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)

Five phages which are morphologically similar to coliphage T7 but attack other host bacteria have been compared to T7 and to its relative, T3, by the following criteria: (a) cross-reactivity with antisera against T7 and T3, (b) DNA base sequence homologies, as determined by the C0t technique, (c) synthesis of two phage-coded enzymes: RNA polymerase and SAMase, (d) patterns of phage-directed protein synthesis, as determined by SDS-polyacrylamide gel electrophoresis of phage coat subunits. As judged by all these criteria, Pseudomonas phage PX3 is not related to T7; thus, morphological similarity was attributed to convergent evolution. The other phages, i.e. Serratia phage IV, Psuedomonas phage gh-1, Citrobacter phage ViIII and Klebsiella phage No. 11, were considered to be related to T7 on the basis of similarities in the patterns of phage-coded proteins and because, early after infection, these phages induced, as T7 does, an RNA polymerase which specifically transcribes the DNA of thehomologous phage. Phages IV and No. 11 also induced the early synthesis of SAMase (previously only known to occur upon T3 infection). With the exception of phage IV, however, DNA base sequence homologies with T7 or T3 seem to be poor or non-existent. The tested phages, again with the exception of phage IV, did not react with antiserum against T3 or T7. It is concluded that a particular pattern of phage-directed protein synthesis (as characterized by polyacrylamide gel electrophoresis and enzyme tests) may provide evidence for phylogenetic relationships between phages, even in cases where other criteria, such as genetic recombination, serological cross-reaction, and DNA base sequence homologies, fail to indicate relatedness.
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PMID:The strategy of infection as a criterion for phylogenetic relationships of non-coli phages morphologically similar to phage T7. 9 Jan 10

Isolated rat liver nuclei were incubated under appropriate conditions in the presence of 0.5 micrograms/ml alpha-amanitin and an RNAase inhibitor prepared from cytosol fraction, together with alpha-32P-UTP or alpha-32P-CTP and three other nucleoside triphosphates. RNA extracted by an SDS-hot phenol procedure was fractionated with sucrose density gradient centrifugation followed by acrylamide gel electrophoresis. Fingerprint analysis of the in vitro synthesized "5S" RNA, which was slightly larger than mature 5S RNA on gel electrophoresis, showed that it contained all the sequences of mature 5S RNA except for the oligonucleotide at the 3' end. Instead, it contained two additional spots which were not present in mature 5S RNA. Analysis of the extra spots revealed that they were derived from the 3' end of the in vitro synthesized "5S RNA, which were sequenced tentatively as -CUUGAUGCUUoh (extra sequence underlined). The 5' end of the product was (p)pGU--. Isolated HeLa cell nuclei synthesized similar sized "5S" RNA under the same conditions. We conclude from these results that in isolated nuclei of these mammalian cells RNA polymerase III starts transcription of 5S RNA gene at the same site as the 5' end of mature 5S RNA, proceeds toward the 3' direction and stops at a site probably 8 nucleotides downstream from the 3' end of mature 5S RNA. Experiments with a short pulse and with various "chases" have demonstrated the presence of a short-lived precursor 5S RNA which is similar in size and sequence to in vitro "5S" RNA, suggesting that 5S RNA is synthesized in vivo as a longer precursor molecular as demonstrated in this in vitro system, and is rapidly processed into mature 5S RNA.
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PMID:In vitro synthesis of a 5S RNA precursor by isolated nuclei of rat liver and HeLa cells. 11 Apr 59

Bacillus subtilis RNA polymerase holoenzyme prepared by several standard methods utilizes bacteriophage T7 DeltaD111 DNA as an efficient template. The major RNA products are specific transcripts from T7 promoters A(1) and C; these promoters are also efficiently utilized by RNA polymerases purified from a wide range of other bacterial species [Wiggs, J., Bush, J. & Chamberlin, M. (1979) Cell 16, 97-109]. In contrast, B. subtilis RNA polymerase preparations purified by a modification of the method of Burgess and Jendrisak (designated fraction 5) utilize T7 DeltaD111 promoters A(1) and C and an additional promoter site, J, which has been located at 90.6% on the standard T7 physical map. This promoter is not used by B. subtilis core RNA polymerase or by RNA polymerase from any other bacterial species we have tested. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of fraction 5 RNA polymerase shows that it contains B. subtilis components sigma and delta and a polypeptide of M(r) 92,000 in addition to the B. subtilis beta, beta', and alpha subunits. Chromatography of fraction 5 on single-stranded DNA-cellulose gives an enzyme fraction, Bs I, that is indistinguishable from B. subtilis RNA polymerase holoenzyme both in its peptide composition (betabeta'alpha(2)sigma) and in the selective transcription of only T7 RNAs A(1) and C. Chromatography of fraction 5 on phosphocellulose yields an enzyme fraction, Bs II, devoid of sigma subunit but containing the M(r) 92,000 peptide and traces of delta. This fraction synthesizes predominantly T7 J RNA in vitro together with traces of T7 A(1) and C RNAs. Hence, B. subtilis RNA polymerase fraction Bs II appears to contain a form of RNA polymerase that can transcribe selectively without detectable amounts of B. subtilis sigma subunit and that utilizes a promoter site not used by other known bacterial RNA polymerases. The structural basis for this specificity is not yet known.
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PMID:Altered promoter selection by a novel form of Bacillus subtilis RNA polymerase. 11 48

Using purified yeast mitochondrial DNA as a template for E. coli RNA polymerase (holoenzyme) complementary mitochondrial RNA has been synthesized in vitro. This RNA has been used to direct a low background E. coli S-30 protein-synthesizing system. The synthesis of mitochondrial polypeptides has been detected by using antiserum raised against purified cytochrome c oxidase holoenzyme and shown to be specific for this antigen. The antiserum-antigen complex was dissociated and subject to SDS-polyacrylamide gel electrophoresis and the presence of 3 polypeptides of 39, 31, and 26 X 10(3) daltons molecular weight demonstrated, which correspond to the subunits synthesized by mitochondria in whole cells which are inhibited with cycloheximide.
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PMID:Synthesis of cytochrome c oxidase polypeptides in an Escherichia coli cell-free system directed by Saccharomyces cerevisiae mitochondrial DNA. 18 80

14C-labeled SV40 DNA has been transcribed with E. coli RNA polymerase using 3H-labeled ribonucleotide triphosphates as precursors. The resulting transcription complexes were then photochemically crosslinked with the psoralen derivative, 4'-aminomethyl-4,5', 8-trimethylpsoralen (AMT), at 37 degrees C and analyzed in SDS-sucrose gradients. It was found that the photochemical crosslinking procedure caused the nascent RNA chains to co-sediment with their double-stranded (helical) SV40 templates in the denaturing sucrose gradient. This result and several control experiments suggest that covalent linkages have formed between nascent RNA and helical DNA after the photochemical reaction. The crosslinking phenomenon was observed to be independent of the superhelical state of the DNA used as the template. Prior addition of EDTA to stop the transcription is not required for successful crosslinkage.
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PMID:Photochemical crosslinking of transcription complexes with psoralen. I. Covalent attachment of in vitro SV40 nascent RNA to its double-stranded DNA template. 20 76

E. Coli RNA polymerase binding to different DNAs (from E. Coli, 5-bromodeoxyuridine (BrdUrd) substituted DNA and poly [d(BrU-A)] was induced with ultraviolet (U.V.) light to form protein-DNA crosslinked complexes. Two independent methods of analysis, polyacrylamide gel electrophoresis in SDS and chloroform extraction indicated the formation of a stable complex between the enzyme and DNA. The complexes were formed under different ionic strength conditions, at low enzyme to DNA ratios in order to approach the conditions of specific binding. In contrast there was no crosslinking of the complex in 1 M KCl solution which dissociates the enzyme from DNA. The efficiency of formation of strongly bound complex was found to be much higher with holoenzyme than with core enzyme. The following results were obtained : 1) The large subunits beta and beta' were found to be bound to DNA. 2) Relatively small amount of sigma subunit were bound to DNA while alpha subunits were essentially not attached to DNA. The high binding affinity of beta and beta' subunits was also observed in the studies of isolated subunits. These results lead to a model of enzyme-DNA complex in which the large beta and beta' subunits provide the contacts between the RNA polymerase and the DNA.
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PMID:Subunit topography of RNA polymerase (E. coli) in the complex with DNA. 35 32

A new procedure for the purification of B. subtilis RNA polymerase, based on mild lysis of cells, low speed centrifugation, gel filtration, DEAE-Sephadex chromatography and affinity chromatography on DNA-cellulose, yields three forms of enzyme referred here as enzyme A, B and C. As revealed by SDS gel electrophoresis, enzyme A has the subunit structure of core polymerase plus some small polypeptides. Its catalytic properties are similar to those of core polymerase. Enzyme B has the composition of core polymerase. Both enzymes A and B can be stimulated by the addition of beta factor. Enzyme C has the holo-enzyme composition. The pattern of sensitivity of the three forms of enzyme towards KCl are very different: enzymes A and B, even at low concentration of salt, are inhibited with all the DNA templates tested, whereas enzyme C shows a pattern of stimulation specific for each DNA tested. The transcripts of the three enzymes on phage SPP1 DNA template have been analyzed by hybridization to the separated strands. Only enzyme C selectively transcribed the H strands.
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PMID:RNA polymerase from Bacillus subtilis: isolation of core and holo enzyme by DNA-cellulose chromatography. 40 60

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

The earliest stages of mouse embryogenesis, from fertilisation to the two-cell stage, are characterised by an extremely low level of RNA synthesis. Indeed, during this period, RNA polymerase II activity and incorporation of labelled precurosrs into heterogeneous RNA are not detectable, and there is no increase in the poly(A) content of the embryo, but rather a slight decrease. The rate of protein synthesis remains low and relatively constant throughout the one- and two-cell stages. However, qualitative analysis of the protein synthetic profile on SDS gels has revealed changes which appear around the late one-cell to early two-cell stage. This early change in the pattern of polypeptide synthesis represents the first major qualitative molecular change found so far in development. We present evidence which suggests that the increased synthesis at the early two-cell stage of a small number of polypeptides of molecular weight 35,000 is not dependent on transcription, but rather represents control at a post-transcriptional level using mRNAs synthesised before fertilisation.
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PMID:Post-transcriptional control in the early mouse embryo. 50 84

Embryos and larvae of the brine shrimp, Artemia salina, provide a useful biological system for biochemical studies of animal development. Dormant encysted embryos can be cultured readily in the laboratory to provide large quantities of free-swimming nauplius larvae. The rate of synthesis of all classes of RNA in swimming larvae declines markedly between 24 and 72 h after immersion of dormant embryos in sea water. Nuclei were isolated from 24-72 h larvae and RNA polymerase activity was measured under conditions in which the nuclei remained intact. Total RNA polymerase activity of isolated nuclei decreased in parallel with RNA synthesis in vivo. RNA polymerases were solubilized from nuclei and fractionated by chromatography on DEAE-cellulose. The levels of both RNA polymerases I and II also decreased in parallel with RNA synthesis in vivo. The specific activity of highly purified RNA polymerase II was determined by comparison of enzyme activity with the mass of RNA polymerase II subunits displayed on SDS gels. The specific activities of RNA polymerase II preparations from 24 and 72 h larvae were identical. The number of polymerase II molecules was estimated from the mass of the subunits. The number of molecules per nucleus declined from 20,000 at 24 h to 3500 at 72 h.
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PMID:DNA-dependent RNA polymerases from Artemia salina: decreasing polymerase activities and number of polymerase II molecules in developing larvae. 72 50


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