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 crude RNA polymerase preparation was made from HeLa cells infected for 3 h with poliovirus. All virus-specific RNA species labeled in vitro (35S RNA, replicative intermediate RNA [RI], and double-stranded RNA [dsRNA]) would bind to poly(U) filters and contained RNase-resistant stretches of poly(A) which could be analyzed by electrophoresis in polyacrylamide gels. After incubation for 45 min with [3-H]ATP in the presence of the other three nucleoside triphosphates, the labeled poly(A) on the RI and dsRNA migrated on gels as relatively homogenous peaks approximately 200 nucleotides in length. In contrast, the poly(A) from the 35S RNA had a heterogeneous size distribution ranging from 50 to 250 nucleotides. In the absence of UTP, CTP, and GTP, the size of the newly labeled poly(A) on the dsRNA and RI RNA was the same as it was in the presence of all four nucleoside triphosphates. However the poly(A) on the 35S RNA lacked the larger sequences seen when the other three nucleoside triphosphates were present. When [3-H]ATP was used as the label in infected and uninfected extracts, heterogeneous single-stranded RNA sedimenting at less than 28S was also labeled. This heterogeneous RNA probably represents HeLa cytoplasmic RNA to which small lengths of poly(A) (approximately 15 nucleotides) had been added. These results indicate that in the in vitro system poly(A) can be added to both newly synthesized and preexisting RNA molecules. Furthermore, an enzyme capable of terminal addition of poly(A) exists in both infected and uninfected extracts.
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PMID:Polyadenylic acid on poliovirus RNA. III. In vitro addition of polyadenylic acid to poliovirus RNAs. 16 94

Rhinovirus type 14 RNA-dependent RNA polymerase complexes were isolated from microsomal and soluble fraction of infected KB cells. Maximum activities were measured at at 6 and 7 hours post inoculation (p.i.) for microsomal and soluble polymerases, respectively. Both polymerase activities are considerably reduced by 8 to 9 hours, p.i., and interval in which the in vivo rate of synthesis of viral RNA is maximal. In vitro RNA products of RNA polymerases in both fractions consist of ribonuclease-sensitive and ribonuclease-resistant RNA of heterogeneous sizes. Detergent treatment of the microsomal RNA polymerase(s) did not affect the total amount of RNA synthesized, the proportion of ribonuclease-sensitive RNA synthesized or the size of the RNA products. The data suggest that RV14RNA polymerase complexes are intially associated with membranes but are then irreversibly released into the soluble phase of the cytoplasm; possible explanations for this phenomena are discussed.
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PMID:Rhinovirus type 14 RNA polymerase complexes. 16 77

We describe a method for linking RNA and DNA covalently to finely divided cellulose through a diazotized aryl amine, which reacts primarily with guanine and uracil (thymine) residues of single strands. The high efficiency of coupling and high capacity of the cellulose for nucleic acid make possible a product with as much as 67 mug of nucleic acid per mg of cellulose. The product is especially suitable for hybridization experiments where very low backgrounds are important, and it is stable in 99% formamide at 80 degrees C so that hybridized nucleic acid can be recovered easily. Full length linear Simian Virus 40 (SV40) DNA, produced by cleavage of SV40(I) DNA with S1 nuclease, can be coupled to diazo cellulose with an efficiency of 80-90%, and is effective in hybridization experiments with SV40 DNA, complementary RNA synthesized in vitro from SV40(I) DNA with E. coli RNA polymerase, and the SV40-specific fraction of total RNA from SV40-infected and transformed cells. In these experiments an excess of cellulose-bound DNA was used, and the efficiency of hybridization was about 90% when ribonuclease treatment of the hybrids was omitted.
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PMID:Nucleic acid hybridization using DNA covalently coupled to cellulose. 16 82

The binding characteristics of [125I]-labeled L-triiodothyronine (T3) to chromatin isolated from rat liver nuclei were investigated. Binding of T3 to chromatin showed temperature-, incubation time-, and DNA concentration-dependence. According to Scatchard analysis, the apparent equilibrium dissociation constant was 225 pM, with a maximum binding capacity of about 0.2 pmoles per mg DNA. Displacement studies with unlabeled thyroxine (T4) and T3 showed that the binding sites for T4 might be the same as T3 but the binding affinity of the former was less than that of the latter. The binding was completely inhibited by the eukaryotic RNA polymerase inhibitor, rifampicin AF/021, but not by the prokaryotic inhibitor, rifampicin and alpha-amanitin. These observations indicate that the receptors for T3 have certain properties in common with RNA polymerase or other enzyme proteins which are sensitive to the rifampicin derivative. The hormone--chromatin fragments complex was solubilized from residual chromatin by digestion with DNase, but not with RNase, suggesting that the T3 receptors localize in the DNase-sensitive regions of DNA in the chromatin. This provides a useful method to use to investigate the localization of the receptor proteins in the chromatin and the interaction of the hormone-receptor complex with DNA.
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PMID:Binding characteristics of L-triiodothyronine to isolated rat liver chromatin. 19 15

Simian virus 40 (SV40) DNA I was transcribed with Escherichia coli RNA polymerase in the presence of gamma-32P-labeled ribonucleoside triphosphates in order to investigate the specificity of initiation of in vitro transcription. ATP and GTP served as predominant initiating nucleotides, the former being incorporated about twice as much as the latter. Cleavage of [gamma-32P]ATP-labeled SV40 complementary RNA (cRNA) with T1 RNase followed by homochromatographic analysis of the resultant 5' initiation fragments revealed the presence of four specific initiation fragments 6 to 9 nucleotides in length, designated AI, AII, AIIIa, and AIIIb. By means of hybridization of [gamma-32P]ATP-labeled SV40 cRNA to DNA from specific adenovirus 2-SV40 hybrids and specific restriction endonuclease fragments of SV40 DNA before chromatographic analysis, it was possible to identify and determine approximate localizations of five [gamma-32P]ATP initiation sites on the SV40 genome: one in Hin-G close to the Hin-G-B junction, giving rise to the AII fragment, two in the overalpping fragment Hin-A-Hae-A,giving rise to AI and AIII fragments, and two in the fragment Hin-A-Hae-E, also giving rise to AI and AIII fragments. All five sites either fall within or lie near regions of the genome that are cleaved by S1 nuclease and subject to partial alkaline denaturation. These five sites lie on the minus strand of SV40 DNA and initiate RNAs that are copied in a leftward direction. Cleavage of [gamma-32P]GTP-labeled cRNA with pancreatic RNase liberated three major 5' initiation fragments of short length, GI, GII, and GIII, suggesting the presence of three principal GTP initiation sites.
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PMID:Specificity of initiation of transcription of simian virus 40 DNA I by Escherichia coli RNA polymerase: identification and localization of five sites for initiation with [gamma-32P]ATP. 19 61

A nucleoprotein complex that is an intermediate in viral transcription has been isolated from simian virus 40 (SV40)-infected BSC-1 cells after lysing infected nuclei with Sarkosyl. It contain DNA, DNA-dependent RNA polymerase II, and nascent RNA chains. RNA chain elongation continues for several hours in vitro and is dependent on exogenous ribonucleoside triphosphates. The complex sediments in neutral sucrose gradients with a main peak at about 24 to 26S. When the nascent RNA on the complex is treated with RNase A, a fraction of the RNA remains resistant to RNase and is hydrogen bonded to the DNA template. The pulse-labeled RNase-resistant RNA can be chased into RNase-sensitive RNA, indicating that it is located at the 3' terminus of the RNA chain. The rate of RNA displacement from the DNA template is consistent with an average rate of RNA chain elongation of 15 to 30 nucleotides per min. At least 70% of the RNA synthesized in this in vitro system is SV40 specific. Hybridization with the separated strands of SV40 DNA and with fragments of SV40 DNA generated with endonucleases HindII + III indicates that this RNA is complementary to all regions of the "late" SV40 DNA strand. Studies of SV40 RNA synthesis in this partially purified preparation at early and late times after infection should provide a way of locating promoter sites for transcription and identifying the form of SV40 DNA that serves as a template for late transcription.
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PMID:Properties of simian virus 40 transcriptional intermediates isolated from nuclei of permissive cells. 19 3

Comparisons were made of the T1 ribonuclease digests of 32P-labeled nucleolar 45S RNA of intact Novikoff hepatoma cells and the RNA synthesized in vitro by isolated nucleoli. Approximately 200 oligonucleotide spots were found in the two-dimensional chromatogram of 45S nucleolar RNA labeled in vivo, which includes fragments of 18S and 28S rRNA and nonconserved spacer regions; four spots containing 2'-O-methyl nucleotides were not found in the corresponding pattern of RNA labeled in vitro. This high degree of fidelity was retained in the patterns of spots from the RNA produced with nucleolar chromatin as template. This specific expression of rDNA was lost when the nucleolar chromatin was completely deproteinized. Specific spots found in the control patterns were absent and many nonspecific oligonucleotides were found to be labeled. A similar nonspecific chromatogram pattern was found when nucleolar chromatin was transcribed with RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) of Escherichia coli. These results show that specificity of genetic expression in vitro of isolated chromatin of eukaryotic systems is dependent on the chromatin-associated proteins and the type of RNA polymerase present.
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PMID:Fidelity of synthesis of preribosomal RNA in isolated nucleoli and nucleolar chromatin. 19 90

Simian Virus (SV40) transcriptional intermediates (T.I.) were isolated from infected cell nuclei incubated in vitro in the presence of the four ribonucleoside triphosphates. The nascent mRNA strands in the viral DNA-RNA hybrid molecules were hydrogen bonded to their template by 200-250 nucleotides on the average, as judged from the extent of their RNase resistance and the aspect of T.I. under electron microscope after treatment with 50 per cent formamide. The RNA polymerase involved (RNA polymerase II) synthesized up to full length transcripts at a rate of approximately 150 nucleotides/min. at 25 degrees C. Each SV40 infected cell was found to contain about 200 active T.I. molecules at the peak of late transcription. The DNA in the T.I. molecules was exclusively form I DNA only in cell infected with the tsA30 mutant of SV40 that had been transferred to non-permissive temperature in order to arrest DNA replication, but both form I DNA and molecules behaving as replicative intermediates (R.I.) in wild type infected cells.
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PMID:Characterization of simian virus 40 transcriptional intermediates in infected CV1 cell nuclei. 23 Aug 57

The synthesis of ovalbumin mRNA sequences was studied in isolated nuclei from hen oviduct. Two different methods of analysis were used to distinguish in vitro synthesized from preexisting mRNA sequences: (i) Mercurated ribonucleotides were used for in vitro RNA synthesis, and the newly synthesized RNA was purified by chromatography on sulfhydryl-agarose and hybridized to radioactive ovalbumin cDNA. (ii) [3H]UTP was used to label the in vitro synthesized RNA. Hybridization to unlabeled mercurated cDNA, RNase A digestion, and subsequent purification of the hybrids on SH-agarose allowed the quantitation of newly synthesized ovalbumin mRNA sequences. Approximately 0.1% of the newly synthesized RNA was identified as ovalbumin RNA by both methods. The synthesis of ovalbumin RNA progressed during the incubation of nuclei and was sensitive to actinomycin D and low concentrations of alpha-amanitin. The preferential in vitro transcription of the ovalbumin gene (1000-fold over random transcription of the chicken genome) by RNA polymerase B (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) suggests that the specificity of in vitro RNA synthesis is retained in isolated nuclei.
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PMID:Preferential transcription of the ovalbumin gene in isolated hen oviduct nuclei by RNA polymerase B. 27 31

The tRNATyr precursor molecule, synthesized from phi 80 psu3+ DNA (containing a single tRNA gene) by DNA-dependent RNA polymerase and q factor, was about 205 nucleotides long. The main product of its digestion with a ribonuclease tii preparation from Escherichia coli showed the same electrophoretic mobility as tRNAtyr precursor isolated in vivo and was found to be identical to it when analysed using fingerprint techniques. This intermediate precursor synthesized in vitro was converted further by processing with ribonuclease P into an RNA identical size to mature tRNATyr. It was concluded that the initiation of transcription of the tRNATyr gene in vitro occurs at the same site as that of transcription in vivo and a termination occurs at about 80 nucleotides beyond the CCA end of tRNATyr.
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PMID:Processing by ribonuclease II of the tRNATyr precursor of Escherichia coli synthesized in vitro. 32 7


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