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)

T3 and T7 phages package homologous DNA more efficiently than heterologous DNA and recombinant plasmids carrying DNA sequences necessary for DNA packaging (pac sequence). The pac sequence contains a promoter for phage RNA polymerase and transcription from the promoter is necessary for DNA packaging. T3 and T7 RNA polymerases are stringently specific for their own promoters. To examine the relationship between DNA packaging and transcription, we constructed a cleared in vitro system for packaging T3 or T7 DNA containing an ammonium sulfate fractionate of a high-speed supernatant of phage-infected cells. In the system, DNA packaging required GTP and was inhibited by the 3'-deoxy analog of GTP, ATP, or CTP. The DNA packaging activity paralleled the transcriptional activity, assayed by incorporation of [32P]UTP into acid-insoluble material. In the system, homologous DNA was packaged more efficiently than heterologous DNA, but heterologous DNA was packaged as efficiently as homologous DNA by the addition of heterologous phage RNA polymerase, demonstrating that the transcriptional specificity determines the DNA packaging specificity of T3 and T7.
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PMID:Transcription dependence of DNA packaging of bacteriophages T3 and T7. 141 5

Escherichia coli RNA polymerase has two subsites, i and i + 1, for the binding of the first two substrates, and the first phosphodiester bond is formed between them during the initiation of transcription. Various studies have shown earlier that the inhibitor rifampicin has little effect, if any, on the formation of this phosphodiester bond. On an earlier occasion, we measured the distance of the i nucleotide from the rifampicin binding site on RNA polymerase using Forster's energy-transfer mechanism [Kumar & Chatterji (1990) Biochemistry 29,317]. In this paper, the 1-aminonaphthalene-5-sulfonic acid (AmNS) derivative of UTP in the presence of 10 mM MgCl2 was used as an energy donor, and its distance from rifampicin was estimated. The modified nucleotide (gamma-AmNS)-UTP binds to RNA polymerase with a Kd of 3 microM and has one binding site in the presence of Mg(II) ion. Fluorescence titration studies performed with or without an initiator indicated that (gamma-AmNS)-UTP exclusively binds to RNA polymerase at the (i + 1) site in the presence of Mg(II). Rifampicin was found to form a 1:1 complex with RNA polymerase bound to labeled UTP. Rifampicin and (gamma-AmNS)-UTP have a substantial spectral overlap with an energy-transfer efficiency close to 50%. Labeled UTP shows a decrease in its excited-state lifetime when bound to the enzyme; the transfer efficiency calculated from lifetime measurements was found to be lower than that estimated from steady-state spectral analysis. Time-resolved emission spectral analysis was carried out to differentiate between the free and bound UTP over the enzyme surface.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Proximity relationship between the active site of Escherichia coli RNA polymerase and rifampicin binding domain: a resonance energy-transfer study. 151 Sep 38

The action of rifampicin on the RNA chain initiation catalysed by E. coli RNA polymerase over different templates has been studied. The steady-state formation of dinucleoside tetraphosphate under the condition of abortive initiation reaction was assayed. It was observed that rifampicin shows a spectrum of inhibitory effects on transcription initiation at different promoters. At two different promoters with a pyrimidine nucleotide at the 5'-initiation site, e.g. rrnB P2 having CTP and lacP2 having UTP, the effect of rifampicin on the abortive synthesis of the first phosphodiester bond was found to be total, even at low concentrations of the antibiotic. On the other hand, in most cases the effect of rifampicin on the abortive synthesis with a purine nucleotide at the 5'-initiation site was found to be only partial, with the exception of the T7A2 promoter, where rifampicin stimulates the abortive synthesis of pppGpC. It was also noticed that if there was a purine nucleotide at the second position of a dinucleotide which had already been synthesised by the enzyme, then further addition of the third nucleotide was not blocked in the presence of rifampicin. It appeared that a purine nucleotide at the initiation site or at the product terminus site of a translocated dinucleotide behaved similarly towards rifampicin. In the same way, if this position was occupied by a pyrimidine, rifampicin would inhibit further phosphodiester synthesis, even at a very low concentration. The stimulatory effect of rifampicin at the T7A2 promoter was presumably because here a ternary complex containing the promoter, enzyme and the abortive transcript pppGpC was initially stable, but dissociated upon addition of rifampicin, resulting in the rapid turn-over of the product.
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PMID:Differential inhibition of abortive transcription initiation at different promoters catalysed by E. coli RNA polymerase. Effect of rifampicin on purine or pyramidine-initiated phosphodiester synthesis. 162 42

A thorough mutational analysis of U6 RNA in combination with a functional reconstitution assay, revealed that three domains are important for U6 function in pre-mRNA splicing. In order to further analyze why these regions are so critical for splicing, we make use of phosphorothioate substituted U6 RNAs. Wild-type U6 RNA was transcribed in vitro with T7 RNA polymerase in the presence of either phosphorothiate (alpha-S) ATP, GTP, UTP or CTP. The functionality of the transcripts was monitored by in vitro reconstitution. While substitution with alpha-S ATP, GTP or UTP blocked splicing, substitution with alpha-S CTP had little or no effect on splicing. We made use of this alpha-S CTP effect in an attempt to elucidate which phosphates in the U6 RNA molecule play a role in the first or in the second step of splicing. U6 mutants in which a change of an A, G or U to C does not have any significant effect on splicing were transcribed in the presence of alpha-S CTP. Observed effects on splicing thus have to be attributed to the presence of the thio-substituted phosphate group rather than the nucleotide change. The results of in vitro reconstitution give a clear answer for at least three phosphates; two of them play a role in the first step, while one of them is involved in the second step of splicing.
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PMID:Thiophosphates in yeast U6 snRNA specifically affect pre-mRNA splicing in vitro. 164 31

New spin-labeled analogs of nucleoside triphosphates, 8-amino(2,2,6,6-tetramethylpiperidine-N-oxyl)adenosine 5'-triphosphate ((8-AmTEMPO)ATP) and 5-amino(2,2,6,6-tetramethylpiperidine-N-oxyl)uridine 5'-triphosphate ((5-AmTEMPO)UTP), with the probe 4-amino(2,2,6,6-tetramethylpiperidine-N-oxyl) (4-AmTEMPO) attached to C-8 of ATP and C-5 of UTP via a secondary amine bond, were synthesized in 50 and 40% yield, respectively. These analogs showed a single spot by thin layer chromatographic analysis. The absorption spectra of (8-Am-TEMPO)ATP and (5-AmTEMPO)UTP exhibit maxima at 310 and 265 nm, respectively; their X-band EPR spectra have a typical three-line pattern with lines at 3,221, 3,239, and 3,257 Gauss. The intensity ratios for mid to high field lines of the EPR derivative lines were found to be 1.03 +/- 0.02, 1.08 +/- 0.04, and 1.15 +/- 0.07 for 4-AmTEMPO, (8-AmTEMPO)ATP, and (5-AmTEMPO)UTP, respectively. The immobilization of 4-AmTEMPO bound to C-8 of ATP or bound to C-5 of UTP was observed to be 5 and 11%, respectively, as compared with free 4-AmTEMPO. The initial velocity (s-1) of [3H]UMP incorporation into RNA in the presence of [3H]UTP, CTP, GTP, and (8-AmTEMPO)ATP or ATP was measured. The percent incorporation of (8-AmTEMPO)ATP into RNA product by Escherichia coli RNA polymerase using various DNA templates is 68, 66, and 61% for pAR1435 (plasmid containing A1 promoter from T7 DNA), calf thymus DNA, and poly(dA-dT) respectively, as compared with ATP incorporation. The polymerase-catalyzed reaction of (8-AmTEMPO)ATP with (3'-OCH3)UTP yielded 5'-triphosphate delta-amino(2,2,6,6-tetramethylpiperidine-N-oxyl)adenylyl (3'-5')3'-methoxy uridine in the presence of poly(dA-dT). The structure of this spin-labeled dinucleotide was identified by paper chromatographic analysis of the products of phosphodiesterase digestion. These analogs also can be used for the study by EPR spectroscopy of the dynamics of gene transcription catalyzed by RNA polymerases or of other nucleotide-utilizing enzymes.
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PMID:Spin-labeled nucleotide substrates for DNA-dependent RNA polymerase from Escherichia coli. 165 31

Poliovirus RNA encodes an RNA-dependent RNA polymerase, designated 3Dpol, that catalyzes the synthesis of both plus and minus strand viral RNA. This enzyme was purified to near homogeneity from poliovirus-infected HeLa cells, recombinant baculovirus-infected insect cells, and from Escherichia coli transformed with an expression plasmid containing poliovirus 3D sequences. The two recombinant expression systems produced significantly higher yields of active enzyme than could be attained from virus-infected HeLa cells. All preparations contained a 52-kDa protein, recognized by antisera raised against 3D expressed as a fusion protein in E. coli. Immunoreactive protein resolved into 3-4 species on isoelectric focusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis two-dimensional gels. Efforts to demonstrate that the multiple spots resulted from phosphorylation were negative. Furthermore, no evidence for autophosphorylation of purified 3Dpol was obtained. Purified 3Dpol from recombinant sources manifested the same specific activities as enzyme from poliovirus-infected HeLa cells in both a poly(A)-dependent poly(U) polymerase assay and a poliovirus RNA-dependent RNA polymerase assay. The products of the latter reaction reached the length of the template (7.5 kilobases) in 20-30 min, indicating an elongation rate of approximately 300 nucleotides/min at 30 degrees C. No products exceeded the length of the template. Intermediate length products were detected, which presumably resulted from pauses in transcription due to template structure. All transcription was dependent on primer. The kinetic parameters of all three purified enzyme preparations were the same; the Km for UTP was 2.4 +/- 0.1 microM in an RNA polymerase activity assay. Product formation was linear for up to 45 min, except for a 3-5-min lag before synthesis began. The lag was independent of enzyme concentration, and independent of the template used. The lag was eliminated by preincubating enzyme, template, primer, and three of the four nucleotide triphosphates, but not by preincubating any subset of these components. This suggested that a preinitiation complex must form as a prerequisite to RNA synthesis. Partially purified preparations of 3Dpol from the three sources showed significant differences in activities and products, including the appearance of primer-independent polymerase activity and production of dimer-length RNA products. These variable properties are likely due to different contaminating activities provided by the different cellular hosts, since upon further purification, all three enzymes exhibited identical properties.
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PMID:Purification, characterization, and comparison of poliovirus RNA polymerase from native and recombinant sources. 166 Aug 94

We have combined immunogold labeling with the Miller spreading technique in order to localize proteins at the electron microscope (EM) level in whole mount nuclei from mouse and human fibroblasts. Anti-histone H1 antibody labels nuclei uniformly, indicating that the nuclear interior is accessible to both antibodies and gold conjugates. Anti-topoisomerase I antibody labels nucleoli intensely, in agreement with previous immunofluorescent and biochemical data. Two different antibodies against the large subunit of RNA polymerase II (pol II) show preferential labeling of the nuclear periphery, as do antibodies against lamin, a known peripheral nuclear protein. Treatment of cells with alpha-amanitin results in loss of virtually all RNA polymerase II staining, supporting the specificity of labeling. Finally, when nuclei are incubated in the presence of biotin-UTP (bio-UTP) under run-off transcription conditions, incorporation is preferentially located at the nuclear periphery. These results support the conclusions that transcriptionally active pol II molecules are non-uniformly distributed in fibroblast nuclei, and that their differential distribution mirrors that of total pol II.
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PMID:Preferential distribution of active RNA polymerase II molecules in the nuclear periphery. 166 44

Tagetitoxin, a chlorosis-inducing phytotoxin produced by Pseudomonas syringae pv. tagetis, inhibits RNA synthesis directed by chloroplast RNA polymerase. In isolated chloroplasts, tagetitoxin quickly and specifically reduced the incorporation of [3H]uridine into RNA. When it was added to transcriptionally active chloroplast protein extracts, the toxin directly inhibited incorporation of [32P]UTP into RNA. In addition, tagetitoxin inhibited in vitro RNA synthesis directed by the RNA polymerase from Escherichia coli. In vitro transcription reactions directed by chloroplast RNA polymerase or E. coli RNA polymerase are inhibited at tagetitoxin concentrations less than 1 microM. Nuclear RNA polymerase II purified from wheat germ was only affected at tagetitoxin concentrations greater than 100 microM during in vitro transcription. Tagetitoxin concentrations as high as 1 mM did not affect in vitro transcription reactions directed by RNA polymerase from bacteriophage T7 or SP6.
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PMID:Tagetitoxin inhibits RNA synthesis directed by RNA polymerases from chloroplasts and Escherichia coli. 168 34

In vitro transcription studies with a trp leader DNA template derived from a double deletion mutant of Serratia marcescens revealed that the transcription complex pauses synthesis of part of the RNA antiterminator, structure 2:3. Pausing was enhanced by NusA protein and was dependent on the concentration of UTP in the transcription reaction mixture. A weak antiterminator pause also was detected during transcription of the wild-type S. marcescens trp leader template in the presence of NusA protein and 1 microM UTP. Transcription pausing following synthesis of the antiterminator also was observed in a cell-free transcription-translation system. Antiterminator-induced pausing may play an important role in vivo by delaying synthesis of RNA segment 4. This delay may influence basal level control in cells with an excess of tryptophan. In addition, formation of the antiterminator pause structure may introduce a more stringent tryptophan starvation requirement for RNA polymerase to read through the attenuator.
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PMID:The RNA antiterminator causes transcription pausing in the leader region of the tryptophan operon. 169 Jul 21

Escherichia coli RNA polymerase transcription elongation complexes have been prepared that contain a photo-cross-linking uridine analog at only the 3' end, or one or two nucleotides removed from the 3' end, in the nascent RNA chain. Additionally, complexes have been isolated in which the analog has been substituted for every UMP residue, at positions ranging from 20 to 140 nucleotides from the 3' end. The RNA has been photochemically cross-linked to the RNA polymerase to identify the subunits that form the binding site(s) for these regions in the nascent RNA. The photo-cross-linking nucleotide analog used for these studies was 5-[4-azidophenacyl)thio)uridine-5'-triphosphate (5-APAS-UTP), which acts as a 10-15 A probe. With 5-APAS-UMP positioned only at the 3' end of the RNA, or one or two nucleotides from the 3' end, only the beta subunit appeared to be contacted. When the analog was positioned throughout the RNA, both the beta and beta' subunits were contacted. No labeling of the sigma or alpha subunits was observed with any RNA length. In addition to placing this analog at specific positions in short RNAs, we have carried out transcription studies with 5-APAS-UTP to determine the optimal UTP to analog ratio for production of full length, photoreactive transcripts. Surprisingly, we found that when transcription complexes were stalled shortly after initiation, by deletion of one ribonucleoside triphosphate to synchronize transcription, changes in transcriptional pausing occurred downstream. These results suggest that events that occur early in transcription can affect the elongation and/or termination events that occur far downstream from the promoter. This effect occurred even with UTP but was greatly enhanced by replacement of UTP with either this analog or 4-thio-UTP. By enhancing the normal transcriptional pausing event, these analogs can serve as probes of the conformational changes that may exist in paused transcription complexes.
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PMID:Active site labeling of Escherichia coli transcription elongation complexes with 5-[4-azidophenacyl)thio)uridine 5'-triphosphate. 169 25


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