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)

DNA-directed RNA polymerase is responsible for gene expression. Despite its importance, many details of its function and higher-order structure still remain unknown. We report here a local sequence similarity between the second largest subunit of RNA polymerase II and bacterial RNases Ba (barnase), Bi, and St. The most remarkable similarity is that the catalytic sites of the RNases are shared with the eukaryotic RNA polymerase II subunits of Drosophila melanogaster and Saccharomyces cerevisiae. Several amino acids conserved among the RNases and the RNase-like domains of the RNA polymerase subunits are located in the neighborhood of the catalytic sites of barnase, whose three-dimensional structure has been resolved. This observation suggests the functional importance of the RNase-like domain of the RNA polymerase subunits and indicates that the RNase-like domain may have RNase activity. The location of the RNase-like domain relative to the region necessary for RNA polymerization is similar to the relative proximity of 5'----3' or 3'----5' exonuclease and the region of polymerase activity of DNA polymerase I. The RNase-like domain might work in proofreading, as in RNA-directed RNA polymerase of influenza virus, or it may contribute to RNA binding through an unknown function.
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PMID:RNase-like domain in DNA-directed RNA polymerase II. 192 68

Glucocorticoid-induced muscle atrophy is associated with a decrease in the level of protein synthesis and a loss of RNA. This paper reports the behaviour of RNA polymerase I- and RNA polymerase II-directed transcription (EC 2.7.7.6) in nuclei isolated from skeletal muscles of rats given a catabolic dose of dexamethasone acetate (5 mg per Kg body weight) over a period of 4 days. Both activities were altered by the dexamethasone treatment. In the case of RNA polymerase I-mediated transcription there was a loss of template-engaged enzymes indicating the existence of an inhibition of initiation of transcription while the rate of elongation of bound enzymes was unaltered. The number of RNA polymerase II-chromatin bound enzymes was increased, but the mean polynucleotide elongation rate was reduced. The possibility that glucocorticoids may impair the elongation stage of transcription in skeletal muscle by increasing the frequency of premature termination of transcripts is discussed. No evidence was obtained for any increase in ribonuclease activity in muscle nuclei of dexamethasone-treated animals.
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PMID:Glucocorticoid-mediated muscle atrophy: alterations in transcriptional activity of skeletal muscle nuclei. 193 39

We have developed an assay that uses phenyl boronate agarose column chromatography to measure the capping efficiencies of RNA polymerases used for in vitro transcription of cloned cDNAs. Capped 32P-labeled ovalbumin mRNAs were synthesized by in vitro run-off transcription with SP6 or T7 RNA polymerase in the presence of cap analogs and digested to completion with T1 and T2 RNase. The resulting 3'-nucleoside monophosphates (NMPs) and cap structures were separated by chromatography on phenyl boronate agarose, and the ratio of radioactivity between the two was used to estimate the extent of transcript capping.
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PMID:A simple assay for determining the capping efficiencies of RNA polymerases used for in vitro transcription. 226 28

Reconstitution of influenza virus nucleoprotein (NP)-RNA complexes was performed with segment 8 RNA, which was synthesized in vitro from cDNA, and NP purified from virions. Under optimum conditions established using a filter binding assay and a gel retardation assay, NP was found to bind any RNA longer than 15 nucleotides. NP-RNA complexes formed at 30 degrees C are more resistant to high concentrations of NaCl than those formed at 0 degrees C. Treatment of NP with N-ethylmaleimide gave no effect on its RNA binding activity, whereas treatment with alkaline phosphatase enhanced its RNA binding activity. The newly developed "reverse-printing" method of RNase V1-treated complexes revealed that reconstituted NP-RNA complexes carry RNase V1-sensitive sites as do native ribonucleoprotein (RNP) cores (RNA polymerase-NP-RNA complexes), implying that RNA-NP complexes structurally similar to native RNP cores are reconstituted from isolated components.
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PMID:Reconstitution of influenza virus RNA-nucleoprotein complexes structurally resembling native viral ribonucleoprotein cores. 235 55

We examined the effects of convulsive seizures on in vitro RNA synthesis by cerebral cortex nuclei in El mice. The rate of incorporation of [3H]uridine-5'-triphosphate by intact nuclei during seizures was decreased to 47.4% compared with the rate during the interictal period, but gradually recovered. During the 30-min period after onset of seizures, the rate of RNA synthesis was significantly lower in El mice than in identically stimulated ddY mice. Seizures in El mice had no effect on liver RNA synthesis, suggesting that the alteration of RNA polymerase activity is specific to the brain. Analysis of gel electrophoresis of polyadenylated RNA synthesized in the presence of ammonium sulphate revealed a marked decrease in high-molecular weight RNA species 15 min after seizures in El mice compared with the pattern in nonstimulated ddY mice. This shift from high- to low-molecular weight RNA species was not attributable to RNase activity, but it appeared to be related RNA polymerase.
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PMID:Alteration of RNA synthesis in vitro in intact cerebral cortex nuclei induced by convulsions in seizure-susceptible El mice. 243 22

B lymphocytes from a patient with systemic lupus erythematosus (SLE) and several circulating autoantibodies (including antinucleolar antibodies) were immortalized by fusion with a hypoxanthine/guanine phosphoribosyl transferase (HGPRT)-deficient human B cell line. Multiple human monoclonal antibodies (mAb) were obtained which, in solid-phase enzyme immunoassay, were reactive with DNA. One mAb was of special interest because it reacted strongly with both single-stranded DNA and an extractable nuclear antigen found in rabbit thymus extract (RTE). In an immunofluorescent assay using fixed human cells, the latter mAb also bound predominantly to cell nucleoli. A combination of enzyme digestion and metabolic inhibitor studies of the target cells in this immunofluorescent assay suggested that the antigen(s) bound by the mAb was an RNA-associated protein or a ribonucleoprotein that is distinct from intact RNA polymerase I and not associated with the transcriptional units of the nucleolus. In other experiments, using fractions of RTE isolated by ion-exchange chromatography, the antigens bound by the mAb were shown to be highly negatively charged molecules. Immunoprecipitation and SDS-PAGE analyses of labeled cell extracts bound by the mAb revealed a doublet of 17 and 18 kD. Since the original patient's serum autoantibodies also bound to both an RNase-sensitive, acidic, extractable nuclear antigen and to nucleoli, and immunoprecipitated proteins of similar molecular masses in SDS-PAGE, it appears that the described mAb is a product of an immortalized autoantibody-producing B cell clone from the SLE patient's peripheral blood. This mAb probably defines a novel RNA-associated autoantigen residing predominantly in the nucleolus or, less likely, a variant of either RNA polymerase I or the ribosomal autoantigens (P proteins).
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PMID:Description and partial characterization of a nucleolar RNA-associated autoantigen defined by a human monoclonal antibody. 243 34

A method for the normalization of multiple RNA samples is described. This method exploits the recently developed technology which allows the synthesis of single-stranded, specific RNA molecules in vitro using either SP6 or T7 RNA polymerase to prepare an external standard cRNA. When this external standard cRNA is added to cell samples at the time of lysis, it becomes a stable, integral part of the RNA content of each sample, which can easily and reproducibly be detected and quantitated by either Northern blot or RNase protection. The feasibility of this approach to normalization has been tested in a mouse 3T3 cell model system. In multiple samples, the relative levels of this externally added standard transcript are shown to closely parallel the relative levels of an internal standard control transcript as well as the amount of RNA determined by spectrophotometric analysis. The data obtained demonstrate that an externally added, in vitro-synthesized transcript can serve as an accurate, universal means of normalizing multiple RNA samples, since it is not dependent on sample RNA concentration, species, cell or tissue type, or experimental manipulation.
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PMID:Normalization of multiple RNA samples using an in vitro-synthesized external standard cRNA. 244 10

Dinucleotides (3'-5')-ApU and UpA and their 3'-O-phosphonylmethyl and 5'-O-phosphonylmethyl analogues were studied as substrates in the primed abortive synthesis catalysed by Escherichia coli DNA-dependent RNA polymerase on poly[d(A-T)] template. All phosphonate analogues of dinucleotides containing the anomalous sugar-phosphate backbone are substrates for the holoenzyme as verified by RNase A and RNase T2 digestion of the trinucleotide analogues obtained. The finding that phosphonate dinucleotides act as primers for transcription indicates that steric requirements at the initiation site are not as specific as previously supposed. Analysis of kinetic constants of ordered bibi reaction Kia, KmA, KmB and Vmax suggests that the instability of short RNA-DNA hybrids contributes to the abortive release of trinucleotides formed.
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PMID:Phosphonate analogues of dinucleotides as substrates for DNA-dependent RNA polymerase from Escherichia coli in primed abortive initiation reaction. 248 57

Oligoribonucleotide duplexes containing one to four 2'-deoxynucleotide residues were used as substrates for ribonuclease V1 and RNase H. Either deoxyadenosine and/or deoxythymidine were incorporated into the duplex, 5'GGCCGGAUCCGCGC3'-5'GCGCGGAUCCGGCC3' by substitution of the appropriate deoxynucleoside triphosphate into a transcription reaction with T7 RNA polymerase. The melting temperature, Tm, of the duplex (1.8 microM in strands in 50 mM NaCl) containing only ribonucleotides was 79.9 degrees C. Substitution of deoxyadenosine in both strands of the duplex lowered the Tm by 2.4 degrees C. Substitution of deoxythymidine had no measurable effect on the Tm. Comparison of RNase V1 digestion patterns of fully ribonucleotide and deoxy-substituted duplexes suggest that any distortion is localized to the site of the substitution. An oligoribonucleotide containing two deoxy residues directs specific cleavage of RNA by E. coli RNase H. Structural requirements for cleavage are proposed for RNase V1 and RNase H.
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PMID:Deoxynucleotide-containing oligoribonucleotide duplexes: stability and susceptibility to RNase V1 and RNase H. 255 16

Only three forms of Kunjin virus-specified RNA were isolated from cytoplasm early after the latent period (about 15 hr) viz., 44 S genomic-sized single-stranded RNA, 20 S double-stranded "replicative form" (RF), and 20-28 S partially ribonuclease-resistant (about 70%) "replicative intermediate" (RI). The RF and RI were resolved by electrophoresis in aqueous-agarose gel only following LiCl fractionation. The RI did not enter urea-polyacrylamide gels. After denaturation of untreated or RNase-treated RI and RF, only 44 S RNA was present in electropherograms. RNA polymerase activity at 8 hr postinfection was detected by in vitro assays of cytoplasmic extracts and reached a maximum at 24 hr, the only major labeled product being RF; a trace amount of free 44 S RNA was also produced. These results, and the kinetics of incorporation of [3H]uridine into RI, RF, and 44 S RNA in pulse and pulse-chase experiments, formed the basis of a model in which flavivirus RF functions as a recycling template for semiconservative and (mainly) asymmetric replication, on which only one nascent strand is synthesized per cycle.
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PMID:Replication strategy of Kunjin virus: evidence for recycling role of replicative form RNA as template in semiconservative and asymmetric replication. 257 39


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