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 procedure has been developed for the purification of soluble DNA-dependent RNA polymerase (EC 2.7.7.6) from rye embryos. The enzyme solubilized by high salt extraction with sonication and resolved by DEAE-cellulose chromatography yields two activities. Enzyme I eluted at 0.15 M (NN4)2SO4, was insensitive to alpha-amanitin and was extremely labile. Enzyme II eluted at 0.25 M (NH4)2SO4 was inhibited by alpha-amanitin. However, DEAE-Sephadex chromatography yields three DNA-dependent RNA polymerases. Enzyme I is resistant to amanitin, while II and III enzymes are inhibited by this poison. Partially purified on DEAE-cellulose, polymerase II was further purified by hydrophobic chromatography on an omega-aminobutyl-Sepharose column. After omega-aminobutyl-Sepharose chromatography, enzyme II was stable and was more active with denatured than with native DNA as template. The activity of purified RNA polymerase II is dependent on the DNA, Mn-2+ and Mg-2+ added and requires ATP, GTP, CTP and UTP for its maximum activity. Transcription is inhibited besides by alpha-amanitin, by chromomycin A3, daunomycin, ethidium bromide and actinomycin D. Rifampin and rifamycin SV do not inhibit the enzyme. Synthetic copolymers were also effective as templates.
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PMID:Isolation and purification of RNA polymerases from rye embryos. 112 11

Under specific binding conditions RNA polymerase forms complexes at several sites of the replicative form DNA of bacteriophage fd. One of these complexes becomes stable to both high salt and low temperature after incubation with GTP. None of the complexes is stabilized by ATP. The stabilization by GTP results from the synthesis of an oligo(G) chain, which is bound in the complex. Size and pyrimidine fingerprints of the DNA segment protected by the enzyme against digestion with DNase are not changed upon initiation of oligo(G) synthesis. This result indicates that binding site and initiation site are identical parts of a promoter region.
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PMID:Stabilization of promoter complexes with a single ribonucleoside triphosphate. 117 37

RNA polymerase activities of whole nuclei, of isolated and purified nucleoli and of the nucleoplasmic fractions obtained from cerebral hemispheres, cerebellum and brain stem of rat at different days of postnatal development have been determined. In the whole nuclei the fraction of RNA polymerase which is sensitive to alpha-amanitin, is strongly affected by salt concentration; at low ionic strength most of the activity is resistant to the drug while at high ionic strength the enzymatic activity shows a greater sensitivity to the drug. In isolated nucleoli RNA synthesis is not inhibited at all by alpha-amanitin. The biosynthesis of RNA, at low ionic strength, is inhibited by low doses of actinomycin D, whereas at high ionic strength it is remarkably inhibited only by higher doses of the drug. The sensitivity of the reaction to alpha-amanitin and actinomycin D provide good evidence that UTP or GTP incorporation into RNA in purified nuclei and nucleoli, is dependent on RNA polymerases acting on DNA template and is not dependent on homopolymer formation. These results show that in the whole brain nuclei at low ionic strength there is a preferential synthesis of rRNA, whereas at high ionic strength the synthesis of heterogenous RNA predominates. In isolated nucleoli the synthesis of RNA is restricted to rRNA.
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PMID:Nucleolar and nucleoplasmic RNA polymerase activity in different regions of rat brain during postnatal development. 121 80

1. At 3 weeks after ovariectomy, mammary glands (5th pair) of adult Swiss mice show (i) no significant decrease in weight, (ii) 20% of the original rate of incorporation of [(3)H]-uridine into RNA (after a 30min pulse), and (iii) 90% of the original rate of incorporation of l-[(3)H]leucine into protein (after a 15min pulse). 2. A single injection of oestradiol-17beta into these ovariectomized mice produces, during the next 17h, a series of discrete bursts of increased incorporation of [(3)H]uridine into mammary-gland RNA; the bursts, which are variable in height, reach peaks at approx. 1, 9, 12 and 16h after hormone administration; an increase is already detected at 15min, the earliest time-point investigated; each burst lasts for approx. 2h. There is no significant stimulation of [(3)H]uridine incorporation into RNA of liver and quadriceps femoris muscle. 3. Nuclear incorporation of [(3)H]UTP into RNA of mammary gland in vitro is linear with time for up to 20min at 15 degrees C; it requires CTP, GTP and ATP and is inhibited by actinomycin D. Also, the incorporation is strongly inhibited by alpha-amanitin in high salt concentrations but only weakly in low salt concentrations, a result indicating that RNA polymerase II activity predominates in high salt, whereas RNA polymerase I activity predominates in low salt concentrations. Injection of oestradiol-17beta in vivo followed by measurement of nuclear RNA synthesis in vitro shows a definite increase in both RNA polymerase activities 30min after oestradiol-17beta injection, the earliest time-point investigated, a higher increase at 1h, a decline at 4h, and again a large increase at 12h. These results in general agree with the changes in precursor incorporation into RNA measured directly in the animal and suggest that changes in [(3)H]uridine uptake into RNA are not precursor-pool-dependent.
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PMID:Polyphasic changes in incorporation of precursors into ribonucleic acid of oestradiol-stimulated mammary gland. 122 Jun 81

DNA-dependent RNA polymerase from Escherchia coli was used to transcribe chromatin from human leukocytes and purified human DNA. RNA was labeled at the 5' terminus with either [gamma-32P]ATP or [gamma-32P]GTP and internally with [3H]UTP. Determination of the average chain length of the RNA molecules by the ratio of moles of 3H-labeled nucleotide incorporated to moles 32P-labeled nucleotide incorporated showed that the size of the transcript of purified DNA was about 2 1/2 times greater than those from chromatin. The percentage of chains initiated with ATP and GTP was observed to vary with the template, the ATP to GTP ratio being greater on chromatin. The kinetics of 3H and 32P hybridization of transcripts of purified DNA showed hybridization primarily to nonrepetitive sequences. Transcripts from the chromatin templates when hybridized to DNA showed a larger proportion of RNase resistance of the 32P-termini at low Cot's.
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PMID:Template restriction in human chromatin. 126 Aug 58

A photoactive nucleotide analogue of UTP, 5-azido-uridine 5'-triphosphate (5-N3UTP), has been demonstrated to interact with the RNA polymerase of the vesicular stomatitis virus (VSV) transcription complex. Kinetic studies indicated that 5-N3UTP served as an efficient replacement for UTP in in vitro polymerase reactions. The Km for the azido analogue was 27 microM and that of the natural substrate, UTP, was 7 microM. Photolysis of [gamma-32P]5-N3UTP in the presence of VSV transcription complexes resulted in selective radio-labelling of the L protein. This photolabelling was saturable with an apparent Kd of 28 microM. The L protein was protected from [gamma-32P]5-N3UTP-mediated photolabelling by competing natural substrates (UTP, CTP, ATP, GTP). The stoichiometry of photoprobe incorporation into the transcription complex was close to unity with respect to the L protein. These data provide evidence that the nucleotide-binding domain of the VSV RNA polymerase contains amino acid residues of the L protein.
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PMID:The L protein of vesicular stomatitis virus transcription complexes is specifically photolabelled by 5-azido-uridine 5'-triphosphate, an analogue of the RNA polymerase substrate uridine 5'-triphosphate. 130 62

A photoaffinity analogue of ATP, 8-azido-adenosine 5'-triphosphate (8-N3ATP), was used to probe ATP-binding sites in native transcription complexes of vesicular stomatitis virus (VSV) (New Jersey serotype). The analogue was found to be a substrate for a serine/threonine protein kinase that phosphorylated both the NS and L proteins of native complexes. The analogue failed to interact with the RNA polymerase, another ATP-utilizing activity associated with the transcription complex. Kinetic analyses of both ATP and 8-N3ATP utilization by the protein kinase yielded biphasic saturation curves. Photolysis of 8-N3ATP in the presence of VSV transcription complexes resulted in selective labelling of the L protein. The photolabelling of L was saturable and apparently biphasic. Photolabelling of the L protein was significantly reduced by competition with ATP whereas other nucleoside triphosphates (GTP, UTP and CTP) were ineffective competitors. The stoichiometry of photolabelling was 0.2 at 10 microM-8N3ATP and 1.3 at 100 microM-ATP. These data provide chemical evidence for a virus-encoded serine/threonine protein kinase which resides on the L protein.
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PMID:Identification and characterization of serine/threonine protein kinase activity intrinsic to the L protein of vesicular stomatitis virus New Jersey. 130 63

We previously purified RNA polymerase II transcription factor delta from rat liver and found that it has an associated DNA-dependent ATPase (dATPase) activity. In this report, we show that delta is also closely associated with a protein kinase activity that catalyzes phosphorylation of the largest subunit of RNA polymerase II. Kinase activity copurifies with transcription and DNA-dependent ATPase (dATPase) activities when delta is analyzed by anion- and cation-exchange HPLC as well as by sucrose gradient sedimentation, arguing that delta possesses all three activities. Phosphorylation of the largest subunits of both rat and yeast RNA polymerase II is stimulated by DNA, whereas phosphorylation of a synthetic peptide containing multiple copies of the carboxyl-terminal heptapeptide repeat is not. Although both ATP and GTP appear to function as phosphate donors, GTP is utilized less than 10% as well as ATP. These findings suggest that delta may exert its action in transcription at least in part through a mechanism involving phosphorylation of the largest subunit of RNA polymerase II.
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PMID:A carboxyl-terminal-domain kinase associated with RNA polymerase II transcription factor delta from rat liver. 138 28

Chimaeric chloramphenicol acetyltransferase (CAT) mRNA, containing the leader sequences of genomic 42S RNA and subgenomic 26S RNA of Semliki Forest virus (SFV) were synthesized by in-vitro transcription. These transcripts were translated with different efficiencies, as the authentic mRNA in SFV-infected cells. Therefore, they can be used as model mRNA species to study the mechanism underlying SFV-directed shut off of host protein synthesis. The interaction of translation initiation factors with the 5' cap structure was studied. Transcripts prepared in vitro using T7 RNA polymerase were capped and methylated posttranscriptionally with [32P]-GTP and S-adenosyl-L-methionine to yield cap-labelled mRNA species. Irradiation with ultraviolet light of 26S CAT and 42S CAT transcripts, together with crude rabbit reticulocyte initiation factors, resulted in the cap-specific cross-linking of eukaryotic initiation factors (eIF) eIF-4E and eIF-4B. The relative binding efficiency of these two factors to the cap structure of the various transcripts was, however, markedly different; the cap structure present in 26S CAT mRNA interacted efficiently with cap-binding proteins, whereas the cap structure of 42S CAT mRNA hardly bound to these proteins. Comparable results were obtained under competitive conditions. Data are presented that the secondary structure close to the 5' cap structure determines the efficiency of recognition of the mRNA by these initiation factors. Using a chemical cross-linking assay, it was demonstrated that eIF-4F, and also eIF-4E, differentially interacted with the cap structure of the various transcripts. The data are discussed with respect to the possible mechanisms involved in SFV-induced shut off of host cell protein synthesis.
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PMID:Interaction of initiation factors with the cap structure of chimaeric mRNA containing the 5'-untranslated regions of Semliki Forest virus RNA is related to translational efficiency. 139 64

The high affinity branched-chain amino acid transport system (LIV-I) in Pseudomonas aeruginosa is composed of five components: BraC, a periplasmic binding protein for branched-chain amino acids; BraD and BraE, integral membrane proteins; BraF and BraG, putative nucleotide-binding proteins. By using a T7 RNA polymerase/promoter system we overproduced the BraD, BraE, BraF, and BraG proteins in Escherichia coli. The proteins were found to form a complex in the E. coli membrane and solubilized from the membrane with octyl glucoside. The LIV-I transport system was reconstituted into proteoliposomes from solubilized proteins by a detergent dilution procedure. In this reconstituted system, leucine transport was completely dependent on the presence of all five Bra components and on ATP loaded internally to the proteoliposomes. Alanine and threonine in addition to branched-chain amino acids were transported by the proteoliposomes, reflecting the substrate specificity of the BraC protein. GTP replaced ATP well as an energy source, and CTP and UTP also replaced ATP partially. Consumption of loaded ATP and concomitant production of orthophosphate were observed only when BraC and leucine, a substrate for LIV-I, were added together to the proteoliposomes, indicating that the LIV-I transport system has an ATPase activity coupled to translocation of branched-chain amino acids across the membrane.
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PMID:Solubilization and reconstitution of the Pseudomonas aeruginosa high affinity branched-chain amino acid transport system. 140 Apr 43


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