotic RNA polymerase II and Escherichia coli RNA polymerase possess an intrinsic ribonuclease activity that is stimulated by the polymerase-binding proteins SII and GreB, respectively. This factor-activated hydrolysis of nascent RNA has been postulated to be involved in transcription elongation as well as removal of incorrect bases misincorporated into RNA. Little is known about the frequency of misincorporation by RNA polymerases in vivo or about the mechanisms involved in improving RNA polymerase accuracy. Here we have developed a luciferase reporter system in an effort to assay for base misincorporation in living Saccharomyces cerevisiae. The assay employs a luciferase open reading frame that contains a premature stop codon. The inactive truncated enzyme would become active if misincorporation by RNA polymerase II took place at the stop triplet. Yeast lacking SII did not display a significant change in reporter activity when compared with wild-type cells. We estimate that under our assay conditions, mRNAs with a misincorporation at the test site could not exceed 1 transcript per 500 cells. The reporter assay was very effective in detecting the previously described process of nonsense suppression (translational read-through) by ribosomes, making it difficult to determine an absolute level of basal (SII-independent) misincorporation by RNA polymerase II. Although these data cannot exclude the possibility that SII is involved in proofreading, they make it unlikely that such a contribution is physiologically significant, especially relative to the high frequency of translational errors.
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PMID:Use of an in vivo reporter assay to test for transcriptional and translational fidelity in yeast. 1200 89

During evolution the promoter elements from prokaryotes and eukaryotes have developed differently with regard to their sequence and structure, implying that in general a transfer of eukaryotic promoter sequences into prokaryotes will not cause an efficient gene expression. However, there have been reports on the functionality of the 35S promoter from cauliflower mosaic virus (CaMV) in bacteria. We therefore decided to experimentally investigate the capability of plant promoter sequences to direct gene expression in various bacteria. Accordingly, we tested ten different plant-specific promoters from Solanum tuberosum, Nicotiana tabacum, CaMV, Agrobacterium tumefaciens, and A. rhizogenes for their ability to initiate transcription in five different eubacterial species (Escherichia coli, Yersinia enterocolitica, A. tumefaciens, Pseudomonas putida, and Acinetobacter sp. BD413). To monitor the strength of the plant-specific promoters in bacteria we created fusions between these promoters and the coding region of the luciferase genes from Vibrio harveyi and measured the luminescence in the bacteria. Heterologous gene expression was observed in 50% of the combinations analysed. We then mapped the transcription start site caused by one of the plant-specific promoters, the ST-LS1 promoter from S. tuberosum, in these bacterial species. The location of the mapped transcription start site indicated that the sequences of the plant promoter themselves were recognised by the bacterial transcription apparatus. The recognition of plant-specific promoter sequences by the bacterial RNA polymerase was further confirmed by site-directed mutagenesis of the ST-LS1 promoter and the analysis of the effects of the mutations on the strength of gene expression in E. coli. Using these mutants in our reporter assays we could localise the sequences of the ST-LS1 promoter serving as -10 region in E. coli. The results of our study show that promoter sequences are much less specific than is generally assumed. This is of great importance for our knowledge about the evolution of gene expression systems and for the construction of optimised expression vectors.
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PMID:Plant-specific promoter sequences carry elements that are recognised by the eubacterial transcription machinery. 1211 61

Phosphodiesterase 4D (PDE4D), part of the complex cAMP-specific PDE4 family, plays a pivotal role in the regulation of airway smooth muscle relaxation by catalyzing the hydolysis of cAMP. Its gene on chromosome 5q12 encodes 5 splice variants, which show tissue-dependent expression and regulation. The genomic arrangement of PDE4D was determined using in silico methods, and a putative promoter of one of the protein kinase A-activated, long isoforms, PDE4D5 was identified. Promoter-luciferase constructs, transiently transfected into a beta(2) adrenoreceptor-expressing CHO-K1 cell line, were used to demonstrate that the PDE4D5 promoter up-regulated reporter gene expression in response to increased cell cAMP. Site-directed mutagenesis of the cAMP-response element (CRE) at position -201 identified this as the principal component of the mechanism underlying this cAMP responsiveness. In the second part of this study, cAMP-dependent induction of PDE4D5 transcript in primary cultured human airway smooth muscle cells (hASMs) was demonstrated using both qualitative reverse-transcriptase PCR and quantitative real-time PCR. Isolated PDE4D5 isoenzyme activity, measured after selective immunoprecipitation from hASMs, confirmed that this increase in expression led to an up-regulation of functional activity. We present evidence for cAMP-driven PDE4D5 up-regulation in hASMs and suggest a CRE-containing, isoform-specific promoter as the primary mechanism.
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PMID:Cyclic AMP-dependent transcriptional up-regulation of phosphodiesterase 4D5 in human airway smooth muscle cells. Identification and characterization of a novel PDE4D5 promoter. 1212 97

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

Cold, hyperosmolarity, and abscisic acid (ABA) signaling induce RD29A expression, which is an indicator of the plant stress adaptation response. Two nonallelic Arabidopsis thaliana (ecotype C24) T-DNA insertional mutations, cpl1 and cpl3, were identified based on hyperinduction of RD29A expression that was monitored by using the luciferase (LUC) reporter gene (RD29ALUC) imaging system. Genetic linkage analysis and complementation data established that the recessive cpl1 and cpl3 mutations are caused by T-DNA insertions in AtCPL1 (Arabidopsis C-terminal domain phosphatase-like) and AtCPL3, respectively. Gel assays using recombinant AtCPL1 and AtCPL3 detected innate phosphatase activity like other members of the phylogenetically conserved family that dephosphorylate the C-terminal domain of RNA polymerase II (RNAP II). cpl1 mutation causes RD29ALUC hyperexpression and transcript accumulation in response to cold, ABA, and NaCl treatments, whereas the cpl3 mutation mediates hyperresponsiveness only to ABA. Northern analysis confirmed that LUC transcript accumulation also occurs in response to these stimuli. cpl1 plants accumulate biomass more rapidly and exhibit delayed flowering relative to wild type whereas cpl3 plants grow more slowly and flower earlier than wild-type plants. Hence AtCPL1 and AtCPL3 are negative regulators of stress responsive gene transcription and modulators of growth and development. These results suggest that C-terminal domain phosphatase regulation of RNAP II phosphorylation status is a focal control point of complex processes like plant stress responses and development. AtCPL family members apparently have both unique and overlapping transcriptional regulatory functions that differentiate the signal output that determines the plant response.
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PMID:C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development. 1214 34

Translation initiation in many eukaryotic mRNAs is modulated by an interaction between the cap binding protein complex, bound to the 5' end of the mRNA, and the polyadenosine binding protein, bound to the 3'-terminal polyadenosine sequences. A few cellular and viral mRNAs, such as the hepatitis C virus (HCV) mRNA genome, lack 3'-terminal polyadenosine sequences. For such mRNAs, the question of whether their 3'-end sequences also regulate the initiation phase of protein synthesis via an interaction with their 5' ends has received intense scrutiny. For HCV mRNA, various experimental designs have led to conflicting interpretations, that the 3' end of the RNA can modulate translation initiation either in a positive or in a negative fashion. To examine the possibility of end-to-end communication in HCV in detail, mRNAs containing the HCV internal ribosome entry site linked to a luciferase coding region, followed by different 3' noncoding regions, were expressed in the cytoplasm of cultured cells by T7 RNA polymerase. The intracellular translation efficiencies, steady-state levels, stabilities, and 3'-end sequences of these chimeric RNAs were examined. It was found that the HCV 3' noncoding region modulates neither the translation nor the stability of the mRNAs. Thus, there is no detectable end-to-end communication in cytoplasmically expressed chimeric mRNAs containing the HCV noncoding regions. However, it remains an open question whether end-to-end communication occurs in full-length HCV mRNAs in the infected liver.
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PMID:Cytoplasmic expression of mRNAs containing the internal ribosome entry site and 3' noncoding region of hepatitis C virus: effects of the 3' leader on mRNA translation and mRNA stability. 1243 71

Enzymes are used widely as labels in binding assays for protein analytes, because they provide signal amplification. Efforts at improving the assay sensitivity have been focused mainly on the synthesis of novel substrates, e.g. fluorogenic and chemiluminogenic ones. We report the investigation of T7 RNA polymerase (T7RP) as a label with unique characteristics for antigen quantification. In an in vitro, coupled (one-step) transcription/translation reaction, T7RP catalyzes the expression of an enzyme-coding DNA template to produce free enzyme (luciferase) in solution. We demonstrate that the generated luciferase is linearly related to the input T7RP in a range covering over four orders of magnitude. It is also shown that T7RP exhibits a significant level of self-replication (100-fold) in vitro by acting on a DNA template comprising the T7RP cDNA downstream of a T7 promoter. By combining the self-replication reaction with the expression of luciferase DNA, as low as 1400 T7RP molecules are detectable. Furthermore, the T7RP is biotinylated, complexed with streptavidin and used for antigen quantification in a microtiter well-based assay with high sensitivity and reproducibility.
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PMID:T7 RNA polymerase as a self-replicating label for antigen quantification. 1249 Jul 31

A specific t(21;22) chromosomal translocation creates the chimeric EWS/ERG gene in some cases of Ewing's sarcoma. In the resultant EWS/ERG fusion protein, the N-terminal part of the ETS family protein ERG is replaced by the N terminus of the RNA-binding protein EWS. We found that both the EWS/ERG and COL11A2 genes are expressed in the Ewing's sarcoma cell line, CADO-ES1. To investigate a potential role for EWS/ERG in COL11A2 gene expression, we characterized the COL11A2 promoter and tested the ability of wild-type ERG and EWS/ERG sarcoma fusion protein to transactivate COL11A2 promoter using a luciferase assay. We found that expression of EWS/ERG, but not wild-type ERG, transactivated the COL11A2 promoter and that this transactivation required not only the N-terminal region of EWS but also an intact DNA-binding domain from ERG. Electrophoretic mobility shift assay using COL11A2 promoter sequence showed involvement of EWS/ERG in the formation of DNA-protein complexes, and chromatin immunoprecipitation assay revealed direct interaction between COL11A2 promoter and EWS/ERG fusion protein in vivo. EWS/ERG, but not wild-type ERG, bound to RNA polymerase II. Treatment of cells with the histone deacetylase inhibitor trichostatin A enabled ERG to transactivate the COL11A2 promoter, therefore abolishing the differential effects of EWS/ERG and ERG. Taken together, these findings indicate that the COL11A2 gene is regulated both by potential ERG association with a histone deacetylase complex and by direct EWS/ERG recruitment of RNA polymerase II.
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PMID:COL11A2 collagen gene transcription is differentially regulated by EWS/ERG sarcoma fusion protein and wild-type ERG. 1255 43


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