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)

Reverse transcriptase (RT; RNA-dependent DNA nucleotidyltransferase) from Rauscher leukemia virus is synthesized in infected cells by way of a read-through poly- rotein of 200,000 molecular weight. This polyprotein (Pr200(gag-pol)) was precipitated by antiserum to RT; in a previous study all the monospecific antisera to gag proteins recognized Pr200(gag-pol). Pr200(gag-pol) contains both p30 and RT peptide sequences. Intermediate RT-related precursors of 145,000 (Pr145(pol)), 135,000 (Pr135(pol)), and 125,000 (Pr125(pol)) molecular weights were specifically recognized by precipitation from infected cell extracts by antiserum to RT. These proteins shared methionine-containing tryptic peptide sequences with a virion polypeptide of 80,000 molecular weight (p80(pol)) precipitate by antiserum to RT. Purification of active RT enzyme from virions labeled with [(3)H]methionine showed that p80(pol) was the major component, based on analysis by gel electrophoresis and tryptic peptide mapping experiments. A polypeptide (Pr80(pol)), similar in size to mature viral p80(pol), was also precipitated from infected cells by antiserum to RT. Its peptide map was nearly identical to that of virion p80(pol). Pulse-chase studies showed that Pr80(pol), Pr125(pol), and Pr135(pol) were stable polypeptides, whereas Pr200(gag-pol) and Pr145(pol) were unstable precursors. Pulse-chase studies with the protein synthesis inhibitor, cycloheximide, showed that the processing of Pr200(gag-pol) occurred for a short time in the absence of protein synthesis.
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PMID:Biosynthesis of reverse transcriptase from Rauscher murine leukemia virus by synthesis and cleavage of a gag-pol read-through viral precursor polyprotein. 7 22

Nucleoli isolated from Novikoff hepatoma cells of the rat were previously shown to carry out synthesis of predominantly ribosomal precursor RNA and methylation of this RNA in vitro. In order to develop in vitro systems for further detailed study of these processes and their interrelationships, isolated nucleoli were incubated in a complete RNA-synthesizing medium using (5-3H)cytidine 5'-triphosphate or S-adenoxyl(methyl-3H)methionine to measure the activities of RNA synthesis and methylation, respectively, under the same reaction conditions. Methylation of the ribose of the nascent ribosomal precursor RNA predominated. It occurred in close coordination with the transcriptional step by RNA polymerase as shown by the kinetic data, the analysis of labeled RNA in sucrose gradients, the inhibition by increased ionic strength or actinomycin D, and the release of labeled nucleotides by a 3'-exonuclease, venom phosphodiesterase. Methylation of the RNA bases occurred more slowly, continued longer after transcription ceased, and appeared to follow later in the processing of the RNA. Certain divalent cations (Mg2+, Mn2+, and Ca2+ at higher concentrations, and Zn2+ and Cu2+) inhibited both RNA synthesis and methylation to similar extents. RNase inhibitors (bentonite and dextran sulfate) at low concentration inhibited methylation while stimulating RNA synthesis, and pyrophosphate greatly decreased RNA synthesis with relatively little effect on methylation. These results indicated that RNA polymerase and ribosomal RNA methylases can function independently despite their close relationship. An exogenous substrate for the nucleolar rRNA methylases was found: nuclear RNA prepared from Novikoff hepatoma cells, cultured in the absence of methionine, served as a good substrate for methylation of both ribose and bases. Other exogenous RNAs, including cytoplasmic ribosomal RNA from these methionine-starved cells, nucleolar RNA from normal cells, and wheat germ ribosomal RNA were almost devoid of methyl-acceptor activity. A description of these parameters helps establish isolated nucleoli as a suitable system for further study of interaction of RNA polymerase, methylases, and nucleases in control of synthesis of ribosomal RNA.
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PMID:Interrelationships between synthesis and methylation of ribosomal RNA in isolated Novikoff Tumor nucleoli. 16 25

Polyoma virus complementary RNA, synthesized in vitro by using highly purified Escherichia coli RNA polymerase and nondefective form I polyoma DNA, was translated in a wheat germ cell-free system. Polypeptides were synthesized that comigrated on sodium dodecyl sulfate-polyacrylamide gels with the polyoma capsid proteins VP1 and VP2, although most of the cell-free products were of smaller molecular weights. The VP1-size protein specifically immunoprecipitated with anti-polyoma virus serum, and upon digestion by trypsin yielded [35S]methionine-labeled tryptic peptides that co-chromatographed with the [3H]methionine-labeled tryptic peptides of virion-derived VP1 on both cation-exchange and anion-exchange resins. The VP2-size in vitro product contained all the virion VP2 methionine-labeled tryptic peptides, as shown by cation- and anion-exchange chromatography and two-dimensional fingerprinting on cellulose. We conclude that full-length polyoma VP1 and VP2 are synthesized in response to complementary RNA and consequently that the viral capsid proteins VP1, VP2, and VP3 are entirely virus coded.
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PMID:Polyoma virus complementary RNA directs the in vitro synthesis of capsid proteins VP1 and VP2. 20 20

Polyacrylamide gel electrophoresis and tryptic peptide fingerprint analysis of the proteins made in a cell-free system derived from L-cells and immunoprecipitated with simian virus 40 (SV40) anti-T serum demonstrated that both SV40 large-T and small-T antigens are synthesized in vitro in response to mRNA isolated from productively infected CV1 CELLS. Sucrose density centrifugation in gradients containing 85% formamide showed that the mRNA's for both forms of T-antigen sediment at about 17.5S, with the mRNA for small-t sedimenting marginally, but reproducibly, ahead of the mRNA for large-T. Hybridization experiments using restriction endonuclease fragments Hae III-E and Hind II/III-B showed that all fractions active in the cell-free synthesis of both forms of T-antigen hybridized equally to both fragments. This suggests that the mRNA's for SV40 T-antigens are at least partly virus coded and that the bulk of the early SV40 mRNA contains sequence information from both ends of the early region. The data are consistent with the suggestion that the large-T mRNA is spliced. SV40 complementary RNA (the product of transcription of SV40 DNA using Escherichia coli RNA polymerase) was also translated in the L-cell system and gave two families of polypeptides which specifically immunoprecipitate with anti-T serum. One family (the small-t family) includes a polypeptide indistinguishable by gel electrophoresis and tryptic peptide fingerprinting from small-t isolated from cells. The other family (the 60K family) has a major component with molecular weight approximately 60,000 and includes other polypeptides with molecular weights ranging from approximately 14,000 to about 70,000. The 60K family has petides in common with large-T but not with small-T. Together, the peptides of the small-t and 60K families account for virtually all of the methionine peptides of SV40 large-T. We conclude from these results (i) that small-t is probably entirely, and large-T at least predominantly, virus coded; (ii) that the small-t and 60K families represent the translation products of two different portions of the early region of SV40 DNA (approximately 0.65 to 0.55 map units and 0.54 to 0.17 map units); and (iii) that although most, if not all, of the large-T and small-t peptides are present in the cell-free product, some feature of sequence arrangement of SV40 complementary RNA prevents the translation of full-length large-T and results instead in the synthesis of fragments. We suggest that the absence of a splice in the complementary RNA is responsible for this result.
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PMID:Cell-free synthesis of simian virus 40 T-antigens. 21

The free 4S RNA of avian RNA tumor viruses is greatly enriched in one of the four methionine tRNAs of the host cells, tRNA4Met. On the assumption that viral tRNAMet forms are identical to the corresponding tRNAs of mouse or chick cells, the following conclusions were drawn concerning the tRNAMet content of oncornaviruses: (1) tRNAMet species may be compartmentalised within the host cells, and the viral tRNA pool could reflect the cellular compartment in which viral maturation takes place since tRNAMet forms distribute unevenly between different fractions of a cell homogenate. (2) tRNA4Met appears to have no special role in the modulation of protein synthesis in as much as no functional difference between tRNA2Met and tRNA3Met, tRNA4Met could be demonstrated in in vitro protein synthesising systems. (3) tRNA4Met differs in nucleotide sequence from all other host cell tRNAMet forms except possibly tRNA2Met. The nucleotide sequences of two tRNAMet species, tRNA1Met and tRNA4Met, have already been determined and the sequence of another host cell tRNAMet, tRNA3Met, was derived from the analogy of its sequence to that of tRNA4Met since the two molecules differ in only 6 nucleotides out of 76. (4) Avian myeloblastosis virus reverse transcriptase has been shown to bind specifically tRNA4Met and tRNATrp in whole cell tRNA and therefore the free tRNA4Met in the virion particle may exist substantially bound to virion-associated transcriptase.
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PMID:Selection of methionine tRNAs by avian oncornaviruses. 21 69

Upon addition of excess one carbon metabolites (including serine)bacteria stop growing because of isoleucine starvation. After such treatment stringent bacteria rapidly resume normal growth whereas relaxed mutants remain unable for some time to grow. We show here that this is due to a lack of derepressibility of ilv genes after the starvation period. Results are also presented which show that RNA polymerase structural mutants may be selected among the clones resistant to a mixture of serine, methionine and glycine, in relA- strains. Finally circumstancial evidence suggests that the one carbon metabolism may be involved in a process controlling isoleucine metabolism.
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PMID:Correlation between the serine sensitivity and the derepressibility of the ilv genes in Escherichia coli relA- mutants. 36 63

An improved purification procedure is described for the sigma subunit of escherichia coli DNA-dependent RNA polymerase [ribonucleoside triphosphate:RNA nucleotidyl-transferase, EC 2.7.7.6]. The method involves chromatography of purified RNA polymerase on single-stranded DNA-agarose, Bio-Rex 70, and finally Ultragel AcA44. The sigma factor obtained is electrophoretically pure with a yield of about 40%. A number of the chemical--physical properties of sigma are presented. A molecular weight of 82,000 was determined by phosphate buffered sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Ultraviolet absorption spectra were used to determine an E280nm 1% of 8.4. The amino acid composition and 12-residue N-terminal sequence (Met-Glx-Glx-Asx-Pro-Glx-(Ser or Cys)-Glx-Leu-Lys-Leu-Leu) of sigma have been determined. The isoelectric focusing properties of sigma are presented. Denaturation--renaturation studies indicate that sigma is capable of an unusually rapid and complete recovery of activity after being subjected to denaturing conditions. A stable, 40,000-dalton fragment is generated from sigma by mild trypsin treatment.
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PMID:Purification and properties of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase. 37 77

Minicells segregated from Escherichia coli chi925 carrying a drug-resistance plasmid were separated from nucleated cells by differential centrifugation and purified by rate-zonal centrifugation in sucrose gradients. Minicells purified in this way were capable of donating the plasmid to nucleated cells. They also incorporated thymidine, uridine and methionine into macromolecules. Methods are described for purification of plasmid-containing minicells on a scale large enough to allow isolation of DNA, DNA polymerase and RNA polymerase in sufficient quantities for studies of enzymes involved in replication and transcription of plasmid DNA.
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PMID:Isolation by differential and zonal centrifugation of minicells segregated by Escherichia coli. 38 90

The fixation of tRNA to Escherichia coli RNA polymerase has been investigated. Bound and free tRNA have been separated and quantified after filtration through cellulose nitrate filters, centrifugation or sucrose gradients or electrophoresis in polyacrylamide gels. We detect no differences between the fixation of E. coli fMet-tRNAfMet, Met-tRNAmMet or uncharged unfractionated tRNA to RNA polymerase. Tight complexes, with a long residence time, are formed between core enzyme and tRNA with a dissociation constant of less than 1 nM. Complexes exist between tRNA and both monomer and dimer forms of the core enzyme. In the monomer complex, one tRNA is bound per alpha 2 beta beta' unit, whereas in the dimer complex only 0.5 tRNA molecule is fixed per alpha 2 beta beta' unit. In contrast to the core enzyme, very little tRNA fixes tightly to the holoenzyme at salt concentrations greater than 80 mM. At lower salt concentrations tRNA fixation results in a loss of sigma subunit from the holo enzyme to the resulting core enzyme where it binds tightly. DNA fixation reduces the binding of tRNA to RNA polymerase and tRNA fixation reduces the binding of DNA. However, binding of DNA to polymerase is not competitive with binding of tRNA, and ternary complexes between RNA polymerase, DNA and tRNA are shown to exist. Our results are discussed in relation to other studies concerning the effects of tRNA upon RNA polymerase.
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PMID:On the binding of tRNA to Escherichia coli RNA polymerase. 38 19

RNA polymerase II polypeptides present in [35S]methionine-labeled Chinese hamster ovary (CHO) cell extracts have been quantitatively immunoprecipitated with an anti-calf thymus RNA polymerase II serum. Analyses of the immunoprecipitates on sodium dodecyl sulfate polyacrylamide gels indicated that the immunoprecipitated polymerase II of both wild type CHO cells and the alpha-amanitin-resistant mutant Ama1 had polypeptides of molecular weight 214,000, 140,000, 34,000, 25,000, 23,000, 20,500, and 16,500. In heterozygous alpha-amanitin-resistant/alpha-amanitin-sensitive hybrid CHO cells, growth in the presence of alpha-amanitin results in the inactivation of the alpha-amanitin-sensitive RNA polymerase II activity and a compensating increase in the activity of the alpha-amanitin-resistant enzyme. Determination of the rates of synthesis and degradation of RNA polymerase II polypeptides using [35S]methionine labeling and polymerase II immunoprecipitation demonstrated that this increase in activity of alpha-amanitin-resistant polymerase II resulted from a co-ordinate increase in the rate of synthesis of at least three polypeptides of RNA polymerase II. At the same time, there was an enhanced rate of degradation of the alpha-amanitin-inactivated RNA polymerase II polypeptides.
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PMID:Regulated synthesis of RNA polymerase II polypeptides in Chinese hamster ovary cell lines. 43 82


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