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)

The addition of a single nucleotide to a short oligonucleotide, catalyzed by RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) in the presence of synthetic DNA templates, has been studied. The reactions A-U + ATP leads to A-U-A and U-A + UTP leads to U-A-U occur in the presence of poly[d(A-T)], while the reactions G-C + GTP leads to G-C-G and C-G + CTP leads to C-G-C take place in the presence of poly[d(I-C)]. These reactions proceed with a turnover of enzyme. The products U-A-U and C-G-C are formed rapidly, while A-U-A and G-C-G are formed much more slowly. Another poly[d(A-T)]-dependent reaction, which occurs with a turnover of enzyme, is U-A-U + ATP leads to U-A-U-A. All of these reactions are only partially inhibited by rifampicin. ATP can be replaced by 3'-deoxyadenosine 5'-triphosphate in the reactions A-U + ATP leads to A-U-A and U-A-U + ATP leads to U-A-U-A, though the rate of formation of the products becomes somewhat slower. The reactions involving 3'-deoxyadenosine 5'-triphosphate are almost completely inhibited by rifampicin, indicating that the 3'-hydroxyl group is necessary for these reactions to occur in the presence of rifampicin.
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PMID:DNA-dependent single-step addition reactions catalyzed by Escherichia coli RNA polymerase. 34 47

A soluble extract prepared from cells of an Escherichia coli strain carrying the antibiotic resistance plasmid R6K is capable of carrying out the complete process of R6K DNA replication. DNA synthesis in vitro is dependent on the four deoxyribo- and ribonucleotide triphosphates and is sensitive to rifampin and streptolydigin, inhibitors of DNA-dependent RNA polymerase. The incorporation of deoxyribonucleotides into R6K DNA also is sensitive to actinomycin D, novobiocin, arabinofuranosyl-CTP, and N-ethylmaleimide. Kinetics of synthesis are linear for 60 to 120 min. Replication proceeds semiconservatively and supercoiled closed-circular DNA molecules are synthesized. Analysis by alkaline sucrose gradient centrifugation indicated that the early R6K DNA products contain DNA fragments of approximately 18 S in size, corresponding to the length between the R6K alpha origin of replication and the terminus of replication observed in vivo. Addition of exogenous supercoiled R6K DNA is inhibitory to the in vitro system, whereas the addition of R6K DNA in the form of relaxation complex stimulates R6K DNA synthesis to a small extent.
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PMID:Replication of antibiotic resistance plasmid R6K DNA in vitro. 35 90

Conditions are described for the replication of exogeneous R6K DNA in an in vitro system prepared from Escherichia coli cells. Replication of plasmid DNA in this system is semiconservative and sensitive to actinomycin D, novobiocin, arabinofuranosyl-CTP,N-ethylmaleimide, and inhibitors of DNA-dependent RNA polymerase. An ammonium sulfate fraction prepared from cells carrying the R6K plasmid is required for replication. A direct role in replication for a plasmid-encoded protein, designated pi, in this fraction is indicated by the inactivity of this fraction when prepared from cells carrying a temperature-sensitive mutant plasmid and the thermolability of this fraction when prepared from cells carrying a partial revertant of the mutant plasmid. This plasmid-encoded protein is necessary for the initiation of R6K DNA replication and functions before or during the formation of nascent RNA in the initiation process. The results of titration assays of this protein using various template DNAs suggest that the protein interacts with the plasmid DNA at the region essential for DNA replication.
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PMID:Requirement of a plasmid-encoded protein for replication in vitro of plasmid R6K. 36 78

Transcription was determined in liver chromatin from rats fed for 6 days, an optimal (20%) or suboptimal (3%) amount of high-quality protein. Transcription by Escherichia coli RNA polymerase (EC 2.7.7.6) was lower after prolonged incubation with chromatin from rats fed 3% as compared with 20% protein. Differences were detected in the transcripts of the two types of chromatin after analysis by sucrose density gradient centrifugation. But no measurable differences were found in the melting profiles at low ionic strength of the two chromatin preparations. Transcription per milligram chromatin DNA was 25-fold higher using E. coli RNA polymerase instead of rat liver RNA polymerase II. The use of UTP as radioactive precursor in the absence of ATP, GTP and CTP resulted in a low labelling of RNA. One [lambda32P]UTP nucleotide was incorporated/8 UMP nucleotides. The product obtained was sensitive to ribonuclease treatment. In the presence of ATP, GTP and CTP [lambda-32P]UTP nucleotide incorporation was reduced and that of UMP nucleotide was increased giving a ratio of 1:188.
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PMID:Transcription of rat liver chromatin by Escherichia coli RNA polymerase: template properties after protein restriction. 36 67

An enzyme has been isolated from Escherichia coli strains harboring the I-like plasmid R64drd11, which is capable of initiating DNA synthesis on the circular, single-stranded DNA of phages phi X174, fd, and G4. In the conversion of these templates to duplex forms in vitro, the enzyme can substitute for the functions of E. coli dna B-dnaB-dnaC-dnaG proteins, E. coli RNA polymerase, and E. coli dnaG protein, respectively. The enzyme requires all four ribonucleoside triphosphates for optimal activity, although a combination of ATP, CTP, and GTP can almost completely satisfy the rNTP requirement. The enzyme appears to cooperate specifically with DNA polymerases III because single-stranded DNA-dependent synthesis takes place in extracts deficient in DNA polymerases I and II but not in extracts from a dnaZ mutant. Highly purified enzyme preparations consist mostly of two major polypeptides, Mr 140,000 and 180,000, when analyzed by sodium dodecyl sulfate gel electrophoresis. These polypeptides cosediment with the enzyme activity through a glycerol gradient with a sedimentation coefficient of 3.6 S. DNA priming activity in extracts of E. coli strains harboring the mutant plasmids R64drd11 or ColIdrd1, which are derepressed in functions of conjugational DNA transfer, severalfold higher than the activity from strains carrying the corresponding wild-type plasmid. This correlation suggests that the enzyme may play a role in conjugational DNA synthesis.
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PMID:A DNA primase specified by I-like plasmids. 38 43

A new class of fluorescent nucleotide analogs which contain the fluorophore 1-aminonaphthalene-5-sulfonate attached via a gamma-phosphoamidate bond has been synthesized. Both the purine and pyrimidine analogs have fluorescence emission maxima at 460 nm. Cleavage of the alpha-beta-phosphoryl bond produces change in both the absorption and fluorescence emission spectra. The fluorescence of the pyrimidine analogs is quenched; cleavage of the alpha-beta-phosphoryl bond of the UTP analog produces about a 14-fold increase in fluorescence intensity at 500 nm. Under the same conditions the fluorescence of the CTP analog increases about 8-fold, whereas the fluorescence of the purine analogs shows only a slight change. These derivatives are good substrates for Escherichia coli RNA polymerase with only slightly increased Km values and with Vmax values about 50 to 70% that of the normal nucleotides. They are used less efficiently by wheat germ RNA polymerase II. The ATP analog can be used by E. coli RNA polymerase to initiate RNA chains.
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PMID:Synthesis and properties of fluorescent nucleotide substrates for DNA-dependent RNA polymerases. 38 81

This paper presents the location and nucleotide sequence of a strong promoter of ColE 1. This promoter is of interest because of its greatly enhanced activity in the supercoiled state of the plasmid DNA (3) and its possible role in the maintenance of the plasmid replicon (4). This strong promoter is located at the restriction endonuclease Hae III f-h site 0.13 map units from the single EcoR 1 site proximal to the origin of DNA replication. The nucleotide sequence of the Hpa II l fragment of ColE 1 which contains this promoter has been determined. Initiation of transcription at this promoter occurred at two positions. Limited transcription by omitting one of the four nucleotide triphosphates allowed transcription to proceed to the fourth (-UTP) and to the twelfth (-CTP) nucleotides respectively. This was used to probe the interaction between RNA polymerase and the ColE 10.13 promoter by means of restriction cutting at the Hae III site at =27 and the Hha I site at +17. RNA polymerase binding alone blocks restriction cutting at the HAE III site but not at the Hha I site. Limited transcrption to the fourth nucleotide resulted in blocking at both sites. Transcription to the twelfth nucleotide resulted in partial cutting at the Hae III site and blocking at the Hha I site.
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PMID:Nucleotide sequence determination of a strong promoter of the colicin E 1 plasmid. Analysis of restriction sites protected by RNA polymerase interactions before and after limited transcription. 39 Apr 98

Metaphase chromosomes from the Chinese hamster cell line M3-1 were separated by means of a flow sorter. Two chromosome fractions were used for this study: A, which consisted of 95% pure chromosome no. 1, and B, which was 90% pure chromosome no. 2. The DNA of 10(6) chromosomes of each type was purified, and a 125I-cRNA transcript was synthesized in a reaction containing E. coli RNA polymerase and carrier-free 125I-CTP (1.7 Ci/mumole). The cRNA product synthesized with template DNA from 10(5) sorted chromosomes contained more than 10(6) dpm. The electrophoretic mobility profiles of the cRNAs on 7.5% SDS acrylamide gels demonstrated that more than 50% of the ribo-polymers were equal to or longer than marker E. coli met-tRNAf. In hybridization reactions 21% and 17% of the transcripts from Chinese hamster whole cell and sorted chromosome DNA hybridized to Chinese hamster DNA and did not hybridize significantly over background in reactions containing calf DNA at Crt values of 1.3 and 1.9 x 10(2) mole sec/l. Labelled cRNAs transcribed from the DNA of sorted chromosomes hybridized with the DNA of each sorted chromosome fractions at a Crt of 0.6 mole sec/l. This study demonstrated that the DNA can be (1) recovered from small numbers of highly purified flow sorted chromosomes, (2) used as template by E. coli RNA polymerase and (3) used to prepare a cRNA in reactions containing polymerase and carrier-free 125I-CTP to yield a product which can be employed for hybridization analysis.
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PMID:Transcription and hybridization of 125I-cRNA from flow sorted chromosomes. 42 70

Some properties of unprimed poly(A)-poly(U) synthesis by DNA-dependent RNA polymerase from Caulobacter crescentus were examined. The reaction required ATP and UTP as substrates and manganese as a divalent cation. Rifampicin completely inhibited the reaction at a concentration of 1 micron/ml, and the enzyme catalyzed the polymer synthesis well regardless of the presence of GTP, CTP or both. The chain length of the poly(A)-poly(U) synthesized was about one hundred base pairs, as estimated from a sedimentation velocity and the molar ratio of [3H]AMP to [gamma-32P]ATP incorporated into the poly(A)-poly(U). The reaction was dependent on the square of the enzyme concentration and the enzyme dimers formed complexes with poly(A)-poly(U) during the reaction.
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PMID:Properties of unprimed poly(A)-poly(U) synthesis by Caulobacter crescentus RNA polymerase. 42 57

A DNA . protein complex of about 150 S is isolated from purified spinach chloroplasts by Sepharose 4B gel filtration. A DNA-dependent RNA polymerase activity is found associated with the complex. This DNA protein complex is able to initiate RNA chains in vitro. The RNA synthesis is more dependent on CTP than other nucleoside triphosphates. 50% of the activity is still present with 0.6 M KCl. The temperature optimum occurs between 30 degrees C and 35 degrees C. Rifampicin and rifamycin SV have no inhibitory effect. TNA products have been characterized by gel filtration and by hybridization with chloroplast DNA (ctDNA). At the beginning of transcription DNA products are linked to the transcription complex and are later detached. The molecular weight of the product ranges between 0.07 X 10(6) and 2 X 10(6). A part of the product (3--4%) has a molecular weight higher than 2 X 10(6). No endogenous RNase activity was present during the molecular weight determinations experiments. Hybridization experiments show that at least 75% of the RNA products are hybridizable with ctDNA and that 40% of these products are composed of chloroplast ribosomal RNA, showing that rDNA is preferentially transcribed.
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PMID:Transcription activity of a DNA-protein complex isolated from spinach plastids. 46 44


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