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)

Linear simian virus 40 DNA has been transcribed in vitro with wheat germ RNA polymerase II. Transcription products have been fractionated on polyacrylamide gels and several discrete sized RNA bands are seen. The RNA band pattern is affected dramatically by deoxyribonuclease treatment during RNA isolation. This is because most of the RNA synthesized is covalently linked to DNA. This linkage has been demonstrated by density analysis in formaldehyde-CsCl gradients and by incorporation of alkali-stable ribonucleotides into DNA. The linear DNA templates transcribed were generated by treatment of circular DNA with restriction enzymes which, in addition to cutting once at a single primary site, were found also to produce single strand nicks at specific secondary sites. The discrete sized RNA bands observed result from initiation at these nicks and terminated at DNA ends. There are two modes of nick-dependent initiation. In one mode the 3'-hydroxyl terminus of the DNA at a single strand nick serves as a primer for the extension of an RNA chain. In a second mode de novo initiation of an RNA chain is promoted at the nick. RNAs which are not primed initiate predominantly with GTP. The catalytic action of wheat germ RNA polymerase II is similar to that of Escherichia coli core RNA polymerase which has also been shown to synthesize primarily RNA which is covalently linked to DNA.
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PMID:Transcription of simian virus 40 DNA by wheat germ RNA polymerase II. Priming of RNA synthesis by the 3'-hydroxyl of DNA at single strand nicks. 624 89

We have determined the nucleotide sequence of a 4.0-kilobase DNA fragment containing the genes of the PstI restriction-modification system. Two large open reading frames were identified within the sequence and were ascribed to the restriction enzyme and methylase by the analysis of a series of deletion mutants. The two genes are encoded on opposite DNA strands, and hence must be transcribed from separate promoters rather than as a polycistronic message. The sequence of the first 10 amino acids of the restriction endonuclease was determined by sequential Edman degradation of the purified protein, permitting the alignment of the polypeptide with the DNA sequence. The NH2 terminus of the modification enzyme was established by sequential Edman degradation of the protein synthesized in bacterial minicells with different radiolabeled amino acids. The initiation codons of the two genes are separated by 130 base pairs. The deduced amino acid sequences indicate that the restriction endonuclease contains 326 amino acids with a calculated Mr = 37,370; the modification enzyme is composed of 507 amino acids with a calculated Mr = 56,830. There is no significant homology between the two proteins at the level of the primary structure. Antibody raised against the purified restriction endonuclease did not immunoprecipitate the modification enzyme. The transcription initiation sites were mapped using mung bean nuclease. Both of the transcripts begin with adenosine. The initiation sites are separated by only 70 base pairs. This close proximity suggests that the promoters for the two divergent genes overlap. DNase I protection experiments show that Escherichia coli RNA polymerase has a higher affinity for the methylase promoter than for the restriction enzyme promoter.
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PMID:The organization and complete nucleotide sequence of the PstI restriction-modification system. 633 92

Further research on bacteriophage T7 DNA penetration mechanism into E. coli cells during the infection was carried out. The DNA-RNA-hybridization on nitrocellulose filters revealed that in the presence of chloramphenicol the T7 DNA penetration from the virion into a host cell was coupled with its transcription by the bacterial RNA polymerase. The data obtained indicate that in the absence of antibiotics the penetration of a part of T7 genome which correspondes to class II and III genes is coupled with its transcription by a phage-specific RNA polymerase. Along with this the host restriction-modification system when its activity is not inhibited by the phage-induced proteins will be able to cleave the penetrated T7 DNA just after its transcription was accomplished. Considering these data along with our conception on direct involvement of transcription in T7 DNA penetration process during the infection one can suggest that E. coli RNA polymerase molecules which provide the phage DNA transport, are localized at the inner surface of cytoplasmic membrane.
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PMID:[Coupling of bacteriophage T7 DNA penetration with its transcription, during infection]. 635 19

Essentially all of the DNA polymerase alpha activity in CV-1 monkey cells could be extracted as an enzyme complex that used DNA substrates with a low primer:template ratio, such as denatured DNA, at least 25 times more efficiently than did purified alpha polymerase. This form of the enzyme was rapidly dissociated either by the nonionic detergent Triton X-100 or by chromatography on phosphocellulose to generate alpha polymerase and its protein cofactor complex, C1C2. Both alpha polymerase and C1C2 were then independently purified free of deoxyribonuclease, RNA polymerase, DNA ligase, and ATPase activities, and the C1C2 complex was shown to consist of at least two proteins. Purified C1C2, which exhibited no DNA polymerase activity, completely restored the ability of alpha polymerase to use denatured DNA. Although high concentrations of denatured DNA inhibited the activity of C1C2, which binds tightly to single-stranded but not double-stranded DNA, low concentrations catalyzed reconstitution of alpha polymerase with C1C2. The resulting enzyme complex was chromatographically distinct from alpha polymerase on DEAE-Bio-Gel, was no longer dependent upon addition of C1C2 in order to utilize denatured DNA as effectively as DNase I-activated DNA, and was not inhibited by high concentrations of denatured DNA. These properties of the purified reconstituted enzyme were indistinguishable from those native alpha X C1C2-polymerase.
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PMID:Preparation of DNA polymerase alpha X C1C2 by reconstituting DNA polymerase alpha with its specific stimulatory cofactors, C1C2. 688 71

High-resolution S1 nuclease mapping of mRNA synthesised in vivo, in vitro run-off transcription with RNA polymerase from Streptomyces lividans and gene fusions were used to analyse the transcriptional organization of the SalI restriction-modification system of Streptomyces albus G. The salIR and salIM genes that encode the restriction endonuclease and its cognate methyltransferase constitute an operon which is mainly transcribed from sal-pR1, a promoter located immediately upstream of salIR, with two possible minor promoters further upstream. Another promoter, sal-pM, is within the 3' end of the salIR coding region, and allows expression of the modification gene in the absence of sal-pR1. The sal-pM promoter might be involved in the establishment of modification prior to restriction endonuclease activity. Sequences upstream of the apparent transcriptional start sites for sal-pR1 and sal-pM show similarity with the -10 region of typical vegetatively expressed eubacterial promoters, but appropriately centered -35 regions are absent.
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PMID:Complex transcription of an operon encoding the SalI restriction-modification system of Streptomyces albus G. 831 78

Expression of recombinant herpes simplex virus type 1 (HSV-1) deoxyribonuclease (DNase) was analyzed in BHK-21 cells, a standard cell line for virus propagation, by using mammalian cell expression systems based on vaccinia virus and on Semliki Forest virus (SFV)1. Although the establishing of recombinant vaccinia virus failed due to the apparent toxicity of the herpesviral enzyme, soluble and functional HSV-1 DNase was efficiently expressed in BHK-21 cells by the vaccinia virus/T7 RNA polymerase hybrid system as well as by recombinant Semliki Forest virus. Using rabbit antiserum ExoC, directed against the C-terminal residues 503-626, or mouse monoclonal antibody (MAb) Q1, raised against the type 2 enzyme, a major 85-kDa protein with the identical size of the enzyme from HSV-1-infected cells was identified to be induced in both expression systems. With recombinant SFV functional HSV-1 DNase coincided with the overproduction of a single major 85-kDa protein reaching an optimum between 16 h and 36 h after infection. At later times of infection the enzymatic activity vanished. Thus, recombinant SFV may be an appropriate expression vector for biochemical studies of the enzyme when (i) packaged recombinant virus particles are used for infection and (ii) infection does not exceed 24 h. Due to the limitations of transient expression systems, the vaccinia/T7 RNA polymerase hybrid system is suited for expression analysis on a small scale, and for studying intracellular interactions of the enzyme as demonstrated by immunofluorescence microscopy studies. Using vector pTM1, recombinant HSV-1 DNase was efficiently overproduced in BHK-21 cells at 6 h after transfection and was shown to colocalize with the cellular chromatin at sites apparently distinct from the bulk of the herpesviral replication sites the way it is observed for the enzyme of lytically infected cells. The deleting of the 123 C-terminal amino acid residues did not alter this nuclear localization of HSV-1 DNase, suggesting that the latter sequences and other herpesviral factors are not required for the chromatin association.
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PMID:Expression analysis of recombinant herpes simplex virus type 1 DNase. 985 86

After synthesis of short, nascent oligonucleotide in the presence of (32P)DNA, GTP, CTP, UTP and 3'dATP, one can excise with deoxyribonuclease a ternary complex of RNA polymerase, protected DNA and oligonucleotide, while the enzyme simply bound to the template is removed by increasing the ionic strength. This ternary complex is retained on nitrocellulose membranes. On polyacrylamide gel electrophoresis it migrates faster than RNA polymerase alone. The protected portion of the DNA is constituted of about 75 nucleotides. It might represent the sites for RNA initiation.
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PMID:??? 1194 67

Sen1p in Saccharomyces cerevisiae is a Type I DNA/RNA helicase. Mutations in the helicase domain perturb accumulation of diverse RNA classes, and Sen1p has been implicated in 3' end formation of non-coding RNAs. Using a combination of global and candidate-specific two hybrid screens, eight proteins were identified that interact with Sen1p. Interactions with three of the proteins were analyzed further: Rpo21p(Rpb1p), a subunit of RNA polymerase II, Rad2p, a deoxyribonuclease required in DNA repair, and Rnt1p (RNase III), an endoribonuclease required for RNA maturation. For all three interactions, the two-hybrid results were confirmed by co-immunoprecipitation experiments. Genetic tests designed to assess the biological significance of the interactions indicate that Sen1p plays functionally significant roles in transcription and transcription-coupled DNA repair. To investigate the potential role of Sen1p in RNA processing and to assess the functional significance of the Sen1p/Rnt1p interaction, we examined U5 snRNA biogenesis. We provide evidence that Sen1p functions in concert with Rnt1p and the exosome at a late step in 3' end formation of one of the two mature forms of U5 snRNA but not the other. The protein-protein and protein-RNA interactions reported here suggest that the DNA/RNA helicase activity of Sen1p is utilized for several different purposes in multiple gene expression pathways.
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PMID:Multiple protein/protein and protein/RNA interactions suggest roles for yeast DNA/RNA helicase Sen1p in transcription, transcription-coupled DNA repair and RNA processing. 1512 1

Partially purified nuclei from citrus exocortis viroid (CEV)-infected Gynura aurantiaca are able to synthesize linear and circular viroid molecules. Pretreatment of the nuclei with actinomycin D or deoxyribonuclease did not affect viroid synthesis, whereas the synthesis of other cellular RNAs was severely reduced. These observations support the essential role of CEV complementary RNA sequences in viroid replication. However, when alpha-amanitin was included in this in vitro synthesis system, CEV replication was markedly reduced by concentrations of 10 nM or greater. Taken together, these data support the proposition that viroid synthesis is catalyzed by a DNA-dependent RNA polymerase acting on a RNA template.
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PMID:Properties of a cell-free system for synthesis of citrus exocortis viroid. 1659 39

Adenovirus deoxyribonucleic acid (DNA) was used as template for the in vitro synthesis of viral-specific ribonucleic acid (RNA). When the kinetics of the reaction were compared by using native and heat-denatured DNA templates, the latter synthesized RNA at a slower rate. The fate of the DNA after acting as template and physical characteritstics of the RNA product were studied. The DNA template, according to its sedimentation rate, was not significantly degraded by the Micrococcus lysodeicticus RNA polymerase. The products of the RNA polymerase reaction had the following properties. (i) Hybridization experiments revealed a high degree of complementarity (50 to 70%) for its homologous DNA. (ii) A very low complementarity (6 to 7%) was found for its heterologous DNA. (iii) The sedimentation rate of the synthetic RNA in a sucrose gradient was 5 to 10S when native DNA was used as the template. When heat-denatured DNA was used, the resulting RNA product, free of the template, sedimented at a rate of 3 to 16S. A rapidly sedimenting (>30S) DNA-RNA complex resulted when denatured DNA was the template. The DNA moiety of the complex was sensitive to 125 mug of deoxyribonuclease per ml. The RNA of the complex, however, was fully refractory to 50 mug of ribonuclease per ml. When the adenovirus DNA was sonically treated and then used as template, the RNA product sedimented at 3 to 9S. The heat-denatured sonically treated DNA template yielded a DNA-RNA complex that also sedimented at an unusually fast rate (>18S).
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PMID:Properties of adenovirus messenger ribonucleic Acid synthesized in vitro. 1678 98


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