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)

The in vitro transcription of viral specific DNA sequences in nuclei and chromatin isolated from mouse cells chronically infected with Moloney murine leukemia virus (Mo-MuLV) has been studied. The in vitro RNA synthesized by Escherichia coli RNA polymerase has been isolated by sulfhydryl affinity column following reaction in the presence of 5-mercuriuridine triphosphate. By comparison of the Crt curves of the in vitro RNA with that of 70S viral RNA, the content of viral sequences is found to be 1.3% in nuclei product and 0.24% in chromatin product which is lower than the 2.5% found in chromatin associated RNA. This latter value, however, is very close to the in vivo viral RNA content in pulse-labeled [3H]RNA of the infected cells. Unexpectedly, it is observed that over 20% of the chromatin associated RNA prelabeled in vivo with [5-3H]uridine is elongated and tagged with Hg atoms during RNA synthesis catalyzed by the exogenous E. coli RNA polymerase in the presence of Hg-UTP. The elongation reaction is dependent on the presence of all four nucleotide triphosphates and appears to be due to E. coli RNA polymerase per se. It is suggested that most of the viral specific sequences observed in the in vitro RNA products are very likely initiated and derived from the chromatin associated species. The implication of the present findings for in vitro RNA synthesis in nuclei and chromatin as related to regulation of gene expression is discussed.
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PMID:In vitro transcription of Moloney leukemia virus genes in infected cell nuclei and chromatin: elongation of chromatin associated ribonucleic acid by Escherichia coli ribonucleic acid polymerase. 32 7

In the presence of RNA polymerase, RNase H, discriminatory factors alpha and beta, Escherichia coli binding protein, DNA elongation factor I, DNA elongation factor II preparation, DNA polymerase III, and ATP, UTP, GTP, CTP, dATP, dTTP, dGTP, and dCTP, fd viral DNA can be quantitatively converted to RFII containing a unique gap in the linear minus strand. This gap, mapped with the aid of restriction endonucleases HinII and HpaII, is located within Fragment Hpa-H of the fd genome. The discrimination reaction has been resolved into two steps: Step A, fd viral DNA, E. coli binding protein, and discriminatory factors alpha and beta form a protein DNA complex; Step B, the complex isolated by agarose gel filtration selectively forms fd RFII when supplemented with RNase H, RNA polymerase, and the DNA elongation proteins. The omission of any of the proteins described above during the first reaction resulted in either no discrimination or a decrease in discrimination when the missing protein was added during the second step. Results are presented which indicate that E. coli binding protein, discriminatory factors alpha and beta, and RNase H must be present during the time RNA synthesis occurs in order to selectively form RFII from fd DNA and not phiX RFII. The amount of fd and phiX174 RNA-DNA hybrid formed in vitro is directly related to the DNA synthesis observed. Thus, under discriminatory conditions, only fd viral DNA leads to fd RNA-DNA complexes and no phiX RNA-DNA hybrid is formed. Under nondiscriminatory conditions, both DNAs yield RNA-DNA hybrids and DNA synthesis. In the absence of discriminatory factor alpha, no RNA-DNA hybrid is formed with either DNA, and in turn, no DNA synthesis is detected with either DNA template.
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PMID:Selective inhibition of phiX RFII compared with fd RFII DNA synthesis in vitro. II. Resolution of discrimination reaction into multiple steps. 32 48

Lutropin and human choriogonadotropin stimulated the endogenous chromatin-associated polymerase activity in purified chromatin prepared from nuclei of bovine corpus luteum. Chromatin was incubated in two different buffer systems: one that mainly supports the activity of polymerase I, another that supports the activity of polymerase II and is largely alpha-amanitin sensitive. The hormones lutropin and chorigonadotropin stimulated an increase in the rate of incorporation of [14C]ATP or [14C]UTP into RNA in both buffer systems. Follitropin, prolactin and beta-corticotropin had no stimulatory effect. Neither the alpha nor beta subunit of lutropin stimulated RNA synthesis. When premixed, the subunits rapidly formed the active molecule. A maximum response to RNA synthesis was achieved by a 10(-9) M concentration of human choriogonadotropin. Considerable activity was obtained at 10(-11) M human choriogonadotropin. There was no lutropin stimulation to RNA synthesis using calf thymus DNA and Escherichia coli RNA polymerase.
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PMID:Lutropin stimulation of RNA synthesis in corpus luteum chromatin. 32 86

We have employed mercury-substituted UTP to study the transcription of duck reticulocyte chromatin in vitro by Escherichia coli RNA polymerase. We find that the use of this method results in large overestimates of the amount of de novo synthesis of globin-specific RNA sequences. The artefact arises because endogenous globin RNA can serve as a template for the RNA polymerase, resulting in the formation of a duplex product in which one strand is the endogenous message, and the other is the mercury-labeled complementary strand. Subsequent purification of the mercury-substituted RNA on thiol-agarose results in copurification of endogenous globin sequences. We document the details of this mechanism and describe methods which will eliminate the artefact.
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PMID:Analysis of in vitro transcription of duck reticulocyte chromatin using mercury-substituted ribonucleoside triphosphates. 33 58

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 new assay yielding mechanistic information on the initiation reaction of Escherichia coli RNA polymerase has been developed. It was found to be useful in characterizing the promoters of bacteriophage DNA templates. The binding of the first two triphosphates in an RNA sequence was determined to be equilibrium ordered with ATP binding first followed by UTP on the lambda promoters PL. and PR. The products resulting from phosphodiester bond formation, pppApU and PPi, dissociated rapidly in the absence of the other triphosphates required for RNA synthesis. The resulting steady state conversion of ATP and UTP into pppApU was the basis for the new assay. The rate-limiting step in the initiation reaction was not precisely determined, but it was argued not to be entirely the release of product. The Zn2+ chelator, 1,10-phenanthroline, was partially characterized and found to be an uncompetitive inhibitor of ATP in the reaction (Ki = 100 micrometer). The unique advantage of this steady state assay is that several steps in the RNA initiation process are amplified kinetically and thus can be examined separately with techniques applicable to any other two-substrate, two-product enzyme reaction.
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PMID:A steady state assay for the RNA polymerase initiation reaction. 36 12

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

E. coli DNA dependent RNA polymerase was modified by diethylpyrocarbonate. Optical and kinetic properties of the reaction were studied. More than 90% of RNA polymerase activity is inhibited by introduction of 9--11 ethoxyformyl groups per enzyme molecule without loss of its ability to bind DNA template. Furthermore the modified enzyme is able to form tight complexes with DNA and to compete with native enzyme for the formation of rifampicin-resistant complex. The ratio of the complex formation constants for the native and modified enzyme was determined to be equal to 10. The enzyme modified to such extent loses the activity in DNA dependent RNA as well as pppApU synthesis. Vmax value rather than Km value for both ATP and UTP decreases following the modification reaction. Incubation of the enzyme modified to the 10% of residual activity with 0.2 M hydroxylamine for 2 hours results in restoration of RNA polymerase activity. Most but not all of the modified histidyl residues restore their native structure. Two of 13 histidyl residues were modified irreversibly due to Bamberger's cleavage reaction, but these two residues were found to be unessential for RNA polymerase activity. Reaction with higher concentration of the diethylpyrocarbonate induces modification of more than 15--20 histidyl residues and leads to irreversible inactivation of the enzyme. Nevertheless the modification of the additional histidyl redidues was reversible as well as the modification of the first 11 residues. RNA polymerase modified to such extent loses the ability to bind DNA. Preformation of the initiated ternary complex of RNA polymerase with template and product fails to protect the enzyme from reversible inactivation at a low reagent concentration, but markedly decreases the rate of the irreversible and unspecific modification of sulfhydryl or amino groups of the enzyme. Reaction with the ternary complex results in reversible inactivation of the enzyme with respect to elongation of RNA chains as well as the pyrophosphate exchange reaction. The complex itself was, however, completely stable under the reaction conditions and the enzyme subunit structure was also conserved after the reaction. Evidently, the mild modification of the histidyl residues with diethylpyrocarbonate selectively inhibits RNA chain elongation.
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PMID:[Modification of the RNA-polymerase of Escherichia coli by diethylpyrocarbonate]. 37 63

dUflTP was tested as substrate in the E. coli RNA polymerase system using poly(dAT) as template. dUflTP could replace UTP when Mn++ was utilized as divalent cation instead of Mg++. The level of transcription with the fluoro analog was then 55% of that with UTP.
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PMID:2'-Deoxy-2'-fluorouridine-5'-triphosphates: a possible substrate for E. coli RNA polymerase. 37 93

We describe a new method for quantitatively assaying the omega subunit of Escherichia coli RNA polymerase. The assay is based on the ability of RNA polymerase holoenzyme to catalyze the continuous synthesis of the dinucleotide pApU on a poly[d(A-T)] . poly[d(A-T)] template when supplied with AMP and UTP as substrates. Core enzyme, lacking omega subunit, catalyzed this reaction at a rate less than 1% that of holoenzyme. The omega subunit was not released from the enzyme/DNA complex during dinucleotide synthesis. Using this assay, a titration of a fixed concentration of core enzyme was observed with increasing concentrations of added omega subunit. Below a 1:1 omega:core ratio the measured activity increased linearly with omega concentration, whereas above a 1:1 ratio the activity remained constant. An immediate application of the assay is in determining the concentration of active omega, or equivalently of active holoenzyme, in any RNA polymerase preparation.
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PMID:A noncycling activity assay for the omega subunit of Escherichia coli RNA polymerase. 37 16


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