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 phi29 early mRNA's synthesized in infected Bacillus subtilis were studied by using sedimentation velocity analysis, polyacrylamide gel electrophoresis, and hybridization of phi29 DNA fragments generated by the restriction endonuclease Eco RI. Viral RNAs synthesized in vivo in the resence of chloramphenicol were found to hybridize to Eco RI-A, -C, and -D fragments, but not to Eco RI-B and -E fragments, of the viral genome. Major early mRNA sedimenting as 16S material in neutral sucrose gradients was examined in detail. Radioactive phi29 RNA, purified by sucrose gradient centrifugation, was hybridized to either the Eco RI-A or Eco RI-C DNA fragment. The RNA was eluted from the hybrids and then tested for complementary hybrid formation with Eco RI-A and -C fragments. RNA eluted from the Eco RI-A fragment annealed only to the Eco RI-A fragment and not to the Eco RI-C fragment. Similarly, RNA eluted from the Eco RI-C fragment hybridized to the Eco RI-C and -D fragments. Viral RNAs synthesized in vitro using B. subtilis RNA polymerase hybridized to both Eco RI-A and -C DNA fragments. Furthermore, RNA initiated with [gamma-(32)P]GTP also hybridized to both Eco RI-A and -C fragments. These results indicate that there are at least two efficient promotors for early transcription on the phi29 chromosome. In addition, a low-molecular-weight RNA initiated with [gamma-(32)P]ATP was found to hybridize exclusively with the Eco RI-A fragment. Kinetic studies of phi29 mRNA synthesis during the lytic cycle have shown that viral RNAs hybridizable to the Eco RI-A and -C fragments are synthesized immediately after phage infection. On the other hand, mRNA specific for the Eco RI-B fragment was not synthesized for several minutes after phage infection. Based on the results of the in vivo and in vitro transcription studies, a transcription map of the phi29 chromosome is proposed.
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PMID:Transcription of the genome of bacteriophage phi 29: isolation and mapping of the major early mRNA synthesized in vivo and in vitro. 40 15

A modified form of Bacillus subtilis RNA polymerase containing a phage SP01-coded regulatory protein (the gene 28 product) selectively transcribes "middle" genes of the phage genome in vitro. In this paper, we identify a subset of restriction endonuclease fragments of SP01 DNA that promote specific transcription by the phage-modified polymerase. In the absence of nucleoside triphosphates, RNA polymerase containing the gene 28 protein selectively binds to these DNA fragments thereby forming stable binary complexes that can be isolated on nitrocellulose filters. In contrast, unmodified RNA polymerase containing sigma factor selectively binds to and transcribes a subset of phage DNA fragments that contain "early" sequences and that are in large part distinct from the fragments recognized by the phage-modified transcriptase. Our results strongly suggest that phage "early" and "middle" genes are transcribed from distinct promoters and that the RNA polymerase containing the gene 28 protein binds to sites that are located at or near promoters for SP01 "middle" genes.
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PMID:Promoter recognition by phage SP01-modified RNA polymerase. 41 6

Non-glucosylated T4 DNA was restricted with the endonuclease EcoRI and the mixture of DNA fragments separated by gel electrophoresis and transcribed with purified Escherichia coli RNA polymerase. Three purified fragments were shown to act as templates for tRNA synthesis. A smaller fragment, shown to be hybridizable to 32P-labeled T4 tRNA was not transcribable. It was concluded that the promoter for T4 tRNA synthesis had been separated from the structural genes in the smaller fragment by EcoRI and that the distal portion of the tRNA gene cluster lacks internal promoters which display in vitro activity. Preparations of non-glucosylated T4 DNA were never fully restricted with EcoRI and when the larger purified fragments carrying the tRNA were restricted with excess enzyme only a slight cleavage to yield the smaller fragments was obtained. The property of the DNA-limiting complete restriction is not know.
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PMID:Isolation of the transfer RNA genes of bacteriophage T4 and transfer RNA synthesis in vitro. 42 Aug 49

Restriction endonuclease Bgl II cleaves T7 DNA at a unique site (28.76% on the standard T7 map), yielding two fragments of molecular weights 18.9 x 10(6) (A) and 7.6 x 10(6) (B). Fragment B, representing the leftmost portion of the genome, has been purified by zone sedimentation. Transcription of fragment B by T7-specific RNA polymerase gives only r-strand-specific RNA. Analysis of the products by polyacrylamide gel electrophoresis reveals four major RNA species which have apparent molecular weights of 2.1 x 10(6), 1.36 x 10(6), 0.85 x 10(6) and 0.125 x 10(6), respectively. Each of these RNAs is reduced in size when transcription is carried out with fragment B, which has been shortened by treatment with Escherichia coli exonuclease III. Therefore, each of the transcripts must be terminated at the right end of fragment B. Analysis of the molecular weights of the four transcripts produced from whole and from exonucleolytically shortened fragment B suggests that these transcripts are read from promoters located at 13.5, 18.9, 22.6, and 27.9%, respectively, on the standard T7 map. Hence, there are at least four promoters governing the transcription of the class II region. Transcripts initiated at these promoters on intact T7 DNA appear to read through the class II and part of the class III genetic region and terminate at the strong terminator for T7-specific RNA polymerase near 61%. Transcription of fragment B which has been cleaved with the restriction endonuclease Hpa I seems to activate a fifth promoter for T7-specific RNA polymerase. This promoter appears to be identical to the promoter previously described by Oakley and Coleman (Proc. Natl. Acad. Sci. U.S.A. 74:4266-4270, 1977) that maps near 15% on the standard T7 map. Little or no RNA is read from T7 Bgl II fragment B, which has a mobility expected for a transcript read from this promoter. However, upon cleavage with Hpa I, this promoter is utilized approximately 10-fold more efficiently than the other class II promoters. The mechanism of this activation is not yet known.
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PMID:Mapping of class II promoter sites utilized in vitro by T7-specific RNA polymerase on bacteriophage T7 DNA. 43 May 92

Adenosine (beta,gamma-imido)triphosphate (AMP-PNP) and guanosine (beta,gamma-imido)triphosphate (GMP-PNP) are analogs of ATP and GTP with non-hydrolyzable gamma-phosphates. Although both AMP-PNP and GMP-PNP were used in place of ATP and GTP by Escherichia coli RNA polymerase to transcribe vaccinia virus DNA, only GMP-PNP was used by the transcriptase present within vaccinia virus cores. AMP-PNP specifically prevented initiation of transcription, since RNA initiated in the presence of ATP, GTP, and CTP was subsequently elongated by incubating the washed cores in the presence of AMP-PNP, GTP, CTP, and UTP. The RNA formed in this manner, however, was (i) several times longer than normal transcripts, indicating a defect in chain termination and/or cleavage of nascent RNA, (ii) was not polyadenylylated (although free polyadenylic acid formed), and (iii) was not extruded from the virus cores. Nearest neighbor analysis demonstrated that AMP-PNP was incorporated adjacent to all four nucleotides, and hybridization to restriction endonuclease fragments of vaccinia virus DNA indicated that the high-molecular-weight RNA was transcribed from representative fractions of the entire genome. The possibility of a block in processing rather than or in addition to a block in chain termination was suggested by the cleavage of the high-molecular-weight RNA within the core after replacement of AMP-PNP with ATP. Cleavage of purified high-molecular-weight RNA by a soluble endoribonuclease extracted from vaccinia virus cores, however, was not dependent upon ATP, nor was it inhibited by AMP-PNP. The latter results suggest that AMP-PNP blocks a step preceding cleavage.
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PMID:Multiple roles for ATP in the synthesis and processing of mRNA by vaccinia virus: specific inhibitory effects of adenosine (beta,gamma-imido) triphosphate. 69 Nov 15

DNA-dependent RNA polymerase class C (or III) has been solubilized from either uninfected or adenovirus-2-infected HeLa cells and purified by chromatography on phosphocellulose, DNA-cellulose, CM-Sephadex and DEAE-Sephadex. The last column separated the enzyme into three forms CI, CII and CIII, which were completely free of RNA polymerases class A and B and of DNase and RNase. The total and the relative amount of these different enzyme C forms did not vary whether purified from uninfected or infected cells. Irrespective of the stage of purification, the three enzyme forms transcribed deproteinized adenovirus-2DNA very efficiently. This transcription was highly sensitive to elevated ionic strength (especially in the presence of Mg2+) and was accompanied by continuous reinitiation as shown by adding poly(rI), a potent inhibitor of initiation. In addition heparin-resistant initiation complexes could be formed at elevated temperature. The RNA synthesized in vitro on deproteinized intact adenovirus-2 DNA by the different forms of RNA polymerase class C, has been characterized. Analysis of the transcripts by gel electrophoresis, RNA self-annealing, hybridization to separated adenovirus-2 DNA strands and to restriction endonuclease (BamHI, HindIII), adenovirus-2 DNA fragments have demonstrated that restriction endonuclease (BamHI, HindIII), adenovirus-2 DNA fragments have demonstrated that the various regions of the adenovirus-2 genome were randomly transcribed. In addition, hybridization of RNA transcripts labelled at their 5' end by either [gamma32P]ATP or [gamma-32P]GTP indicated that not only elongation but also initiation occurred randomly through the entire adenovirus-2 genome, irrespective of the form of the enzyme and of the origin of the cells (normal or infected). The results are discussed in terms of the components which are possibly involved in specific transcription.
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PMID:Transcription in vitro of adenovirus-2 DNA by RNA polymerases class C purified from uninfected and adenovirus-infected HeLa cells. 71 Apr 51

We estimate that E. coli RNA polymerase is able to form stable, rifampicin-resistant, pre-intiation complexes with Adenovirus 2 DNA at three to six binding sites. The number of RNA chains initiated from such complexes has been determined form the incorporation of gamma-32P-ATP and -GTP at two rifampicin concentrations (7 mug/ml and 24 mug/ml) and after pre-incubation at either 25 or 37 degrees C. The total number of RNA chains initiated ranges from 2.6 per Ad 2 DNA molecule at a rifampicin concentration of 24 mug/ml and pre-incubation temperature of 25 degrees C, to 5.4 per Ad 2 DNA molecule at a rifampicin concentration of 7 mug/ml and pre-incubation temperature of 37 degrees C. Efficient initiation with GTP occurs only after pre-incubation at 37 degrees C whereas initiation with ATP is equally as efficient at either pre-incubation temperature. Promoters for initiation with ATP have been localized to the leftmost 58% of the Ad 2 DNA molecule, defined by the EcoR.RI restriction endonuclease fragment A; promoters for initiation with GTP are located on the remaining 42% of the Ad2 DNA molecule. It is likely that on Adenovirus 2 DNA each RNA chain is initiated from a unique binding site which constitutes a seperate promoter for E. coli RNA polymerase.
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PMID:In vitro transcription of adenovirus 2 DNA. II. Quantification and localization of promoters for E. coli RNA polymerase. 76 52

The 1400 base pair repeat produced by digestion of calf satellite I DNA (phi = 1.714 g/cm3) with EcoRI, was cloned in E. coli. The hybrid plasmid (pGM 214) which contains the ColE1-Ap vector (pSF 2124) and the 1400 base pair fragment replicates stably in E. coli and can be amplified by chloramphenicol treatment. No clone was found in which more than one "repeat unit" of the satellite I DNA was present in the chimaera plasmid. Digestion of the original satellite I and the plasmid pGM 214 with R-SmaI shows that the satellite DNA replicated in E. coli is cleaved by the restriction endonuclease SmaI whereas the original satellite I DNA from calf thymus is not, suggesting that the satellite I contains a large amount of modified cytosine or guanosine, probably 5-methyl-cytosine. R-EcoRI* produces a number of fragments with the satellite I in the range of 300 base pairs to 1400 base pairs. A physical map of pGM 214 (and pSF 2124) with R-EcoRI, R-HincII, R-HindIII, R-SmaI, R-BamI and R-EclI was constructed. The 1400 base pair "repeat unit" in the pGM 214 is efficiently transcribed in vitro by purified RNA polymerase, starting from a pSF 2124 promoter. The restriction enzyme EclI produces a 350 base pair repeat with calf satellite II (phi = 1,722 g/cm3), whereas the satellite I is not cut by this enzyme.
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PMID:Cloning of calf thymus satellite I DNA in Escherichia coli. 79 69

We have determined the nucleotide sequence of 75 nucleotides of the 3'-untranslated portion of normal human alpha globin mRNA which corresponds to the elongated amino acid sequence of the chain termination mutant Hb Constant Spring. This was accomplished by sequence analysis of cDNA fragments obtained by restriction endonuclease or T4 endonuclease IV cleavage of human globin cDNA synthesized from globin mRNA by use of viral reverse transcriptase. Analysis of cRNA synthesized from cDNA by use of RNA polymerase provided additional confirmatory sequence information. Possible polymorphism has been identified at one site of the sequence. Our sequence overlaps with, and extends the sequence of 43 nucleotides determined by Proudfood and coworkers for the very 3'-terminal portion of human alpha globin mRNA. The complete 3'-untranslated sequence of human alpha globin mRNA (112 nucleotides including termination codon) shows little homology to that of the human or rabbit beta globin mRNAs except for the presence of the hexanucleotide sequence AAUAAA which is found in most eukaryotic mRNAs near the 3'-terminal poly (A).
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PMID:Nucleotide sequence of 3' untranslated portion of human alpha globin mRNA. 90 79

A factor stimulating RNA polymerase II from Ehrlich ascites tumor cells was purified. The final preparation appeared almost homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular weight of 38 000. The endonuclease activity of about 10 mug of purified factor, if any was well below the 10(-5) mug equivalent of pancreatic deoxyribonuclease, indicating that the stimulation of RNA synthesis by this factor was not due to contaminating endonuclease. This factor specifically stimulated RNA polymerase II on native DNA as template and did not affect RNA polymerase I at all. The molecular size of RNA synthesized in the presence of this factor increased markedly compared with that synthetized by RNA polymerase II alone.
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PMID:Purification of a factor from Ehrlich ascites tumor cells specifically stimulating RNA polymerase II. 99 Feb 65


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