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)

Vaccinia virus-dependent CAT expression was observed in virus-infected cells cotransfected with a promoterless CAT gene. Restriction endonuclease resection of the CAT plasmid indicated that expression was due to recognition by vaccinia virus RNA polymerase of sequences within the CAT gene itself, probably located within the 5' untranslated region of the gene. This observation is relevant to the design of reverse-genetic systems which use CAT as a reporter gene to detect replication of negative-strand RNA virus pseudogenomes.
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PMID:Tn9 CAT gene contains a promoter for vaccinia virus transcription: implications for reverse-genetic techniques. 131 41

We examined the effects of okadaic acid (OA), a potent and specific inhibitor of serine phosphatases 2A and 1, on the transient expression of an hsp 70 promoter-reporter gene construct in IMR-90 human diploid lung fibroblasts. We showed that OA markedly potentiated the heat-induced but not the basal expression of pHBCAT, a full-length human hsp-70-promoter-driven CAT gene construct. This effect of OA was dose and time dependent and promoter specific. Importantly, the potentiating effects of OA appeared to be independent of the binding of the activated heat shock transcription factor (HSTF) to its consensus DNA sequence, the heat shock element (HSE). Thus, OA had no effect on the HSTF DNA-binding activity as measured by mobility shift assay, and mutation of the HSE sequence did not obliterate the stimulatory effects of OA on reporter gene expression under a heat shock condition, although heat shock by itself was without effect. Analysis of the status of phosphorylation of the largest subunit of RNA polymerase II provided evidence that this effect of OA is attributable, at least in part, to the increased phosphorylation of RNA polymerase II. These results provided evidence that the heat-induced hsp 70 promoter activity is negatively regulated by serine phosphatases. We propose that the heat-induced transcriptional activation of hsps is associated with phosphorylation of component(s) of the transcription complex; one of the likely candidates being the transcriptionally engaged RNA polymerase II. OA, by inhibiting phosphatase 2A and 1 activity, enhanced this phosphorylation and potentiated the transcriptional activation of hsps.
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PMID:The heat-induced hsp 70 promoter activity is negatively regulated by serine phosphatases: evidence from the effects of okadaic acid. 133 79

The variant-specific surface glycoprotein (VSG) gene 221 of Trypanosoma brucei is transcribed as part of a 60 kb expression site (ES). We have identified the promoter controlling this multigene transcription unit by the use of 221 chromosome-enriched DNA libraries and VSG gene 221 expression site specific transcripts. The start of transcription was determined by hybridization and RNase protection analysis of nascent RNA. The 5' ends of the major transcripts coming from the initiation region map at nucleotide sequences that do not strongly resemble rRNA transcriptional starts even though the transcripts are synthesized by an RNA polymerase highly resistant to alpha-amanitin. The cloned VSG gene 221 ES transcription initiation region promotes high CAT gene expression, when reintroduced by electroporation into T. brucei. We show that the activity of this expression site is controlled at or near transcription initiation in bloodstream trypanosomes. The 221 ES is inactivated without any sequence alteration within 1.4 kb of the transcription start site. This excludes mechanisms of promoter inactivation involving DNA rearrangements in the vicinity of the transcription start site, e.g. promoter inversion or conversion.
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PMID:The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei. 169 65

We show that the ribosomal RNA (rRNA) promoter can efficiently direct expression of protein-coding genes in the parasitic protozoan Trypanosoma brucei. The rRNA promoter was characterized by: (i) point mutations at the rRNA transcription initiation site which completely abolished its promoter function in transient CAT transformation assays; (ii) the alpha-amanitin resistance of transcription of rRNA promoter-neomycin phosphotransferase (neo) genes in stably transformed trypanosomes; and (iii) the nucleolar location of neo RNA, synthesized under the control of the rRNA promoter. The rRNA promoter-derived CAT mRNA required a 3' splice acceptor site and the neo mRNA was trans-spliced and polyadenylated. In situ hybridization revealed neo RNA at the nucleolus in stably transformed trypanosomes in which rRNA promoter-neo constructs were integrated either at a rRNA locus or at a locus for the procyclic acidic repetitive protein (PARP) coding genes. We postulate that trans-splicing, by uncoupling the requirement for transcription of protein-coding genes by RNA polymerase II, allows RNA polymerase I mediated protein-coding gene transcription, presumably because a 5' cap can be transferred to the pre-mRNA by trans-splicing.
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PMID:RNA polymerase I can mediate expression of CAT and neo protein-coding genes in Trypanosoma brucei. 191 99

The sequences preceding the albumin mRNA start site are able to direct efficient transcription only upon introduction into cells expressing the endogenous albumin gene. In transient expression assays, the activity of a reporter gene (CAT) linked to this promoter is 100-fold higher in H4II differentiated hepatoma cells than in H5 dedifferentiated cells which no longer express their albumin gene. This tissue specificity depends on the very proximal promoter region, composed of a CCAAT box, the proximal element and a TATA box. Deletion of the CCAAT box leads to a two- to threefold decrease in activity, deletion of the proximal element (PE) results in loss of activity. The PE is a high-affinity binding site for HNF1/APF, a strictly liver specific trans-acting factor. When the affinity of this factor for PE is decreased by bacterial methylation (PE includes a dam methylase site), by mutation, or by its replacement with the homologous element from the alpha-fetoprotein gene (AFP), the activity of the short promoter (PE plus the TATA box) is abolished. This activity can be rescued in the presence of the more upstream elements: DEII, DEI and the CCAAT box (recognized, respectively, by the NF1/CTF, C/EBP and NFY/ACF factors) which are then absolutely required. Our results suggest that the upstream elements contribute to promoter activity by stabilizing the HNF1-PE complex and not by direct interaction with TFIID or the RNA polymerase. It is probable that these elements, essentially dispensable in already differentiated hepatoma cells, play a crucial role during development or differentiation to activate the promoter in cells that contain a low concentration of HNF1 and/or an HNF1 unable to open inactive chromatin alone.
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PMID:Anatomy of the rat albumin promoter. 218 62

Using purified sigma 55 RNA polymerase from Bacillus subtilis in an in vitro transcription system, we have shown that both promoters and terminators of Gram negative origin are recognized by this enzyme. Furthermore, when B. subtilis is transformed with a shuttle vector containing certain of these promoters, synthesis of the Staphylococcus aureus CAT protein is achieved, and levels up to 25% of the total cellular protein can be obtained. These findings indicate a closer evolutionary relationship of the expression machinery of these two bacterial species than has been assumed so far. On the basis of these results, the construction of new expression vectors for B. subtilis is likely to be facilitated, since a variety of well-characterized signal elements from Escherichia coli are available.
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PMID:Efficient utilization of Escherichia coli transcriptional signals in Bacillus subtilis. 241 70

A mutation is described that alters the promoter specificity of sigma 70, the primary sigma factor of Escherichia coli RNA polymerase. In strains carrying both the mutant and wild-type sigma gene (rpoD), the mutant sigma causes a large increase in the activity of mutant P22 ant promoters with A.T or C.G instead of the wild-type, consensus G.C base-pair at position -33, the third position of the consensus -35 hexamer 5'-TTGACA-3'. There is little or no effect on the activities of the wild-type and 23 other mutant ant promoters, including one with T.A at -33. The mutant sigma also activates E. coli lac promoters with A.T or C.G, but not T.A, at the corresponding position. The rpoD mutation (rpoD-RH588) changes a CGT codon to CAT. The corresponding change in sigma 70 is Arg588----His. This residue is in a region that is conserved among most sigma factors, a region that is also homologous with the helix-turn-helix motif of DNA-binding proteins. These results suggest that this region of sigma 70 is directly involved in recognition of the -35 hexamer.
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PMID:A mutant Escherichia coli sigma 70 subunit of RNA polymerase with altered promoter specificity. 266 27

The mobile element jockey is similar in structural organization and coding potential to the LINEs of various organisms. As demonstrated here, two polyadenylated jockey transcripts detected at different stages of Drosophila ontogenesis and in cell cultures have the same length as genomic copies of jockey and correspond to the strand containing ORFs. alpha-amanitin experiments indicate that jockey is transcribed by RNA polymerase II. Analysis of both expression of CAT constructions and initiation of transcription in jockey genomic and transfected copies has shown that jockey transcription is controlled by an internal promoter. Inward location of the promoter allows it to be preserved in the course of replication via reverse transcription and accounts for the distribution of jockey and probably other LINEs throughout the genome. This is the first case of an internal promoter described for RNA polymerase II. The comparison of sequences at the beginning of LINE elements in Drosophila allows one to detect possible core sequences.
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PMID:jockey, a mobile Drosophila element similar to mammalian LINEs, is transcribed from the internal promoter by RNA polymerase II. 284 63

Two polyadenylated transcripts of the jockey are detected at different stages of Drosophila melanogaster ontogenesis and in the cell culture. They have the same length as complete and deleted copies of jockey and correspond to the DNA strand containing open reading frames coding for polypeptides which are homologous to retroviral RNA-(DNA)-binding proteins and to their reverse transcriptases. The results of the experiments, where transcription was inhibited with alpha-amanitin in vivo, indicate that jockey is transcribed by RNA polymerase II. The analysis of expression of CAT constructions made on the basis of jockey, and the detection of a fixed site for transcription initiation in jockey genomic and transfected copies have shown that jockey transcription is controlled by an internal promoter located not farther than 12 nucleotides from the beginning of the element. Such an inward location of the promoter allows it to be preserved in replication via reverse transcription and accounts for the distribution of jockey and probably other LINEs throughout the genome. This is the case of the first internal promoter described for RNA polymerase II. The comparison of starting sequences of LINEs in Drosophila makes it possible to detect core sequences of such a promoter.
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PMID:[The Drosophila mobile element jockey, being a typical LINE, is transcribed from the internal promoter by RNA polymerase II]. 284 76

Bacillus mesentericus DNA fragments carrying promoters were cloned in Bacillus subtilis. The nucleotide sequences of four promoters, the sites of transcription initiation and the levels of promoter-mediated expression of pPL603 CAT gene at different stages of cell growth were determined. It was identified, that the two promoters (P435 and P442) are the typical sigma 43-RNA polymerase promoters. Promoter P462 possessed the unusual nucleotide sequence and induced the expression of indicator CAT gene at the middle of the stationary phase of cell growth. The next P428 promoter was found to be a complex promoter composed of three overlapping ones: P428-1, P428-2 and P428-3. P428-3 is the sigma 43-specific promoter which revealed the high degree of homology with Bacillus subtilis spoOB gene promoter. We suggest the promoters P428-1 and P428-2 to be sigma 43- and sigma 37-RNA polymerase specific, respectively.
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PMID:[Structural-functional characteristics of Bacillus mesentericus promoters cloned in Bacillus subtilis cells]. 311 Jun 6


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