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)

A synthetic gene coding for bovine pancreatic DNaseI has been cloned under the control of a T7 promoter present on the plasmid pET11. This construct yields a stable Escherichia coli transformant only when transcription from this promoter is tightly controlled. Production of recombinant DNaseI (reDNaseI) is achieved by infection of the cells with a mutant lambda phage, CE6, which carries the gene encoding T7 RNA polymerase. Induced bacterial cultures yield in excess of 2 mg per litre of reDNaseI after purification.
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PMID:Overproduction of the toxic protein, bovine pancreatic DNaseI, in Escherichia coli using a tightly controlled T7-promoter-based vector. 829 27

Elongation complexes of RNA polymerase II, RNA-DNA-enzyme ternary complexes, are intermediates in the synthesis of all eukaryotic mRNAs and are potential regulatory targets for factors controlling RNA chain elongation and termination. Analysis of such complexes can provide information concerning the structure of the catalytic core of the RNA polymerase and its interactions with the DNA template and RNA transcript. Knowledge of the structure of such complexes is essential in understanding the catalytic and regulatory properties of RNA polymerase. We have prepared and isolated complexes of purified RNA polymerase II halted at defined positions along a DNA template, and we have used deoxyribonuclease I (DNAse I) to map the interactions of the polymerase with the DNA template. DNAse I footprints of three specific ternary complexes reveal that the enzyme-template interactions of individual elongation complexes are not identical. The size of the protected region is distinct for each complex and varies from 48 to 55 bp between different complexes. Additionally, the positioning of the protected region relative to the active site varies in different complexes. Our results suggest that RNA polymerase II is a dynamic molecule and undergoes continual conformational transitions during elongation. These transitions are likely to be important in the processes of transcript elongation and termination and their regulation.
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PMID:Contacts between mammalian RNA polymerase II and the template DNA in a ternary elongation complex. 844 6

The NIFA protein activates transcription of nitrogen fixation (nif) operons by the sigma 54-holoenzyme form of RNA polymerase. We purified active NIFA from Klebsiella pneumoniae in the form of a maltose-binding protein (MBP)-NIFA fusion; proteolytic release of MBP yielded inactive and insoluble NIFA. MBP-NIFA activated transcription from the nifHDK promoter in a purified transcription system. Like the related transcriptional activator NTRC, MBP-NIFA catalyzed the ATP-dependent isomerization of closed complexes between sigma 54-holoenzyme and a promoter to open complexes. MBP-NIFA had a broader nucleotide specificity than NTRC, being able to utilize pyrimidine in addition to purine nucleoside triphosphates. Both MBP-NIFA and a purified C-terminal fragment of NIFA bound to the upstream activation sequence for the nifHDK promoter, as assessed by DNAse I footprinting. When assays were performed at 37 degrees C instead of the usual 30 degrees C, transcriptional activation, open complex formation, and DNA binding by MBP-NIFA were all abolished, consistent with the known heat lability of NIFA. However, the purified C-terminal fragment of NIFA still bound the upstream activation sequence at 37 degrees C, indicating that the function of the helix-turn-helix DNA-binding motif is not inherently heat-labile.
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PMID:Activity of purified NIFA, a transcriptional activator of nitrogen fixation genes. 846 Jan 32

The KorB protein of broad-host-range plasmid RK2 is a transcriptional repressor involved in the control of genes for plasmid replication, conjugative transfer and stable maintenance. We have purified this protein close to homogeneity from cells harbouring an overexpression vector with the korB gene under the control of the tac promoter. KorB binds to restriction fragments bearing its proposed operator sequence, OB. Its interaction with this palindromic site was confirmed by DNaseI or hydroxyl radical footprinting at two OB sequences from RK2. Comparisons showed that the OB context affects the nature of the footprint. Our evidence suggests that KorB is a tetramer. As such, it may be able to bind two sites simultaneously on the same or on different DNA molecules. Using the korABF promoter, which is subject to KorB repression, we demonstrate by footprinting and restriction protection that KorB and RNA polymerase can bind simultaneously. Permanganate footprinting showed that KorB represses this promoter by preventing isomerization of the RNA polymerase-promoter complex from the closed to open form.
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PMID:Multifunctional repressor KorB can block transcription by preventing isomerization of RNA polymerase-promoter complexes. 846 98

The largest subunit of the RNA polymerase II (pol II) contains at the carboxy-terminus a peculiar repetitive sequence that consists of 52 tandem repeats of the consensus motif Tyr-Ser-Pro-Thr-Ser-Pro-Ser, referred to as the C-terminal domain (CTD). Upon transcriptional initiation/promoter clearance, the CTD becomes extensively phosphorylated and apparently remains so during elongation. While the underphosphorylated CTD plays a role in transcriptional initiation, recent evidence couples the highly phosphorylated CTD to RNA processing, namely polyadenylation and splicing. Using a yeast two-hybrid screen, we have selected for human proteins that interact with the CTD of RNA polymerase II. The CTD-GAL fusion protein used as a bait is highly phosphorylated in yeast and, accordingly, we did not isolate proteins implicated in transcriptional regulation but rather proteins with possible roles in RNA splicing. One major cDNA clone isolated this way encodes SRrp129/CASP11, a protein that contains a conserved CTD-interaction domain at the C-terminus and an internal serine-arginine rich domain (SR domain). Proteins of the SR family have been implicated in RNA splicing, notably in the regulation of alternative splicing. Thus we consider it likely that SRrp129 is an auxiliary splice factor. We also improved our method to quickly map domains involved in protein-protein interaction (Stagljar et al., 1996, BioTechniques 21, 430-432). Instead of using sonication for the production of a random DNA fragment library, we took advantage of the fact that DNAse I in the presence of manganese (II) produces double strand rather than single strand DNA breaks. The DNA fragment library of the SRrp129 clone was then used in the yeast two-hybrid system to identify the 100-amino acid domain that interacts with the CTD of RNA polymerase II.
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PMID:A novel SR-related protein specifically interacts with the carboxy-terminal domain (CTD) of RNA polymerase II through a conserved interaction domain. 922 39

We present a comparative study on epitope mapping of four monoclonal antibodies directed against four different antigens using alternative phage display techniques and peptide scanning: mAb215 reacts with the largest subunit of RNA polymerase II, mAbBp53-11 with the tumor suppressor protein p53, mAbGDO5 with the Hantaan virus glycoprotein G2 and mAbL13F3 with the Hantaan virus nucleocapsid protein. Epitopes were determined (i) by gene-fragment phage display libraries, constructed by DNaseI digested random gene fragments cloned into the 5' terminus of the pIII-gene of fd phage and (ii) by random peptide phage libraries displaying 6mer and 15mer peptides at the N-terminus of the pIII protein. Using the gene-fragment phage display libraries a single round of affinity selection resulted in the determination of the corresponding epitopes for all monoclonal antibodies tested. In contrast, biopanning of 6mer and 15mer random peptide libraries was only successful for two of the antibodies (mAbBp53-11 and mAbGDO5) after three or four rounds of selection. For the anti-p53 antibody we recovered the epitope from both the 6mer and 15mer library, for mAbGDO5 only the 6mer library displayed the epitope sequence. However, screening of the random peptide libraries with mAb215 and mAbL13F3 failed to yield immunopositive clones. Fine mapping of the epitopes by peptide scan revealed that the minimal epitopes recognized by mAbBp53-11 and mAbGDO5 consist of four and five amino acids, respectively, whereas mAb215 requires a minimal epitope of 11 amino acids for antigen recognition. In contrast, mAbL13F3 did not react with any of the synthesized 15mer peptides. The limits of the different methods of epitope mapping tested in this study are compared and the advantages of the gene-fragment phage display system are discussed.
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PMID:Epitope mapping by phage display: random versus gene-fragment libraries. 932 67

Mitochondrial RNA polymerase activity has been isolated from the crustacean Artemia franciscana at two stages of development, dormant embryo and developing larva. The preparations were obtained from purified mitochondria and the polymerase activity was purified by heparin-Sepharose chromatography. The presumed polymerase has a molecular mass of about 120 kDa and a 7.4 S sedimentation coefficient. The biochemical characterization of the enzymatic reaction identified our RNA polymerase preparations as mitochondrial. The transcription initiation sites of Artemia mtDNA were characterized recently in our laboratory (J. A. Carrodeguas and C. G. Vallejo, Eur. J. Biochem. 250, 514-523, 1997). Artemia mtDNA fragments comprising the transcription initiation sites were transcribed by the partially purified polymerase preparation from the two developmental stages, but the transcription turned out to be unspecific. DNAse I footprinting analysis of a main transcription initiation site-containing DNA fragment revealed a protected region around the initiation site +1 position, when using a crude polymerase preparation. However, the protected region was not observed with the purified preparation. The results altogether suggest that a specificity factor is lost during purification. Based on the footprinting data, we suggest that the sequence from positions -6 to +13 of the main transcription initiation site in the Artemia mitochondrial DNA is the binding site of the homologous RNA polymerase holoenzyme.
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PMID:Identification of a mitochondrial RNA polymerase in the crustacean Artemia franciscana. 960 62

There is accumulating evidence that the coordinate transcription of the virulence genes in Listeria monocytogenes constitutes a very complex regulation mechanism which might require other factors in addition to PrfA. We previously described an unknown proteinaceous component from crude bacterial cell extracts, which, together with PrfA, formed a specific complex (CI) in electrophoretic mobility shift assays (EMSA) with an hly promoter probe. Here we identify the RNA polymerase (RNAP) of L. monocytogenes as an essential component of the CI complex. Addition of purified RNAP plus PrfA to the hly promoter probe allowed reconstitution of a complex migrating at the same height as CI. By using EMSA and DNaseI footprint experiments it could be shown that PrfA leads to an enhanced and specific binding of RNAP. Transcriptional activity of RNAP in vitro, using the actA promoter, was strictly dependent on PrfA.
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PMID:PrfA mediates specific binding of RNA polymerase of Listeria monocytogenes to PrfA-dependent virulence gene promoters resulting in a transcriptionally active complex. 1079 34

Transcription by RNA polymerase I in Saccharomyces cerevisiae requires a series of transcription factors that have been genetically and biochemically identified. In particular, the core factor (CF) and the upstream activation factor (UAF) have been shown in vitro to bind the core element and the upstream promoter element, respectively. We have analyzed in vivo the DNAse I footprinting of the 35S promoter in wild-type and mutant strains lacking one specific transcription factor at the time. In this way we were able to unambiguously attribute the protections by the CF and the UAF to their respective putative binding sites. In addition, we have found that in vivo a binding hierarchy exists, the UAF being necessary for CF binding. Because the CF footprinting is lost in mutants lacking a functional RNA polymerase I, we also conclude that the final step of preinitiation-complex assembly affects binding of the CF, stabilizing its contact with DNA. Thus, in vivo, the CF is recruited to the core element by the UAF and stabilized on DNA by the presence of a functional RNA polymerase I.
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PMID:In vivo binding and hierarchy of assembly of the yeast RNA polymerase I transcription factors. 1125 Oct 85

The effects of mutations of --10 T:A to A:T, C:G, or G:C in the lambda P(R) promoter on formation of transcriptionally competent open complexes were studied by DNAse I footprinting, KMnO(4)-sensitivity, and abortive initiation kinetic analysis. The mutations --10A (T:A --> A:T) and --10C significantly reduce k(f), the composite rate constant for conversion of closed complexes (RP(c)) to open complexes (RP(o)) but do not affect K(B), the equilibrium constant for formation of closed complexes. Unlike the other mutants or wild-type P(R), the mutation with the largest effect on open complex formation, --10G (T:A --> G:C), substantially decreases the occupancy of the promoter. When reduced occupancy is taken into account, the calculated effect of the mutation on k(f) is a 20-fold reduction. Analysis of open complex formation by a three-step pathway that includes an additional intermediate, RP(i), indicates that the primary effect of all three mutations is a reduction in the rate of isomerization of RP(c) to RP(i), which precedes DNA strand separation. Thus, RNA polymerase holoenzyme must recognize specific base pairs in the --10 region of P(R) while the DNA is still double-stranded. Comparison of the observed level of stable complexes (RP(i) plus RP(o)) with the level of productive complexes (RP(o)) indicates that the --10G mutation may also affect the equilibrium between RP(i) and RP(o) at 37 degrees. Open complexes formed at the three mutant promoters are approximately 3-5 times less stable at 37 degrees than those formed at wild-type P(R).
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PMID:Mutations at position -10 in the lambda PR promoter primarily affect conversion of the initial closed complex (RPc) to a stable, closed intermediate (RPi). 1132 69


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