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)

Poliovirus (PV)-infected cells undergo extensive proliferation and rearrangement of intracellular smooth membranes to generate vesicles on which viral RNA replication occurs. PV proteins 2C and 2BC are known to be tightly associated with these membranous replication complexes and have been proposed to be involved in the formation of these virus-induced vesicles. We have expressed these proteins, and proteins with mutations in the putative nucleotide (NTP) binding motifs, in human cells using recombinant vaccinia viruses and T7 RNA polymerase-directed transcription. To ascertain the subcellular localization properties of these proteins in the absence of other PV proteins and to determine whether they induced ultrastructural changes, cells expressing 2C and 2BC proteins were examined by immunofluorescence (IF) microscopy, electron microscopy (EM), and immuno-EM (IEM). The cytoplasm of cells expressing either 2C or 2BC exhibited vesicles of 50-350 nm in diameter, which resembled those found in PV-infected cells. Both 2C and 2BC were associated with these vesicles. Mutations in the putative NTP binding motif did not affect vesicle induction by 2C or 2BC. Despite the membrane reorganization and vesicle formation induced by 2C and 2BC proteins, no enhanced synthesis of lipid was observed. Guanidine hydrochloride at a concentration that inhibits PV replication, did not have significant effects on the IF patterns of either 2C or 2BC. An additional prominent alteration in cells expressing 2C, but not 2BC, was the formation of extensive tubular membrane structures with a myelin-like arrangement in the lumen of the rough endoplasmic reticulum. IEM analyses showed that 2C was associated with these structures. In the presence of other PV proteins, the tubular membrane structures induced by 2C were not detected. These structures are not observed in poliovirus-infected cells, but likely indicate a novel property of 2C that induces a complex interaction with intracellular membranes.
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PMID:Membrane rearrangement and vesicle induction by recombinant poliovirus 2C and 2BC in human cells. 800 27

A HeLa cell-free extract has been prepared that efficiently uses full-length poliovirus-specific RNA, transcribed from plasmids with phage T7 RNA polymerase, or poliovirion RNA, for viral protein synthesis in vitro. Extensive proteolytic processing of the polyprotein in the extract produced viral enzymes that led to de novo viral RNA synthesis, and to the formation of infectious particles, as assayed on HeLa cell monolayers. The titre of plaque-forming units (p.f.u.) in the cell-free extract could be increased 70-fold when nucleoside triphosphates were added to the incubation mixture. Formation of infectious material was completely abolished if guanidine hydrochloride, an inhibitor of poliovirus RNA synthesis, but not of viral protein synthesis, was added; it was restored when the template used in the incubation was the RNA of a guanidine-resistant poliovirus mutant. Infectivity was completely inhibited by type-specific neutralizing antisera to poliovirus, and plaques were not formed if the HeLa cell monolayers were first treated with monoclonal antibodies to the poliovirus receptor. These results suggest de novo synthesis of poliovirus in a cell-free extract.
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PMID:In vitro synthesis of poliovirus. 838 31

RAP30 and RAP74 are subunits of RAP30/74 (TFIIF), a general initiation and elongation factor for transcription by RNA polymerase II. Complementary DNA (cDNA) clones have previously been reported encoding human RAP30 and RAP74. Here we report expression of these cDNAs using a T7 RNA polymerase system in Escherichia coli. Production of human RAP30 was very efficient using the expression vector pET11d. RAP30 accumulated within inclusion bodies and was solubilized using guanidine hydrochloride. After removal of the denaturant, RAP30 was soluble and active in accurate transcription. Approximately 44 mg of highly purified and soluble RAP30 was obtained from a 1-liter culture of cells. Production of RAP74 was more problematic, because a mixture of full length RAP74 and RAP74 fragments was produced in E. coli. Most RAP74 fragments were shortened by deletion of the COOH-terminus of the protein and probably resulted from premature translation termination. RAP74 was most successfully produced using a pET23d construct, in which the RAP74 peptide was fused to a short polyhistidine stretch at its COOH-terminus. Addition of the polyhistidine sequence allowed purification using a Ni2+ affinity resin. Full length RAP74 carrying this polyhistidine extension was purified in a single step by Ni2+ affinity chromatography in 4 M urea; the yield of RAP74 was approximately 3 mg from a 1-liter culture of cells. RAP74 derivatized with a polyhistidine stretch at its NH2-terminus, on the other hand, remained contaminated with RAP74 fragments after Ni2+ affinity chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Production of human RAP30 and RAP74 in bacterial cells. 839 Aug 79

RNA polymerase (RNAP) isolated from Staphylococcus aureus is deficient in sigma factor and is poorly active in transcription assays. Based on amino acid sequence homology of the Bacillus subtilis vegetative sigma factor sigmaA and the predicted product of the chromosomally located plaC gene of S. aureus, it was hypothesized that plaC could encode the vegetative sigma factor. We cloned plaC under a T7 promoter and overexpressed it in Escherichia coli strain BL21(DE3)pLysE. The overproduced protein, present in inclusion bodies, was solubilized with guanidine hydrochloride, renatured, and purified by DEAE-Sephacel and Sephadex G-75 chromatography. The purified protein, designated sigmaSA, cross-reacted with the B. subtilis anti-sigmaA antibody. E. coli core RNAP, reconstituted with sigmaSA, initiated promoter-specific transcription from the S. aureus promoters hla, sea, and sec and from the E. coli promoters rpoH P1, rpoH P4, and ColE1 RNA-1, which are recognized by the E. coli sigma70. sigmaSA, when added to the purified RNAP from S. aureus, stimulated transcriptional activity of the RNAP up to 72-fold. As determined by primer extension studies, the 5'-ends of the sigmaSA-initiated mRNAs synthesized in vitro from the agr P2 and sea promoters are in general agreement with the 5'-ends of the cellular RNAs. Disruption of the plaC gene on the S. aureus chromosome was lethal. We conclude that plaC encodes the primary sigma factor in S. aureus.
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PMID:Characterization of the primary sigma factor of Staphylococcus aureus. 870 82

Reverse transcriptase PCR (RT-PCR) is an important tool for Mycobacterium tuberculosis research and diagnostics. A standard procedure using N-acetyl-L-cysteine (NALC) and NaOH has been widely adopted for digestion and decontamination of sputum specimens for mycobacterial culture. The objective of this study was to determine the compatibility of this method with the recovery of RNA for RT-PCR assays. Nineteen sputum specimens were collected from smear-positive, pretreatment tuberculosis patients. After homogenization with NALC and glass beads, specimens were further processed by the addition of either NaOH, as per the standard decontamination protocol, or phosphate buffer. RNA was prepared by using a modified guanidine-phenol extraction method developed specifically for sputum sediments. DNA was isolated from the same specimens. Reverse transcriptions of alpha antigen (85B protein) mRNA and 16S rRNA were performed together, and aliquots were removed for separate PCRs. In all specimens, the 85B mRNA target was greatly diminished by treatment with NaOH; however, the 16S rRNA target remained unaffected. Storing sputum specimens for 48 h at 4 degrees C before processing did not seem to affect the integrity or yield of RNA; however, some degradation occurred by 72 h. Data suggest that the NaOH-NALC method for processing sputum samples is not suitable for detecting mRNA targets in RT-PCR assays.
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PMID:Alkaline decontamination of sputum specimens adversely affects stability of mycobacterial mRNA. 888 Apr 95

The sigma factor sigma 73 of the obligate intracytoplasmic bacterium Rickettsia prowazekii was overexpressed and purified from Escherichia coli. The rickettsial rpoD gene encoding sigma 73 was cloned into a Ndel-BamHI-cleaved pET-15b vector under control of T7 transcription and translation signals. The recombinant plasmid encoded a 75 kDa fusion protein that was overproduced in E. coli BL21(DE3) and purified from inclusion bodies after solubilization with guanidine hydrochloride and using His. Bind metal chelation resin. The N-terminal His. Tag sequence of the 75 kDa fusion protein was removed by thrombin treatment to obtain R. prowazekii sigma 73T. The R. prowazekii sigma 73T as well as the 75 kDa fusion protein had the ability to bind to core DNA-dependent RNA polymerase of both R. prowazekii and E. coli and to stimulate their interaction with a rickettsial promoter.
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PMID:Rickettsia prowazekii sigma factor sigma 73 can be overexpressed in Escherichia coli and promotes RNA polymerase binding and transcription. 893 16

The Rieske 2Fe-2S protein is a distinguishing subunit of the photosynthetic electron transport cytochrome b6f complex in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for overproduction in Escherichia coli of fusion, full-length, and truncated forms of the Rieske (PetC) protein from the cyanobacterium Nostoc sp. PCC 7906. A glutathione S-transferase/Rieske fusion protein was used to prepare specific chicken egg-yolk antibodies against the Rieske protein. Expression of the nonfusion petC gene in a T7 RNA polymerase promoter vector produced copious quantities of the full-length Rieske protein predominantly as inclusion bodies. The highly enriched, Rieske protein from inclusion bodies has been denatured in guanidine hydrochloride and refolded and the characteristic 2Fe-2S cluster reconstituted in vitro by incubation with iron and sulfide under reducing conditions. Purification by chromatography on Whatman DE52 cellulose and ultrafiltration through a 30000 molecular weight cutoff membrane yielded pure and predominantly monomeric Rieske protein. Reconstituted Rieske preparations showed intense and highly characteristic gx = 1.74, gy = 1.89, and gz = 2.03 "Rieske-type" electron paramagnetic resonance signals at 15 K. Two methods of reconstitution yielded Rieske preparations in which 20-60% of the protein contained 2Fe-2S clusters as determined by EPR spin quantitation. The reconstituted Rieske protein was soluble and stable at 4 degrees C in buffers containing nonionic detergents and showed a redox midpoint potential of +321 mV at pH 7.0 as determined by optical circular dichroism (CD) spectroscopy. These data demonstrate the in vitro restoration of a Cys and His liganded 2Fe-2S cluster and provide the basis for mutational and structural analysis of a PetC Rieske protein of oxygenic photosynthesis.
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PMID:Reconstitution of the 2Fe-2S center and g = 1.89 electron paramagnetic resonance signal into overproduced Nostoc sp. PCC 7906 Rieske protein. 895 2

Nonstructural proteins 2C, 3CD, 3C, and 3D, and the cellular protein actin, are present in highly purified preparations of foot-and-mouth disease virus (FMDV) and poliovirus. They remain bound in variable amounts to the RNAs when the RNAs are extracted from the viruses with phenol or phenol-sodium dodecyl sulfate (SDS) and, for FMDV, when the RNA is released from the particles by a lowering of the pH below 7. RNA prepared by these methods is rapidly degraded at 37 degrees C, particularly in the presence of NH4+ ions, but hydrolysis can be prevented by antibody against Escherichia coli-expressed 3D, indicating that it is the RNA polymerase that has nuclease activity. In contrast, virion RNA from which the nonstructural proteins and actin have been removed by extraction with guanidine thiocyanate-phenol-chloroform or proteinase K-phenol is stable at 37 degrees C, although its specific infectivity is lower than that of the RNA extracted with phenol or phenol-SDS. The possible implications of the close association of replication complex proteins with the RNA in virus particles are discussed.
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PMID:Foot-and-mouth disease virus and poliovirus particles contain proteins of the replication complex. 931 48

T7 RNA polymerase shows an increase in processive transcription in the presence of low concentrations of guanidine hydrochloride (GdnCl) upto 60 mM, which is not observed when the enzyme is treated with urea. Higher concentrations of the denaturant lead to a progressive loss in the processive transcriptional activity of the enzyme. We have attempted to explain the above phenomenon in terms of the structural change in the enzyme. Fluorescence and CD studies suggest that the tertiary structure of the native enzyme undergoes an alteration upon addition of low concentration of guanidine hydrochloride. This is also indicated from the decreased susceptibility of the enzyme to limited proteolysis by trypsin.
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PMID:Enhancement of transcriptional activity of T7 RNA polymerase by guanidine hydrochloride. 963 52

The sigmaH of Bacillus subtilis directs transcription of a large number of early sporulation genes, whereas the principal sigma factor, sigmaA, is essential for the transcription of the genes for vegetative growth and early sporulation. We have purified sigmaA and sigmaH proteins, and characterized their properties. The genes encoding sigmaA or sigmaH were separately cloned into an expression vector under the control of T7 promoter. Both proteins were overproduced in Escherichia coli BL21(DE3) and purified from inclusion bodies after solubilization with guanidine hydrochloride. Antigenicities and N-terminal amino acid sequences of the overproduced proteins were used to identify both proteins. Unlike sigmaA protein, sigmaH protein showed a DNA-binding ability. To compare the promoter selectivity of the sigmaA protein with that of the sigmaH protein, transcription in vitro of 16 promoters was performed using RNA polymerase holoenzymes reconstituted from a purified core enzyme with either sigmaH or sigmaA. These holoenzymes correctly recognized each of the cognate promoters; sigmaH-RNA polymerase recognized sigmaH promoters but not sigmaA promoters, and vice versa. A competition experiment for core RNA polymerase using sigmaA and sigmaH revealed that sigmaA had a stronger affinity. We propose that the predicted replacement of a sigma subunit in a holoenzyme from sigmaA to sigmaH in vivo at late logarithmic growth phase may require an additional factor, or the modification of a core enzyme or sigma factor.
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PMID:Promoter selectivity of the Bacillus subtilis RNA polymerase sigmaA and sigmaH holoenzymes. 964 50


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